Download Structure and Function of the Brain Serotonin System

PHYSIOLOGICAL REVIEWS
Vol. 72, No. 1, January
Printed
1992
in U.S.A.
Structure
and Function of the Brain Serotonin System
BARRY
L. JACOBS
AND
EFRAIN
C. AZMITIA
Program in Neuroscience, Department
of Psychology, Princeton
University, Princeton, New Jersey; and
Center for Neural Science, Department
of Biology, New York University, New York, New York
I.
INTRODUCTION
In the course of reviewing the anatomy and physiology of central nervous system (CNS) serotonin [5=hydroxytryptamine
(50HT)] in vertebrates, we focus on
three major themes. First, although 5-HT has been implicated in a vast array of physiological
and behavioral
processes in vertebrates,’ it appears to be essential for
’ This may also apply to invertebrates, but their somatic diversity and the ganglionic organization of their nervous systems make it
0031-9333/92 $2.00 Copyright 0 1992 the American Physiological
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I. Introduction
.........................................................................................
II. Historical Perspective ...............................................................................
III. Anatomy .............................................................................................
A. Introduction ......................................................................................
B. Nuclei ............................................................................................
C. Afferents .........................................................................................
D. Pathways and terminals .........................................................................
E. Distribution of receptors ........................................................................
F. Ultrastructure
...................................................................................
G. Summary .........................................................................................
IV. Development .........................................................................................
A. Introduction ......................................................................................
B. Nuclei ............................................................................................
C. Pathways and terminals .........................................................................
D. Tissue culture ....................................................................................
E. Growth regulatory factors .......................................................................
F. Summary .........................................................................................
V. Adult Plasticity ......................................................................................
A. Introduction ......................................................................................
B. Regeneration .....................................................................................
C. Collateral sprouting ..............................................................................
D. Transplantation
..................................................................................
E. Summary .........................................................................................
VI. Cellular Neurophysiology
...........................................................................
A. Unit activity in anesthetized animals or brain slices ...........................................
B. Unit activity in behaving animals ...............................................................
VII. Postsynaptic Actions ................................................................................
A. Introduction ......................................................................................
B. Inhibition/modulation
...........................................................................
...........................................................................
C. Excitation/modulation
D. Cotransmission ..................................................................................
E. Latency and duration of action ..................................................................
F. Summary .........................................................................................
VIII. Discussion and Speculation .........................................................................
A. Activity and actions of serotonergic neurons ...................................................
B. Growth/plasticity
and physiology ...............................................................
C. Two serotonergic systems .......................................................................
none of them (92a, 236a, 507). This apparent contradiction is reconciled by the concept that the expansive system of serotonergic neurons exerts a tonic modulatory
influence on its widespread targets. This is expressed
primarily
in association with the organism’s motor activity and is in phase with the sleep-wake-arousal
cycle.
difficult to encompass them within this generalization. Nonetheless,
similar conclusions to those made here have been arrived at by a number of investigators working in this field. The reader is referred to
articles by Kravitz (270), Kupfermann et al. (273), and Lent (287).
Society
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BARRY
L. JACOBS
AND
II.
HISTORICAL
PERSPECTIVE
The brief history in this section describes the seminal phases of research in this area, especially as they
pertain to this review.
The central or midline (raphe) location of the large
neurons in the brain stem has attracted the attention of
anatomists
since the time of Ramon y Cajal (396), who
described these cells as large multipolar
neurons with
uncertain
projections.
No one suspected
that they
contained
the same chemical
substance
distributed
throughout
the body. For many years, investigators
had
known of a blood-borne
chemical that produced vasoconstriction
(a “serum”
factor that affected blood vessel
“tonus,”
hence the name serotonin)
and of a substance
present in the gut that increased intestinal motility (enteramine). In the mid-twentieth
century, 5-HT, the single compound producing both these effects, was isolated
and synthesized,
and its molecular structure
was elucidated (204,399,400). Shortly thereafter,
5-HT was found
to be present in the mammalian
CNS in significant
quantities and to be concentrated
in varying amounts in
different regions of the brain (18, 473). This led to the
proposal of 5-HT as a CNS neurotransmitter.
A great
deal of interest soon focused on 5-HT because of its presumed involvement
in the major psychoses. This hypothesis was based primarily
on the finding that the
actions of 5-HT in the periphery
were antagonized by
the powerful
hallucinogenic
(or psychotomimetic)
drug
d-lysergide
(d-LSD)
( an extensive review of the relationship between the action of hallucinogenic
drugs and
5-HT can be found in Refs. 232-234). Around the same
time, Brodie and Shore (90) displayed extraordinary
foresight
in proposing that 5-HT and norepinephrine
might act as opposing central neurochemical
systems,
somewhat akin to epinephrine
and acetylcholine
in the
peripheral autonomic nervous system.
As is common with many advances in neuroscience,
the next significant step in 5-HT research was anatomic
in nature. Employing
the newly refined Falck-Hillarp
histochemical
fluorescence
technique, Dahlstrom
and
C. AZMITIA
Volume
72
Fuxe (117,118,173) described the localization of the cell
bodies and axon terminals of rat brain neurons containing 5-HT. This description
of serotonergic
neurons answered Ramon y Cajal’s question about the extent of the
projections
of the raphe neurons. The information
also
provided a much needed “wiring
diagram” for this neurochemical
system. It is now accepted that this brain
stem raphe system comprises
the most expansive and
complex anatomic/neurochemical
system in the mammalian CNS (35).
The next piece of information
set the stage for
much of the neurophysiological
research on 5-HT over
the ensuing 20 years. On the basis of the aforementioned
histofluorescence
results, that the cell bodies of 5-HT
neurons were clustered along the midline in the brain
stem, microelectrodes
were lowered into this region of
the rat brain. Aghajanian
et al. (6) found that these
neurons consistently
displayed a distinctive
slow and
highly regular discharge pattern, which was established
as a bioelectric
“signature”
for this neurochemical
group of neurons.
The last set of advances relevant to this review conterns the plastic nature of brain serotonergic
neurons
and involves three related research areas: development
of specific chemical neurotoxins,
regeneration
and collateral sprouting
in the CNS, and transplantation
and
culturing of fetal neurons. Neurotoxins
to 5-HT enabled
inputs from this system to be destroyed without producing mechanical injury to other neural and nonneural
systems (57,58). This permitted researchers
to study the
process of regeneration
of damaged serotonergic
axons
and to establish that new sprouts not only formed (70)
but also reestablished
functional contacts (363). By using intracerebral
microinjections
of neurotoxins
into individual serotonergic
pathways,
homotypic
collateral
sprouting of undamaged serotonergic
fibers was shown
to restore the structure
and function of lost serotonergic
axons (41). The transplantation
of fetal serotonergic
neurons into the adult brain provided a method for replacing or augmenting the normal innervation
and demonstrated that both fetal and adult neurons respond to
the same trophic factors (46, 71). The ability of fetal
serotonergic
neurons to grow in tissue culture provided
a controlled environment
for quantifying
the survival
and growth of serotonergic
neurons, making the search
for endogenous trophic and toxic molecules feasible (49).
Finally, the topic of CNS 5-HT receptor subtypes
h as recently attracted a great deal of interest. Sections
of this review describe their anatomic distribution
and
th e postsynaptic
actions associated with them. However, the topics of their associated intracellular
second
messenger systems and ion channels and their molecular biology are beyond the scope of this paper. These
latter two topics have been the subjects of recent reviews (169a 9 357a 9 507) .
III.
ANATOMY
A Introduction
’
Visualization
of neurotransmitters
ch emical fluorescence, radioautography,
in situ by histoand immunocy-
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Thus serotonergic
neurons regulate the expression
of
neural circuits without
being a necessary component of
them. Second, although the firing rate of serotonergic
neurons is unperturbed
by a variety of physiological
variables, the growth and terminal density of these neurons, both during development and after injury, are responsive to many of these same manipulations.
We propose that the degree of serotonergic
innervation
of their
target sites is, in fact, facilitated
by this dissociation.
Finally, there are evolutionary
trends in the anatomy of
this system: in lower vertebrates,
serotonergic
neurons
have fine unmyelinated
axons that are highly collateralized, whereas in higher vertebrates
there is evidence
for significant
myelination
of serotonergic
axons and
fewer collaterals.
In this context, we also discuss the
possibility
of an evolutionary
trend toward subsystems
of serotonergic
neurons with more precise anatomic projections subserving
more specific physiological
functions.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
B. Nuclei
The 5-HT cell bodies in the brain are located in the
brain stem on or near the midline and can be divided
into superior and inferior groups. This division is based
on the early developmental
appearance
of mesencephalic and myelencephalic
groups (see sect. IvB). The
original classification
designated the 5-HT fluorescent
clusters as Bl-B9 (117). The nomenclature
used in this
review has been agreed upon by a subcommittee
of the
Serotonin Club and does differ in some respects from
published studies. We make an attempt in this section to
clearly point out the changes.
The superior group consists of four main nuclei:
caudal linear nucleus (CLN: B8). median raphe nucleus
BRAIN
SEROTONIN
SYSTEM
167
(MRN; B8 and B5, previously referred to as nucleus centralis superior)
and its laterally displaced cells in nucleus pontis centralis oralis, the lateral B9 neurons lying
just dorsal to the medial lemniscus (ML), and the dorsal
raphe nucleus (DRN; B7 and B6). The inferior group
consists of five main nuclei: nucleus raphe obscurus
(NRO; BZ), nucleus raphe pallidus (NRPa; Bl and B4),
nucleus raphe magnus (NRM; B3), the neurons in the
ventrolateral
medulla [lateral paragigantocellular
nucleus (LPGN)
and the intermediate
reticular
nuclei
(IRN)] (Bl/B3), and the area postrema. The anatomy of
these groups has been reviewed
extensively
in many
species, including humans (465), Old and New World
monkeys (45,150, ZZ), cats (54,236), and rats (305,374,
456). In this review, we survey the serotonergic
neurons
in rats, cats, and primates. The general location of these
cell bodies within the mammalian
brain can be seen in
Figure 1, which displays their distribution
in a sagittal
view of the cat brain stem. In addition, Table 1 presents
the approximate
number of serotonergic
neurons in
each of the main clusters for both cats and rats.
1. Superior brain stem group
I)CAUDAL LINEAR NUCLEUS(~8). The most rostra1
group of serotonergic neurons is in the CLN, which extends from the level of the red nucleus caudal to the
anterior border of the superior cerebellar decussation
(SCD). The 5-HT neurons are located between the rootlets of the oculomotor nuclei and extend dorsally from
the anterior edge of the interpeduncular nucleus to the
DRN. These neurons have similar efferents to those described for the DRN but not the MRN (229). Furthermore, there is a different dendritic morphology between
the CLN and the MRN (222). Therefore these cells
should be considered as distinct from the more caudal
cells in the MRN and similar to the dorsal cells in the
DRN (see sect. IvBI).
II)MEDIANRAPHENUCLEUS(B~AND
~5). TheMRN
is a paramedian and median cluster of cells lying below
and caudal to the SCD (Fig. 2). The group has a rostrocaudal oblique orientation (34,305), and the paramedian
columns are separated by as much as 400 pm in cats and
monkeys. Rostrally, the cells from this group end
around and within the caudal and lateral aspects of the
interpeduncular nucleus. Caudally, the ventral border of
this group is the trapezoid body, and the dorsal border
fuses with the ventromedial (interfascicular)
component of the DRN. In cats the MRN begins as a single
cluster of cells at the level of the dorsal tegmental nucleus (236). The cluster lies in paramedian columns extending ventrally from the medial longitudinal fasciculus (MLF). The main cluster of neurons extends ventrolaterally from the SCD to an area just dorsal to the ML.
The small paramedian cluster of 5-HT cells with a
few cells lying directly on the midline formerly known
as B5 (nucleus raphe pontis) can be classified as the
caudal border of the MRN (B8). The cells are located
ventral to the MLF and extend caudally to the rostra1
end of the abducens nucleus. At the caudal portion of
this group, the cells are situated dorsal to the NRM (B3).
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tochemistry
changed our view of the nervous system.
Before their introduction,
brain anatomy could be summarized with black arrows
indicating
the connections
between one group of cells and their targets along a
particular
pathway. With the advent of these new techniques, the dynamics and organization
of specific neurochemical systems could be studied. Among the systems
that have been elucidated, the serotonergic
system is
the most expansive. The serotonergic
cell bodies are restricted to clusters of cells in the brain stem, but their
fibers, using most of the known pathways,
innervate
nearly every area of the brain.
In this section, we review the location of the cell
bodies both of the superior (ascending) and inferior (descending) groups of serotonergic
neurons. The afferents
to these cells are described both anatomically
and neurochemically. Associations
between serotonergic
neurons
and nonneuronal
cells are discussed. The efferents are
reviewed to illustrate
the size and characteristics
of the
axons, the wide number of pathways
used, the general
and precise patterns of innervation,
and the evidence for
both junctional
and nonjunctional
contacts. The anatomic distribution
of the 5-HT receptor subtypes
are
reviewed. In addition there is evidence that the various
serotonergic
nuclei possess individual
characteristics,
such as the presence of cotransmitters,
which may influence their anatomy and physiology.
Before proceeding, it is important
to obtain some
general feeling for the numbers involved when we discuss 5-HT anatomy in the CNS. It may seem somewhat
surprising
that so much has been written
about so little,
i.e., the neurons in the entire mammalian
CNS number
in the billions, whereas serotonergic
cells number in the
thousands.
Therefore
they constitute
-l/l,OOO,OOO of
all CNS neurons. However, their influence on their target sites appears to go far beyond these numbers. In the
rat brain it is estimated that there are -6 X lo6 serotonergic varicosities/mm3
cortical tissue. By extrapolation, this means that each serotonergic
neuron projecting to the cortex may be responsible
for 5 X lo5 serotonergic varicosities,
that each of their cortical target
and that serotonerneurons receives ~200 varicosities,
gic terminals
may account for as many as l/500 of all
axon terminals
in rat cortex (29).
OF
168
BARRY
L. JACOBS
AND
EFRAIN
C. AZMITIA
Volume
72
Scattered 5-HT cells of the MRN are seen ventrolateral to the MLF. These laterally situated cells, especially prominent in monkeys and cats, lie in the nucleus
pontis centralis oralis (367) and form a ring around the
central tegmental tract, one of the most primitive
ascending pathways
carrying reticulothalamic
axons.
III)DORSALRAPHENUCLEUS(B~AND
~6). TheDRN
1. Number (bilateral counts) and location of
serotonergic neurons in cat and rat brains
TABLE
No. of Serotonergic
Brain
Region
Linearis
intermedius
Raphe dorsalis
Raphe medianus
Raphe magnus
Raphe pallidus
Raphe obscurus
Nonmidline*
Total
Cat
2,000
24,000
7,000
2,000
8,000
2,000
15,000
60,000
Neurons
Rat
(25)
(70)
(35)
(15)
(50)
(35)
11,500
1,100
2,600
1,500
1,300
2,500
20,500
Numbers
in parentheses
are serotonergic
neurons
as a % total
neuronal
population.
Linearis
intermedius
numbers
for rat are included in raphe medianus
and nonmidline.
* At all brain stem levels from medulla
oblongata
to caudal mesencephalon,
a band of scattered serotonergic
cells forms a lateral extension
from ventral
raphe.
The most lateral
of these cells are located in relation
to rubrospinal
bundle (which
serves as a good landmark
at its location
lateral
to
lateral
reticular
nucleus,
facial nucleus,
superior
olive, etc.). From
rostra1 medulla
oblongata
to mesencephalon
there exists a lateral
extension also from dorsal raphe, which consists of numerous
serotonergic cells within
periventricular
gray and subjacent
tegmentum.
At
pontine
and most caudal mesencephalic
levels, scattered
serotonergic
cells in lateral
tegmentum
connect lateral extensions
from dorsal and
ventral
raphe. Thus, at pontine
levels, distribution
of serotonergic
cells forms bilateral
“rings”
meeting
in raphe nuclei in the midline.
[From Wiklund
et al. (511).]
is divided into medial, lateral (the wings), and caudal
components. The medial component can be further divided into a mediodorsal (superior) and a medioventral
(interfascicular) component (Fig. 2). The superior component is in the central gray just below the cerebral
aqueduct. It is much larger in its rostra1 aspect (level of
the trochlear nuclei) and becomes smaller as it extends
caudally. The cells extend rostrally to the caudal border
of the oculomotor nuclei. The interfascicular component
surrounds the MLF and is especially prominent between
the fasciculi. It has been called the nucleus annularis,
the interfascicular nucleus, or the pars dorsalis of the
superior central nucleus (34,36). The neurons extend to
merge with B6 caudally and lie just dorsal to MRN behind the SCD. There is developmental and morphological evidence for the caudal part of this group of 5-HT
neurons being more closely related to MRN than the
other components of DRN (see sect. IVES).
The lateral component (the wings) forms the larger
division of the DRN and extends as far rostrally as the
oculomotor nuclei. This can be seen in rats and cats but
is most prominent in primates. The wings are best developed at the level of the trochlear nucleus. This laterally
situated group extends caudally and can be seen in the
central gray of the pons just below the fourth ventricle.
In humans, the lateral wings can be divided into a dorsal
and ventral subdivision (465).
IV) B9. This group is located along the superior surface of the ML (supralemniscal) from the rostra1 border
of the inferior olive to the level of the red nucleus (Fig.
2). These cells are occasionally continuous with the paramedian cells of the MRN. The similar cytological and
fluorescent characteristics of the cells in MRN and B9
were first noted in squirrel monkeys (227). In cats, these
numerous cells are continuous with the MRN and form
the ventral border of the ring of scattered cells that
surrounds the central tegmental tract in the pontine
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FIG. 1. Drawing
of midsagittal
view
of cat brain
stem with 5-hydroxytryptamine
(5-HT)-immunoreactive
cells indicated
by black dots. Scale at bottom
indicates
anteroposterior
levels in stereotaxic coordinates
marked
off in millimeters.
AQ, cerebral
aqueduct;
CLN,
caudal linear nucleus; DRN, dorsal raphe
nucleus;
MRN,
median
raphe
nucleus;
NRM,
nucleus raphe magnus;
NRO, nucleus raphe
obscurus;
NRPa,
nucleus
raphe pallidus;
V4, 4th ventricle.
[From
Jacobs (234b).]
January
1992
STRUCTURE
AND FUNCTION
OF BRAIN
SEROTONIN
SYSTEM
169
reticular formation. These findings confirm the similarity between these two nuclei in rats reported by DahlStrom and Fuxe (117).
There is also evidence for a group of serotonergic
neurons in the dorsomedial
nucleus of the hypothalamus of rats. With the use of histochemical
fluorescence
after intraventricular
infusion of 5-HT in rats pretreated with a monoamine oxidase inhibitor (MAOI), an
additional group of 5-HT cells located in the dorsomedial nucleus of the hypothalamus
was identified (175).
This provided the first evidence that the hypothalamus
contains an endogenous source of 5-HT. Biochemical
support for the hypothalamus
containing 5-HT-producing cells comes from the high concentrations
of 5-HT
and tryptophan
hydroxylase present in the surgically
isolated mediobasal hypothalamus
(91). With the use of
5-rH]HT
in vivo radioautography,
a single cluster of
labeled cells was found in the ventral part of the dorsomedial nucleus (60). A group of small round “immature”
5-HT-immunoreactive
cells in the same area was observed only if the animal was pretreated with tryptophan and MAOI (165,166). Thus these neurons may have
special characteristics
unlike the other “traditional”
groups and at this point should not be considered in the
same category as the main nuclei.
2. Inferior
brain stem group
I) NUCLEUS RAPHE OBSCURUS (B2). The large COlleCtion of 5-HT neurons in monkeys, the NRO, lies in a
symmetrical
paramedian cluster that extends from the
caudal border of the pons to the cervical spinal cord
(Fig. 2). In the spinal cord, the cells are scattered in the
central gray area just ventral to the central canal of the
spinal cord and on the medial border of the ventral horn.
The majority of these neurons are associated with the
fibers of the MLF and the tectospinal tract as these
tracts move from their superior position in the medulla
to an inferior position in the spinal cord. The cells in the
cervical spinal cord lying below the central canal were
described in monkeys (276). In cats, at the level of the
decussation of the pyramidal tract, 5-HT cells are seen
off the midline along the border between the MLF and
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FIG. 2. Adult rat brain coronal sections showing 5-HT-immunoreactive
neurons. A: superior group: DRN, MRN, and B9 in low power (X15).
B: DRN shown with its lateral (L), dorsomedial (DM), and ventromedial (VM) components in high power (~63). C: inferior groups: NRO, NRM,
NRPa, and ventral lateral medulla (VLM) in low power (~15). D: inferior groups in high power (x63).
170
BARRY
L. JACOBS
AND
C. AZMITIA
Volume
7.2
At the rostra1 end of the inferior olive, the 5-HT cells
move medially and become continuous with the NRPa
and NRM.
V) AREA POSTREMA. The 5-HT neurons in the area
postrema of the brain are very small and densely packed
with non-5-HT cells. They are apparent in both monkeys
(45) and rats (305). In rats these cells project to the parabrachial area and appear to play a role in the neural
circuitry involved in cardiovascular, respiratory, and
ingestive control (277, 332).
3. Nonserotonergic neurons in raphe nuclei
A substantial number of non-5-HT cells exist in
many of the nuclei designated as serotonergic, especially the laterally placed neurons in B9 and the ventrolateral medulla. Even the MRN, the lateral wings of the
DRN, and the medullary raphe nuclei have a sizeable
number of non-5-HT neurons (Table 1).
The surprising observation is that most studies find
that nearly all the large cells with long projecting fibers
in the raphe nuclei are serotonergic. For example, electrophysiological studies indicate that the vast majority
of midline midbrain raphe cells can be identified as serotonergic (14). In anterograde tracing studies, the injection of 5,7-dihydroxytryptamine
(5,7-DHT) into the
medial forebrain bundle (MFB) eliminates nearly all the
radioautographically
labeled fibers after [3H]proline injections into the midline mesencephalic raphe nuclei
(47). In retrograde transport studies, the connections to
their forebrain targets from the midbrain raphe nuclei
are nearly all serotonergic (34, 265, 266, 365, 453, 530).
The observation that despite the heterogeneity of neurons in the ascending nuclei the majority of long projecting cells (except for BS-cortical projections) (365) are
serotonergic is also seen with the descending raphespinal projections. For example, the NRM, NRPa, and
NRO project largely to sensory, autonomic, and motor
centers, respectively, in the spinal cord (21, 55, 84, 85,
311). The large percentage of BS-cortical projections
may reflect the dispersed placement of the‘neurons, the
great expansion of the cortical target area, or the methodology used to estimate retrogradely labeled 5-HT neurons.
4. Colocalixation of 5-hydroxytryptamine
with neuropeptides
At least three neuropeptides, substance P, thyrotropin-releasing hormone (TRH), and enkephalin, have
been colocalized to the cell bodies of the medullary serotonergic neurons (217). Enkephalin, besides being localized to serotonergic neurons in the LPGN, is also found
in certain neurons in the DRN (188). Single medullary
neurons projecting to the spinal cord have been found to
contain 5-HT, enkephalin, and TRH (243). It is generally
believed that these neuropeptides function to modify
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the pyramidal
decussation
just ventral to the central
canal.
Further
rostrally
at the exit of nerve VI, the nucleus is less densely packed than the NRPa and NRM.
The neurons are medium sized, and at their most anterior extent they lie dorsal to the NRM. At the anterior
aspect of NRO a smaller number of 5-HT cells have split
dorsally from the main cluster and, in accordance to the
nomenclature
of Olszewski
and Baxter (367), are designated as the extraraphe
component of NRO. This group
of 5-HT cells is seen in monkeys just ventral to the
fourth ventricle at the level of the genu of nerve VII and
extends caudally to join the NRO. This group was previously designated as the nucleus raphe ventricularis
(45).
The cells are small and located on the midline. The extraraphe
component of NRO probably continues caudally to form the group of 5-HT neurons that surrounds
the central canal at upper cervical levels of the spinal
cord.
II) NUCLEUS RAPHE PALLIDUS
(~1). The ventral
midline cluster of 5-HT neurons, the NRPa, extends
from nerve XII to the rostra1 pole of the inferior olive
(Fig. 2). The neurons lie in a compact group between the
pyramidal tracts. The dorsal border is continuous rostrally with the less densely packed NRM. The majority
of the 5-HT cells are found medial to the pyramidal
tract. In cats, this is the largest nucleus in the inferior
group, and the cells form paramedian columns. In rats
the cells are fused on the midline and contain mediumsized multipolar cells.
III) NUCLEUS RAPHE MAGNUS
(~3). The midline
group of large cells, the NRM, extends rostrocaudally
from the emergence of the nerve XII roots to the rostra1
superior olive (Fig. 2). Its location overlaps the trapezoid body and the dorsal border of the ML. It is continuous at certain points with both the NRO and NRPa. In
cats, at the level of the rostra1 half of the facial nucleus,
the NRM is located on the midline between the ML. The
cells of this nucleus remain largely on the midline, but
scattered cells can be seen above the ML and superior
olivary complex and within the trapezoid body. The
group extends laterally into the nucleus reticularis gigantocellularis.
1V)VENTRALLATERAL
MEDULLA
(Bl/B3).
Neurons
in the ventral lateral medulla (VLM) are found in the
reticular formation of the rostra1 and caudal ventrolatera1 medulla and extend laterally along the trapezoid
body, ML, and pyramidal fibers (Fig. 2). Rostrally they
are in the medial aspect of the LPGN and reach caudally
into the ventral part of the IRN. This substantial group
of cells extends from the emergence of the roots of nerve
XII to the rostra1 part of the inferior olivary nucleus.
The original classification proposed by Dahlstrom and
Fuxe (117), in adult rats, assigned the cells to both NRPa
and NRM. In cats, these cells were described as reaching
the floor of the medulla (236,511). The 5-HT-immunoreactive cells in monkeys and cats reach the ventral pia
and in certain cases are associated with the large blood
vessels entering the medulla. The 5-HT cells are
surrounded by a dense plexus of 5-HT fibers in this area.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
5. Nonneuronal
interactions
Associations are also seen with nonneuronal
cell
types and serve to illustrate the possible role of 5-HT as
a general trophic substance in addition to its role as a
neurotransmitter.
Neurons in the DRN and MRN are in
apposition to oligodendrocytes
(33,131), and their fibers
in the MFB are usually in apposition to non-5-HT myelinated fibers (44). 5-Hydroxytryptamine
cells from
the DRN, area postrema, and NRO are associated with
the ependymal cells of the cerebral aqueduct, fourth
ventricle, and central canal. Rare ependymal cells in
adult rats were immunoreactive
to 5-HT antibody (E. C.
Azmitia and P. J. Gannon, unpublished observations).
This relationship
between the DRN and the ventricular
system is established very early in development when
thick dendrites from 5-HT cells extend through the
ependymal layer into the cerebrospinal
fluid (CSF).
Bundles of 5-HT dendrites and ventricular
tanocytes
have been reported (150). 5-Hydroxytryptamine
cells
are commonly associated with large blood vessels, with
the cell body, or with processes in physical proximity to
endothelial cells (33). This is most evident in the MRN
and the LPGN. Tissue culture studies have also found
evidence for a stimulatory
role between astrocytes and
fetal 5-HT neurons (491, 282a). During early development, 5-HT neurons are closely associated with astrocytes heavily stained by S-100 antibodies. Astrocytes
also contain significant amounts of 5-HT, receptors.
The possible implications
of this 5-HT-astrocytic
relationship are discussed in sections 1vE4 and vE.
C. ARerents
In this section the afferent connections of the raphe
nuclei are described, and the neurotransmitter
content
of these afferent neurons is indicated. The first and largest afferent source is from the raphe nuclei themselves.
BRAIN
SEROTONIN
SYSTEM
171
These connections actually begin in the embryo with the
5-HT commissural
links between the paired bilateral
neurons before they fuse along the midline (491). In
adult animals, connections between DRN and MRN, B9,
LPGN and NRM have been described in cats and rats by
a variety of researchers using horseradish peroxidase
(HRP) (13,250,257,343,417,456)
and wheat germ agglutinin (WGA)-HRP
(250). 5-Hydroxytryptamine
autoreceptors have been demonstrated pharmacologically
and
physiologically
(see sect.
III&).
The rest of the afferents to the raphe nuclei can be
classified according to neurotransmitter
type and/or anatomic location. Brain stem afferents to the DRN come
from the superior vestibular nucleus via the MLF (250,
257) using acetylcholine as the neurotransmitter
(327);
the dorsal medulla in either the nucleus propositus hypoglossi (417), perihypoglossi
(250), or nucleus of solitary tract (13) provides a source of epinephrine
(218);
locus coeruleus and subcoeruleus (13,229,250,458) bring
norepinephrine
(105,308,458); the substantia nigra and
ventral tegmental area (13,250,417) provide dopaminergic afferents (252, 337); and finally the inputs from the
periaqueductal
gray (250,417) supply various neuropeptides (271).
The inputs from the hypothalamus
would be expected to complement the inputs derived from the brain
stem. The most important
of these comes from the medial preoptic area (13, 417) and lateral hypothalamus
(13, 250, 417). Fewer labeled cells were reported in arcuate (417) and dorsomedial hypothalamus
(250). These
hypothalamic
nuclei may be the source of neuropeptidergic (271) or histaminergic
(371, 372, 451, 512) afferents to the raphe nuclei. Thus there is little doubt that
the brain stem serotonergic neurons are in direct connection, by both their afferents and efferents, with the
main brain stem and hypothalamic
autonomic centers.
Importantly,
the serotonergic neurons also receive
inputs concerned with the limbic forebrain. A major input from the lateral habenula has been described anatomically
and electrophysiologically
(13, 417) and has
been shown to provide excitatory amino acid-containing
fibers to both the MRN and DRN (250, 254). Some of
these inputs probably end on the 5-HT neurons themselves, since these cells are sensitive to glutamate both
neurophysiologically
(see sect. VI) and metabolically
(see sect. IV@. The physiological significance of this input appears to be complicated by ending on the GABAergic interneurons
surrounding the MRN and DRN
(284,359). The inputs to the GABA interneurons might
provide an inhibitory
feedforward mechanism on the
5-HT neurons excited by the habenular-glutamate
connection. The pivotal position of the habenular nuclei in
the limbic-midbrain
system (355) places the serotonergic neurons in an ideal position to receive information
concerning the activity of motor and limbic areas. Several other nuclei in the forebrain, which are part of the
limbic system and contain cholinergic neurons, have
also been reported to be sparsely labeled after HRP or
WGA-HRP
injections into the DRN (13,250). These include the prefrontal cortex, the diagonal band of Broca,
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the action of the more classic type neurotransmitter
on
the postsynaptic cell (see sect. VIID).
Another important action of neuropeptides is to regulate the growth of serotonergic neurons (49, 1%). Enkephalin
has been shown to function as a negative
growth regulatory factor on cultured fetal serotonergic
neurons (124). Thus the neuropeptide that is colocalized
may influence the innervation pattern of the parent neurons, which, in turn, determines the sites where 5-HT
action will be strongest. This topic is discussed in
greater detail in section V.
There is evidence that nonneuropeptide
neurotransmitters can be colocalized with 5-HT in certain neurons.
For example, in the area postrema in rats, quantitative
studies indicate that a large number of neurons contain
both 5-HT and norepinephrine
(332). In addition, studies of the DRN of rats suggest that certain neurons
may contain both 5-HT and y-aminobutyric
acid
(GABA) (354).
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BARRY
L. JACOBS
AND
D. Pathways
and Terminals
Serotonergic
neurons have extensive projections,
both rostrally and caudally, from the superior and inferior groups, respectively (Fig. 3).
C. AZMITIA
Volume
72
FIG. 3. Summary
diagram
of primate
serotonergic
system. Main
nuclei are shaded. Fiber pathways
are shown as broken
lines. AC,
anterior
commissure;
Am, amygdaloid
nuclei; CB, cingulum
bundle;
CC, corpus callosum;
Cal, calcarine
fissure; Cer, cerebellum;
CLN, cauda1 linear nucleus; CQ, corpus quadrigimini;
CSul, central sulcus; DG,
dentate
gyrus;
DRCT,
dorsal
raphe cortical
tract;
F, fornix;
FCtx,
frontal
cortex; H, hypothalamus;
Hipp, hippocampus;
IC, internal
capsule; IO, inferior
olivary
nuclei; IP, interpeduncular
nucleus; LC, locus
coeruleus;
MB, mammillary
body; MFB, medial forebrain
bundle; OB,
olfactory
bulb, P, pons; S, septum; SM, stria medullaris;
SN, substantia nigra; T, thalamus;
TCtx, temporal
cortex; VAFP, ventro amygdalofugal pathway.
[Modified
from Azmitia
and Gannon
(45).]
1. Descending system
The main nuclei of the inferior serotonergic clusters (NRO, NRM, NRPa, and ventrolateral
medulla) account for the innervations
seen in the spinal cord (Fig.
4). In studies in monkeys, two main descending projections were seen, a ventromedial
pathway to the ventral
horn and a lateral pathway to the central gray area of
the spinal cord (45). The 5-HT projections to the intermediate lateral columns probably arise from the caudal
extension of the extraraphe subnucleus of NRO that
reaches the cervical spinal segments in layer X. Inputs
to the intermediate
columns appear to arrive also from
the NRPa or LPGN (85).
The fibers of NRO project caudally and ventrally to
the motoneurons of the ventral horn via the descending
MLF. These fibers begin dorsally at the level of the NRO
and descend in the ventral columns of the spinal cord.
The fibers reach this inferior position at the level of the
decussation of the pyramidal tract along with the fibers
comprising the MLF. This innervation
of the motoneurons of the ventral horn is consistent with the other
projections from NRO to the motoneurons of the brain
stem nuclei of nerves X and XII (150). This ventral descending tract is called the raphe obscurus spinal tract
(45). The 5-HT cells of the NRM heavily innervate the
substantia gelatinosa of the spinal cord. Interestingly,
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and the bed nucleus of the stria terminalis.
These forebrain nuclei all contribute to the descending MFB (33)
and would provide a direct but minor input from limbic
centers.
Immunocytochemical
and receptor binding studies
have been used to describe a number of peptidergic and
hormonal afferents to the raphe nuclei. Almost this entire literature is based on studies carried out in the rat
CNS (a few of the opioid analyses were conducted in
monkeys). Enkephalin-like
immunoreactivity
has been
reported in the raphe for both Old World monkeys (200)
and for rats (259). The input to the DRN in monkeys was
moderate, whereas that to the inferior raphe groups
was described as sparse. ,&Endorphin-immunoreactive
fibers are found in the DRN of rats, but the inferior
raphe groups and the NRM were reported to be devoid of
such fibers (77). With the use of rH]diprenorphine
to
label opioid receptors in the rat brain, the DRN and
MRN were reported to have a low density of binding
sites (28). Substance P immunoreactivity
and receptor
sites for substance P have been examined in studies of
the rat brain, with surprisingly
consistent results (309,
319,438). The DRN and MRN had moderate substance P
inputs, whereas the inferior 5-HT nuclei (NRM, NRO,
and NRPa) had only weak substance P inputs. Angiotensin II has been examined in the rat brain with immunohistochemistry
(174) and autoradiography
(331). The results of both types of analyses indicate little, if any,
angiotensin
II input to the raphe nuclei. In contrast,
immunohistochemical
analyses of neurotensin fibers in
rats indicate moderate to strong inputs to several of the
raphe nuclei (241, 474). Although the data are limited,
there is some evidence for somatostatin
inputs to the
NRM (but not to any of the other 5-HT nuclei) and for
‘?-labeled
somatostatin
binding in the DRN of rats
(151, 288). Autoradiographic
analyses of cholecystokinin (CCK) indicate little [3H]CCK, binding in the
raphe nuclei in rats (484) and some CCK-immunoreactive fibers in the DRN and NRM (477). Both immunohistological and neurochemical
analyses indicate that in
rats the DRN, but not the other raphe nuclei, receives a
moderate neuropeptide Y input (104a).
Substances related to the pituitary-adrenal
axis
have been studied. Autoradiographic
analyses of receptors for corticotropin-releasing
factor (CRF) (using ‘%ICRF) indicate a low level in the raphe nuclei in rats
(133). Consistent with this, immunohistochemical
analyses of CRF-immunoreactive
fibers indicate that they
are found in the various raphe nuclei of rats (459). All
raphe groups in rats are reported to receive substantial
adrenocorticotropic
hormone (ACTH)-immunoreactive
fibers (411). Two types of receptors for corticosterone
have been studied in the rat brain (404). Those for glucocorticoids are present in moderate to high density in the
raphe groups, whereas those for mineralocorticoids
are
absent in the raphe.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
the substantia
gelatinosa might contain a pathway for
descending serotonergic
fibers to use for innervating
lower spinal levels. This is based on studies of the descending noradrenergic
(NA) fibers from LRN that suggest that once NA fibers innervate the substantia
gelatinosa at the medullary-spinal
cord interface, they descend for long distances within the nucleus itself (171).
There is evidence that some of the 5-HT fibers in
the lateral descending tract may be myelinated. Labeled
myelinated
fibers in the spinal cord in rats were seen
after incubating the tissue with 5-[3H]HT and processing for electron microscopic
radioautography
(415). Labeled by 5-[3H]HT, myelinated fibers are also detected in
the lower brain stem of monkeys (99). The frequency
and distribution
of these descending myelinated
5-HT
fibers remain to be determined.
Finally, the projection
from the ventrolateral
medulla probably innervates the
intermediate
lateral columns of the spinal cord (199).
This projection would provide 5-HT fibers from the autonomic centers of the LPGN to the autonomic centers of
the spinal cord. The fibers may use the lateral descending tract in the lateral funiculus
or travel within the
intermediate
gray column itself.
In the monkey spinal cord, a dense plexus of 5-HTimmunoreactive
fibers is present in the substantia
gelatinosa from the NRM, around the motoneurons
in the
ventral horn from the NRO, and in the intermediate
sensory and motor zone of the autonomic neurons from
the LPGN. Large injections of HRP into the lumbar spinal cord in Macaca fascicuZaris
label cells in all three
nuclei (85). A few cells with efferents to the spinal cord
have been reported in the medial and lateral division of
the DRN in squirrel
monkeys and a baboon (320), although this has not been confirmed. Serotonergic
endings from the NRM make direct contact with the neurons in the dorsal horn that give rise to the spinothalamic tract (228, 414) and are believed to be involved in
modulating transmission
of nociceptive information.
BRAIN
SEROTONIN
SYSTEM
173
The distribution
of 5-HT fibers in the brain stem
has been described for rats (450), cats (511), and monkeys (150). Highest levels of immunoreactive
terminal
plexuses in rats were seen in areas of the interpeduncular nucleus, nucleus of the solitary tract, trigeminal motor nucleus, facial motor nucleus, substantia
gelatinosa,
and the ventral horn of the spinal cord. These studies
have clearly shown that even within
a particular
nucleus there is a great deal of heterogeneity
in fiber density. For example, the distribution
of 5-HT-immunoreactive fibers in the trigeminal
nuclear complex of the rat
showed a dense innervation
in the sensory nuclei, primarily associated with nociceptive activity, and sparse
innervation
in the sensory nuclei related to nonnociceptive afferent activity (114). Furthermore
the marginal
and gelatinosa layers of the spinal subnucleus caudalis
had a dense number of fine fibers. The motor nucleus
contained as many immunoreactive
fibers as the subnucleus caudalis, but the fibers here were thicker and varicosities were more irregularly
spaced than in the caudalis.
2. Ascending
system
Ascending 5-HT-containing
fibers were first visualized with histochemical
fluorescence methods (118,173),
and using anterograde
tracing methods, six ascending
projections
were described (33). Myelinated and unmyelinated 5-HT-containing
axons in the MFB of rats and
monkeys
were visualized
with immunocytochemistry
(44). 5-Hydroxytryptamine
immunoreactivity
was seen
predominantly
in unmyelinated
axons surrounded
by
unlabeled processes. More than half of these 5-HT-immunoreactive
fibers were in apposition with unreactive
myelinated axons in the hypothalamus
of both rats and
monkeys.
In addition to the unmyelinated
fibers, intensely labeled myelinated axons were seen in the MFB
of both rats and monkeys. The percentage of 5-HT-immunoreactive
myelinated
axons was substantially
greater in monkeys than in rats (25.4 vs. 0.7% of the
total number of 5-HT-immunoreactive
fibers, respectively).
The serotonergic
fibers projecting to the forebrain
originate mainly in the superior group. Two main ascending bundles have been described in the primate
brain (360,425,426). In human fetuses, 5-HT fibers were
seen in the central gray near the ependyma of the fourth
ventricle
and the cerebral aqueduct and in a position
between the medial raphe cells and the lateral cell system (360).
In juvenile macaques, two main ascending bundles
were described using histochemical
fluorescence (426).
A dorsal bundle was seen just ventral to the MLF, which
originated
at the level of locus coeruleus and received
fibers from lateral wings of DRN and the ventromedial
subnucleus of the DRN. These fibers appear to turn ventrally at the level of the red nucleus. A second bundle of
ventrally flowing serotonergic
fibers lateral to the MRN
originated at the level of the trochlear nucleus and received fibers from the midline DRN and MRN. These
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FIG. 4. Schematic
drawing
of sagittal
section showing descending
projections
of inferior
serotonergic
neurons
from medulla
to spinal
cord. Serotonergic
soma are depicted
as filled circles.
Serotonergic
cells are seen in VLM, NRM,
and NRO with its dorsal subnucleus,
extraraphe
(NROer).
5-HT fibers are seen in tractus
spinal thalamicus (tst) and spinal olivaris
(tso) to intermediate
gray from VLM, in
medial longitudinal
fasciculus
(mlf) and tractus tectal spinalis (tts) to
ventral
horn from NRO, in substantia
gelantinosa
(SG) to dorsal horn
from NRM, and in central
gray from NROer.
CC, corpus callosum.
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C. AZMITIA
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72
5-HT fibers extended to the external plexiform
layer.
The number of 5-HT-immunoreactive
fibers was also
numerous
in the inner portions
of the granular
cell
layer. In contrast to both rats and cats, the distribution
of 5-HT-immunoreactive
fibers in monkeys was scanty
and partial in the glomerular
layer. In this area, only a
few of the glomeruli (no more than 5%) possessed 5-HTimmunoreactive
axons.
The heterogeneous
distributio In of serotonergic
fibers in the telencephalo n is briefly descr 1 bed for monkeys (45), si rice they show a more heterogeneous
distribution than is seen in rats (144). Highest levels are seen
in limbic area ,s (temporal lobe amygdaloid nucl .ei, and
.
hi ppocam PUS) and in sen sory areas (visual, cal carine;
auditory,
superior
temporal
gyrus;
somatosensory,
postcentral
cortex; olfactory, amygdaloid nuclei and entorhinal cortex). The lowest levels are found in the motor regions in the fro ntal lobe (with the exception of the
ret tus gyrus) . The distribut ion is qu ite varied even
within the subregions of the hippocampal complex (dentate gyrus is greater than cornu Ammonis).
As a general
rule 5-HT levels are highest in those regions of cortex
having more granule cells (layer IV). These cells are associated with sensory-receiving
areas and association
projections.
Therefore
the modulation
of granule cell
activity would affect the activity in all other cortical
layers as well as corticocortical
projections.
Laminar distribution
of 5-HT fibers has been described in the cerebral cortex. Although there are differences in the density of serotonergic
innervation
of
different
laminae of rat neocortex, this is seen most
strikingly
in monkey cortex. In monkeys, a clear lamination was observed with the highest densities in layer I
and layer IV (45,80,342).
The density in layer IV again
reflects a preferential
innervation
of granule cells by
5-HT fibers. It is interesting
that in rats, developing
serotonergic
fibers first project to the granular
layer
(IV), but as the brain matures the fibers become more
localized to layer V (79). The most highly differential
laminar specialization
of all neocortical areas in monkeys is observed in primary visual cortex (also referred
to as Vl and area 17) (268). 5-Hydroxytryptamine-immunoreactive
fibers are present in all cortical layers of
Vl in M fascicularis
but form two especially prominent
broad bands of fibers: one, extending from the deep half
of layer III through IVC, reaches a peak in IVB, and the
other begins in VA and goes through VI (Fig. 5). Between these two dense bands there is a band with a low
density of serotonergic
fibers in layer IVC (268). More
recently, a quantitative
analysis of the laminar density
of innervation
showed that layer IVC contained more
varicosities
per unit area than any other sublayer of
primary visual cortex (126).
In rats, there is evidence for two distinct populations of fibers in the cortex (264). They have been described as fine with many branches
(D from dorsal
raphe nucleus) and thick varicose fibers (M from median raphe nucleus) (267). The density of the fine fibers
is heaviest in layers I and VA in adult rats (81). In cats,
these fine fibers are widely spread throughout
the corti9
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fibers appear to turn rostrally
just dorsal to the nucleus
interpeduncularis.
In lK fascicularis,
dense SHT-immunoreactive
staining was present in the two main fiber tracts described above (45). In addition, fibers near the cerebral
aqueduct in the hypothalamus
were seen projecting
lateroventrally
from the DRN. Thus all six of the 5-HT
fiber bundles observed in rats (47) can be observed in the
primate brain stem. Furthermore,
in monkeys, the main
ascending bundle through the hypothalamus
may not be
the MFB, as it is in rats. Examination
of the caudal
hypothalamic
area shows two main ascending bundles
previously
described in rats [the MFB and the dorsal
raphe cortical tract (DRCT)]. However, in primates, the
latter tract, which ascends to the cerebral cortex via the
internal capsule, is larger than the MFB (44). The DRCT
thus appears to have undergone a significant increase in
absolute size when compared
with the homologous
pathway in rats. This probably represents an increase in
the projection
from the DRN to the cerebral cortex in
primates.
Several studies
have used immunocytochemical
methods to map the terminal distribution
of 5-HT fibers
(45, 304, 450). Highest levels of immunoreactive
terminal plexuses were seen in hippocampus,
suprachiasmatic nucleus of the hypothalamus,
zona compacta of
the substantia
nigra, medial mammillary
nucleus, lateral septum, periventricular
nucleus of the thalamus,
ventrolateral
geniculate, and the medial nucleus of the
amygdala. Lower, but clearly detectable, 5-HT-immunoreactive fibers were seen in virtually
every area of the
brain examined, except for major fiber tracts such as
the corpus callosum and optic tract.
Certain patterns in this vast innervation
network
can be seen. Innervation
is greatest in those thalamic
nuclei associated
with limbic functions
(e.g., nucleus
reuniens and nucleus anterior dorsalis)
and with sensory transmission,
either nociceptive
(e.g., posterior
complex) or visual (e.g., dorsal lateral geniculate nucleus). In monkeys,
5-HT-immunoreactive
axons were
seen in both regions of the substantia
nigra, although
mainly in the pars reticulata
(ZZO), whereas in rats the
highest density was described in pars compacta (450). In
the neostriatum
of monkeys,
5-HT-immunoreactive
fibers were particularly
abundant in the ventromedial
region of the caudate head bordering
the nucleus accumbens where the plexus was heavier (378). The number of fibers in the caudate was reduced in more dorsal
and lateral areas. The fibers were uniformly
distributed
in the putamen. Thus, despite the nearly ubiquitous distribution,
selected areas receive much denser innervation.
A comparative
study in monkeys, cats, and rats of
the olfactory bulb shows clear species differences. Serotonergic fibers were seen in all layers except the olfactory nerve layer in M. fuscata (460). 5-Hydroxytryptamine-immunoreactive
fibers were observed to form a
chain of large varicosities
in the periglomerular
region.
The 5-HT distribution
was predominantly
in the internal plexiform layer. In some portions, a dense plexus of
EFRAIN
January
STRUCTURE
1992
AND
FUNCTION
cal layers with increased branching in the upper layers
(348,349). The large nonvaricose fibers (up to 2 pm wide)
run in layer I and in the lower white matter of the cortex. They appear similar to the large fibers described in
monkeys (342, 461). In cats, these large varicose fibers
give off collaterals that form the basket terminals in the
upper layers of the cortex (I-III) (348). The target cells
for these basket terminals are heterogenous: both pyramidal and nonpyramidal
(e.g., GABAergic) cells are included. A similar distribution
of the fine and large varicose fibers is reported for marmosets (222a). The large
varicose fibers are often seen surrounding certain cell
bodies or proximal dendrites, possibly GABAergic or
peptidergic. These fibers “may have a strong and specific influence on the cortical inhibitory
circuitry, via
relay through cortical inhibitory
interneurons”
(222a).
3. Nuclear
organization
of efferents
The organization
of the superior raphe projections
indicates a rostrocaudal encephalotopy (229). The rostral group (DRN and the CLN) projects to basal ganglia-motor systems (e.g., corpus striatum and substantia nigra). The caudal group (MRN and the interfascicular aspect of the DRN) project to limbic structures (e.g.,
hippocampus and septum). The amygdala receives inputs from both groups, with the basolateral nucleus,
which has projections to corpus striatum, belonging to
the rostra1 group. The raphe nuclei with overlapping
BRAIN
SEROTONIN
SYSTEM
175
terminal areas have a sizeable number of collateralized
neurons to those terminal areas. This confirms and extends previous reports showing that serotonergic neurons are highly collaterized with innervations
to more
than one terminal area (19, 128, 266, 478). The projections to cortex show that the DRN projects heaviest to
frontal cortex from the rostra1 and lateral wings of the
nucleus (365). The lateral wings also project to the caudate putamen (452, 453). These projections would be
compatible with this region being associated with the
basal ganglia motor system. The MRN and B9 projected
equally to parietal, occipital, and frontal cortex (365).
This organizational
pattern supports the idea that functionally related nuclei can be innervated by the same
group of serotonergic neurons (33,229,334,489)
or even
the same individual neuron (498).
The bilateral organization of the serotonergic neurons has been studied. There is general agreement that
most of the serotonergic ascending projections from
dorsal raphe are ipsilateral
(235, 290). However, it
should be noted that during development, a sizable number of serotonergic fibers cross at the midline (see sect.
IvC).
Thus contralateral
or bilateral projecting neurons
should be found. In a study of the dorsal hippocampal
raphe afferents, -50% of the neurons were on or near
the midline in both the DRN and MRN (34). Of the remainder not located on the midline, one-half of these
cells were contralateral
to the injected hippocampus in
the MRN while only 17% were contralateral
in the DRN.
Furthermore,
10% of the labeled MRN neurons projected bilaterally
to the hippocampus. When the projections to cortex were studied, a similar proportion
of
DRN and MRN ipsi- and contralateral
cells to those to
hippocampus were found (365). In the DRN, exclusive of
the lateral wings, there is a predominately
(3:l) ipsilatera1 projection with decreasing numbers of cells projecting to frontal, parietal, and occipital cortex. However,
the MRN and B9 projections are laterally symmetric,
with the MRN projecting throughout the neocortex and
B9 projecting to selective cortical areas.
E. Distribution
of Receptors
The availability
of specific 5-HT ligands has made
possible a study of the distribution
of binding sites in
the brain. The labeling is dependent on the particular
ligand employed, and early studies used either [3H]LSD
(330) or 5-[3H]HT (524). We briefly review both the anatomic and laminar distribution
of the 5-HT,, 5-HT,, and
the 5-HT, receptor families. Details of this classification can be found in recent reviews (169a, 369,385,507).
1. 5Hydroxytryptamine,
The original classification of 5-HT, and 5-HT, receptors used 5-[3H]HT and [3H]spiroperidol
as specific
ligands (386). The radioautographic
binding in rats
showed a wide variation, with the highest levels in ven-
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FIG. 5. Distribution
of 5-HT-immunoreactive
fibers in primary
visual cortex of cynomolgus
monkey.
A: bright-field
photomicrograph
of opercular
surface
of primary
visual cortex
Nissl stained
to show
laminar
boundaries.
B: dark-field
nhotomicrogranh
of same field in
adjacent
section immunostained
for 5-HT visualization.
Bar, 200 Frn.
[From De Lima et al. (126).]
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hippocampus (225). In the cortex, layer II (322 fmol/mg
protein) was higher than layers III and VI (40 and 87
fmol/mg protein, respectively). In the hippocampus, the
pyramidal
layer of CA1 (505 fmol/mg
protein) was
higher than CA3 (102 fmol/mg
protein) with the DG
having intermediate
levels (280 fmol/mg protein).
II)
MINES,,.
5-HYDROXYTRYPTAMINE&-HYDROXYTRYPTA-
The 5-HT,, site has been defined in rat by 5[3H]HT binding in the presence of the 5-HT,, ligand 8OH-DPAT (382) or by the ligand [‘251]iodocyanopindolol
([1251]CYP) (142). Specific 5-HTlB binding was blocked by
the P-adrenergic
antagonist
RU-21009 and RU-24969
(147). The 5-HT,, sites were particularly
rich in the globus pallidus, dorsal subiculum, substantia nigra, and
the olivary pretectal nucleus.
5-Hydroxytryptamine,,
binding was performed on
postmortem human tissue with ( -)-[1251]CYP in the presence of (-)-isoprenaline
to suppress binding to ,&adrenergic receptors (224, 225), and specific sites were not
detected. Other workers, using a similar technique, did
report small nu mbers of these receptors in basal ganglia
region s in hum an pos tmortem brains (68) . A complication in these studies is that 5-HT,, receptors may share
many of the properties of the 5-HT,, receptors.
III) 5-HYDROXYTRYPTAMINElc.
The 5-HT,o site can
be labeled by 5-[3H]HT, [3H]mesulergine,
or ‘%I-LSD
(216, 226, 381, 521). The labeling is almost exclusively
confined to the choroid plexus. For example, in humans,
5-HT,c sites are highest in the choroid plexus (1,700
fmol/mg protein), and much less is found in the hippocampus, with the pyramidal layers of CA1 and CA3 having a higher density than the DG (170 vs. 80 fmol/mg
protein).
2. 5-Hydroxytryptamine,
Morphological
evidence for a distinct low-affinity
5-HT receptor came from the differential anatomic distribution
of 5-HT, receptors in the rat brain. This site
has been studied
using [3H]ketanserin,
[3H]LSD,
[3H]spiperone,
and [3H]mesulergine
(379), with ketanserin being the most selective (301). The 5-HT, sites are
high to intermediate
in the medial and posterior part of
the medial mammillary
nucleus, parafascicular nucleus
of thalamus, inferior olivary nucleus, nucleus accumbens, body and tail of caudate, olfactory tuberc le, anterior olfactory nucleus, insulae Calleja, and in various
areas of the cortex. In the cortex, high levels were seen
in the entorhinal, pyriform, cingulate, frontal, and frontoparietal motor cortex with lower levels in the frontoparietal sensory, striate, and auditory cortex (380). The
highest density reported was in lamina IV of the neocortex.
The 5-HT, receptor distribution
in rat neocortex
was studied with M-methyl-2-1251-LSD
(81). The anatomic distribution
in cortex was frontal > parietal or
occipital. A dense band of labeling was found in the upper part of lamina V and not lamina IV in the somatosensory cortex. In addition to the band seen in lamina
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tral pallidum
(3,600 fmol/mg
protein) and the lowest
levels in reticular and ventroposterior
thalamic nuclei
(180-190 fmol/mg
protein) using 5-[“H]HT as a label.
The density of 5-HT, receptors was high in the choroid
plexus, lateral septal nuclei, dentate gyrus (DG), dorsal
subiculum, olivary pretectal nucleus, substantia nigra,
entorhinal cortex, Rexed layers I and IV of spinal cord,
nucle us of the sol itary tract, gelatinosum
area of nucleus of the spinal tract of V, amygdaloid nuclei (lateral
and cortical highest), hypothalamus
(ventromedial
and
dorsomedial highest), and DRN (67,380). In cortex, the
density of receptors was entorhinal
> anterior cingulate > frontal > somatosensory > motor > striate (382).
This distribution
is generally in good agreement with
the known serotonergic fiber density in rat brain as described.
A study of the distribution
of 5-[3H]HT binding in
rhesus monkey cortex showed higher levels in prefrontal than parietal or prestriate, with the somatosensory
cortex higher than the motor cortex (306). The laminar
distribution
showed 185 fmol/mg tissue in layer II of the
somatosensory cortex compared with 50 fmol/mg tissue
in layers IIIB-V. In general, the 5-HT, receptors were
located supragranularly
(layers I and II). This general
pattern was different in motor and visual cortex. In the
motor cortex, the highest density was in layers I and II,
and in the visual cortex, it was highest in layer IVC with
high levels throughout layers III-VI. The distribution
of
the uptake sites as defined by [3H]paroxetine
binding
did not have strict correlation with the 5-HT, receptor
distribution
in cortex.
The regional density of 5-[“H]HT
binding varied
markedly in the postmortem
human brain: 1,151 fmol/
mg protein in frontal cortical layer I to 74 fmol/mg protein in ventral thalamus (66). Highest levels were seen
in cortex (superior frontal > cingulate > medial frontal), subiculum, globus pallidus, claustrum, and midline
nuclei of the thalamus (66). The laminar distribution
in
cortex showed that layers I and II were highest, with
substantial labeling also in layer VI.
I) 5-HYDROXYTRYPTAMINE,*.
There is long-standing evidence for the existence of multiple high-affinity
5-HT receptors (502). The availability
of specific ligands
has made the study of their differential distribution
in
the brain possible. The 5-HT,, receptors are usually defined using 8-hydroxy-2(di-n-propylamino)tetralin
(8OH-DPAT), either as the radiolabel or as the displacing
(defining) ligand. With the use of this approach, the
highest levels in rats of 5-HT,, receptors were seen in
the midbrain raphe nuclei, DG, and lateral septum (382)
and in the hippocampus and cortex (132, 193, 380,488).
In the frontal cortex, the 5-HT,, receptors were reported to be highest in the deeper cortical layers (382).
The highest binding in spinal cord was seen in layer I,
which corresponds to substantia gelatinosum.
The cellular binding of 8-OH-[3H]DPAT
to midbrain raphe neurons has been shown to be on the soma and dendrites of
5-HT-immunoreactive
neurons (16).
In human postmortem
brain, the binding of 8-OH[3H]DPAT was region specific in both frontal cortex and
EFRAIN
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1992
STRUCTURE
AND
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BRAIN
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SYSTEM
177
(entorhinal
> retrosplenic
= frontal = cingulate = temporal > occipital
= parietal).
Recent studies
with
[3H]ICS 205-930 in the human brain show heavy labeling
in the substantia
gelatinosa
of the spinal cord (219
fmol/mg
protein),
area postrema
(729 fmol/mg
protein), nucleus of solitary
tract (255 fmol/mg protein),
cranial nucleus of the vagus (493 fmol/mg protein), and
the spinal trigeminal
nucleus (178 fmol/mg
protein)
(490). Hippocampus
and amygdala showed some labeling (61 and 72 fmol/mg protein, respectively),
with the
entorhinal cortex having much lower levels (18 fmol/mg
protein).
4. Summary
There is now evidence for at least six separate 5-HT
receptors,
each with a unique pharmacological,
behavioral, and anatomic profile. The 5-HT,* is the cell body
autoreceptor
and is also responsible
for some of the
postsynaptic
actions of 5-HT, especially in the hippocampus and cortex. The 5-HTlB/5-HTlD
receptor
(5HT,, in humans) is the axon terminal autoreceptor
in
rats and primates
and is presumed to be involved in
inhibiting the release of 5-HT from the axon terminals.
The 5-HT,o receptor is most densely located in the choroid plexus, with much lower but detectable levels in the
hippocampus
and cortex. The 5-HT, receptor is postsynaptic and is associated with the fine serotonergic
fibers
in the middle layers of the cortex. It is responsible
for
many of the behavioral
effects of 5-HT and has been
shown to be involved in the action of the major hallucinogenic drugs. Finally, the 5-HT, receptor is found in
limbic areas, where it may serve a role in anxiety and
psychosis;
in the substantia
gelatinosa, where it may
modulate sensory input; and in the area postrema,
where it may inhibit emesis.
The variety of 5-HT receptors
may represent
the
distribution
on the cell body versus the terminal or on
the different population of cells (neurons and glial cells)
that receive a 5-HT signal. Therefore, although the 5HT neurons show a rather simple pattern of firing (see
sect. VIA) and can communicate
through unmyelinated
fibers onto nonspecialized
postsynaptic
sites, the receptors recognizing 5-HT are differentiated
and highly specific.
F. Ultrastructure
3. 5-Hydroxytryptamine,
The most recently described CNS 5-HT receptor is
5-HT,, which is known to bind cocaine (86). This receptor is labeled with [3H]GR 65630A and has been localized
to the brain with the highest levels in the entorhinal
cortex (11.3 fmol/mg
protein) and the lowest levels in
the cerebellum (0 fmol/mg
protein) (260). Other areas
with dense 5-HT, binding are amygdala, hippocampus,
nucleus accumbens/olfactory
tuberculum,
and cortex
Ultrastructure
of serotonergic
neurons has been reviewed (33, 61). 5-Hydroxytryptamine
neuronal perikarya in the DRN are medium-sized
(15-25 pm mean
diam) neurons with a relatively
small but deeply indented nucleus. The cytoplasmic
distribution
of organelles depicts a metabolically
active cell. There are numerous free ribosomes, a well-developed
Golgi apparatus, numerous dense and multivesicular
bodies, small
clear vesicles, and occasional large dense-core vesicles.
This descrintion
cannot be generalized to all serotoner-
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VA, there was also heavy labeling in layer I just beneath
the pia. Lower densities were seen in the deep half of the
cortex. In the motor cortex, the middle band extended
throughout
layer V where the projecting pyramidal neurons are located. This region displayed more 5-HT, receptors than the somatosensory
cortex. Interestingly,
layer VA corresponded
to the fine axons with pleomorphic varicosities
that densely innervate this area from
the DRN (267).
The distribution
of 5-HT, receptors
as seen with
[3H]ketanserin
showed a moderate variation
in rhesus
monkeys (306). Highest levels (100 fmol/mg tissue) were
observed in layers III and IV, and lowest levels (~25
fmol/mg tissue) were seen in layer I of prefrontal
cortex. The density in the motor cortex was greater than in
the somatosensory,
similar to that seen in rats. Labeling
in most cortical areas was heaviest in layers III and IV,
with much lower levels in other laminae. In the visual
cortex, the density of receptors was high in all laminae
but was highest in lamina IVC. Interestingly,
the receptors were higher in the supragranular
layers (I-III)
in
the motor cortex than in other cortical areas.
The localization of SHT, receptors in human postmortem
tissue was studied with [3H]spiroperidol
or
E3H]ketanserin
(66, 146, 226). The distribution
determined by binding studies showed cortex and hippocampus > globus pallidus and caudate nucleus (146). The
results with quantitative
radioautography
(range: 48
fmol/mg protein in lamina IV of cingulate cortex to 0 in
ventral thalamus) showed the highest binding in cortex,
amygdala, DG, anterior ventral nucleus of thalamus,
and caudate/putamen
(66). The cingulate cortex had
more binding than the superior frontal cortex, and lamina IV was much higher than laminas I-III. In the spinal
cord, highest levels (100 fmol/mg protein) were seen on
motoneurons.
A final study showed high levels in the
cortex and hippocampus
(226). Frontal cortex layers III
and IV showed high levels (217-247 fmol/mg
protein),
whereas lower levels were seen in layer II. In the hippocampus, the pyramidal
CA1 showed higher levels (190
fmol/mg protein) than were seen in CA3 or DG. Interestingly, the distribution
of 5-[3H]HT uptake sites is highest in layer IV of primates, which agrees with laminar
distribution
of the 5-HT, receptor (44). The regional distribution
of the uptake sites and the receptor binding
does show many discrepancies
in rank order, which may
reflect the downregulation
of the receptor by 5-HT release.
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443, 444). These percentages should be considered only
rough approximations
since synaptic contacts may be
quite labile. In fact, the number of 5-HT junctional varicosities can be dramatically
altered by changes in the
local neuropil, as shown by studies performed
with
agranular rats where the number of specialized contacts
was reduced (445).
The factors that determine the frequency of junctional versus nonjunctional
contacts are unknown.
It
has been suggested that fine fibers do not, but the thick
fibers do, make specialized contacts (465). However, this
view is not universally
shared. This issue has received so
much interest because the appearance of a specialized
contact suggests relatively
stable and strong associations between an afferent neuron and its target. Conversely, the lack of synaptic specialization
implies a dynamic and possibly indiscriminate
interaction
with target neurons.
Serotonergic
neurons
have long been
described as a global modulatory system consistent with
a nonjunctional
framework.
Furthermore,
the regenerative capacity was initially
said to be limited to these
nonjunctional
fibers (72). This view should be tempered;
5-HT specialized contacts do exist, but they are not rigid
and permanent.
Regenerative
5-HT fibers of the junctional types have been described (167,168). Some 5-HT
fibers do lack specialized regions of contact and release
5-HT to nonsynaptically
related neurons, glial cells,
ependymal cells, endothelial cells, endocrine cells, and
into the CSF (33,36,44,97,131,150).
Thus the types of
contacts made by these neurons throughout
the brain
reflect the varied nature of the action of serotonin.
It seems clear from these ultrastructural
data that,
in most areas of the mammalian CNS, there are at least
some sites where 5-HT is released and no evidence for
synaptic specializations
can be found. This type of arrangement,
where neurotransmitter
is released and
then diffuses over distances as great as several hundred
microns to affect target neurons, is well known in the
mammalian
peripheral
autonomic nervous system. It
implies the existence of a hormonelike
form of neurotransmission,
which is slow, global, and therefore carries only a primitive form of neural information.
One of
the more well-delineated
examples of nonsynaptic
release of 5-HT, with the accompanying
physiological
evidence, is in the spinal cord of a primitive vertebrate.
In
lampreys, serotonergic
axonal varicosities
are located in
proximity
to motoneurons
and premotoneurons,
but no
synaptic specializations
are observed. If 5-HT is applied
during fictive locomotion in the spinal cord of the lamprey, it has profound effects on the motor pattern (for
review see Ref. 492).
Several additional
points deserve discussion
regarding the issue of nonsynaptic
release. First, in a
given species, the percent of 5-HT terminals associated
with synaptic specializations
apparently can vary from
~0 to lOO%, depending on the particular
brain region.
This may have important
implications
for the type of
information
processing
in which 5-HT is involved in
each of these brain areas in a given species. Second, it
will be important
to gather cross-species
data on this
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gic groups, since in the monkey LPGN they are mostly
fusiform
and somewhat
larger than the serotonergic
cells in DRN (98).
The 5-HT neuronal dendrites have been shown to
receive 5-HT terminals
in monkeys (255) but not rats
(131,383) or cats (101). However,
many 5-HT processes
lie in proximity
to the 5-HT dendrites
and provide a
structural
basis for homotypic
interactions.
Dendrodendritic appositions were frequently
seen in all species
with some evidence of specialization
in cats and monkeys. However, with respect to the soma and dendrites,
no unique elements were reported for separating
5-HT
cells from the surrounding
non-5-HT neurons.
The sizes of the 5-HT-immunoreactive
fibers in the
MFB are 0.2-1.25 pm in diameter
for unmyelinated
axons and 1.0-2.1 pm for myelinated axons (44). The intraneuronal
5-HT-immunoreactive
labeling was detected within small vesicles (30-35 nm) and the tubular
profiles of smooth endoplasmic
reticulum.
These two
sites comprise the membrane-enclosed
compartments
of 5-HT immunoreactivity.
Evidence of 5-HT immunoreactivity was also found on the outer membranes of mitochondria (where monoamine oxidase is localized) and in
discrete patches along the inner face of the axolemma.
In the cat solitary
tract, 5-HT immunoreactivity
was
reported in the large granule vesicles (80-150 nm) (317),
and in the monkey it was associated with microtubules
(377). There is a report that substance P and 5-HT can
be contained within
the same large granular
vesicle
(384). The localization of 5-HT to large granular vesicles
is still controversial
since not all studies have seen this
localization.
In certain brain nuclei (locus coeruleus, portions of
the cerebellum, and the inferior olive), some of the 5-HT
varicosities
contain small vesicular
organelles
(15-25
nm) associated with tubular elements. These microcanaliculi (62,99,285) may represent a continuous intra-axonal network similar to the smooth endoplasmic reticulum. These may provide the pathway for the retrograde
transport
of 5-[3H]HT from the terminal to the cell body
(25,43a). The projecting
axons are either fine and varicose or large and smooth (113, 114, 118, 303, 450). The
fibers were described as both unmyelinated
and myelinated (44, 98, 99, 255, 415). There is now general agreement regarding
whether
serotonergic
terminals
make
specialized synaptic contacts or release their transmitter by nonjunctional
means. Both types of terminals
have been clearly documented, suggesting both forms of
release are present in the brain. The relative frequency
of each type is region specific (33, 61). For example, in
rats, there are very few postsynaptic
specializations
associated with serotonergic
varicosities
in layer I of the
cerebral cortex (59,130), but in the deeper layers of somatosensory
and visual cortex, the percent of junctional
contacts can be as high as 50-90% (335,373). In the cat
dorsal horn and in the rat hypothalamus,
there are
abundant specializations
(167, 168, 414), but they are
extremely rare in rat locus coeruleus (285). In the neostriatum,
only 15% of all 5-HT varicosities
made an
asymmetrical
synapse on dendritic shafts or spines (26,
EFRAIN
January 1992
STRUCTURE
AND
FUNCTION
issue to see if there are any phylogenetic trends in this
relationship (similarly, developmental data in a given
species would be of interest). Finally, the dynamics of
release at nonsynaptic sites remain to be delineated,
such as the relative magnitude of these effects and their
temporal characteristics.
G. Summary
BRAIN
SEROTONIN
SYSTEM
179
ation of the primate serotonergic neurons. However,
and not surprisingly, this concept of an evolutionary
stable system does not hold completely when all the comparative evidence is scrutinized.
The nuclear organization in the primate brain does
differ from that of rodents in several important aspects.
First, in the rat brain, the majority of the serotonergic
nuclear groups are indeed raphe nuclei; that is, the cells
lie on the midline seam and extend laterally from there.
In the primate, fewer cells lie directly on the midline,
and most have at best a paramedian organization. “It
appears the serotonin system undergoes a ‘lateralization’ as the phylogenetic scale is traversed towards primates” (441). Lateral cells in rats may in fact have fewer
collaterals than the midline neurons (148) and would
therefore have a more restricted terminal distribution.
Second, the DRN is more highly organized in primates.
The lateral wings have both a dorsal and ventral subdivision, and the ventromedial subnucleus includes the
nucleus annularis rostrally and the interfascicular nucleus and the B6 group caudally. Third, the serotonergic
cells from NRO extend far more caudally in the primate
brain to reach the cervical levels of the spinal cord. The
location of these cells in lamina X and the ventromedial
fasciculus may indicate a more important relationship
between serotonergic cells and spinal cord in the primate brain. Finally, the hypothalamic 5-HT-concentrating cells described by several groups in rats are not
seen in the primate hypothalamus.
Important differences also have been noted in the
projection pathways. Descending fibers to the spinal
cord in rats originate from NRM and NRPa but appear
to originate only from NRM in primates (85). Ascending
fibers to the forebrain in rats use mainly the MFB (33),
but in monkeys, the DRCT may be quantitatively
larger
(44). Finally, fibers in the primate brain have been
noted by several workers to be myelinated. Although a
few myelinated 5-HT fibers have been noted in rodents
(415), the occurrence in monkeys is 36 times greater
than in rats (44).
Differences between primates and subprimates in
the termination pattern suggest that the primate has a
more restricted target innervation. This can be seen in
the distribution of 5-HT uptake sites in hippocampus
where the CA fields are less innervated in monkeys
compared with rats (45). In the cortex of primates, the
laminar distribution is highest in layer IV (45, 80,342),
whereas in rats it is in layer I and VA (81,130). Finally,
in the olfactory bulb, the glomeruli are innervated in
rats and cats but not monkeys (460). Whether this represents a true evolutionary trend, as has been suggested
(36), or species differences in function remains unknown.
To summarize, in higher mammals the cell bodies of
the serotonergic system are more laterally situated (possibly associated with fewer collaterals), have a much
greater proportion of their axons myelinated, and have
a more restricted terminal innervation (type II). These
properties are suited for rapid dissemination of information from the brain stem raphe to the related target
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Serotonergic neurons do not comprise a homogenous collection of cells organized in specific nuclei. The
various 5-HT-designated nuclei contain many nonserotonergic neurons. However, most studies have shown
that the overwhelming majority of neurons within these
nuclei with long projections are serotonergic. Furthermore, even the serotonergic neurons are not homogenous since many of the cells show that 5-HT is localized
with particular neuropeptides, such as substance P,
CCK, and TRH. Finally the axons of these neurons have
been described as myelinated or unmyelinated, fine or
coarse, and as making synaptic or nonsynaptic contacts.
This morphological evidence supports the idea of two
serotonergic systems (see sect. VIII).
Serotonergic cell body distribution and their projection network appear to have remained remarkably
stable across phylogeny. The main cellular nuclei located in the brain stem first described in rats are present in the monkey and the human brain. It has been
concluded that “the pattern of organization of the three
recognized aminergic systems of the brain have been
preserved with remarkable consistency in the evolution
of primates” (230). The stability of the cellular organization is consistent with the descriptions of the main descending and ascending projections. The four descending projection groups (NRO, NRPa, NRM, and ventrolateral medulla) in the primate brain are still believed
to innervate the substantia gelatinosa of the dorsal
horn, the motor neurons of the ventral horn, and the
intermediate autonomic neurons of the spinal cord. The
ascending nuclei (MRN, DRN, and B9) in the primate
brain use multiple tracts to innervate most of the subcortical and cortical areas of the forebrain with a distribution pattern highly consistent with that reported in
rats. Finally, the termination patterns seen are largely
similar to those described in subprimates. The serotonergic fibers enter the various target structures through
similar pathways and appear to innervate the same target neurons. For instance, the labeling patterns seen in
the DG of monkeys are almost indistinguishable from
those seen in rodents (45). Numerous types of morphologically distinct serotonergic terminals seen in the cerebellum and paratrigeminal
nuclei were identified in
both rats and monkeys (96, 99).
The similarities between the primate and subprimate serotonergic systems are apparent in nuclear organization, major efferent pathways, target structures,
and ultrastructural
relationships. Thus it would appear
that anatomic and physiological studies performed in
subprimates would have a direct bearing on our appreci-
OF
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AND
IV.
DEVELOPMENT
A. Introduction
The foundation
of the organization
of the adult
CNS serotonereic
svstem is embedded in the migration
C. AZMITIA
Volume
7.2
and outgrowth
of serotonergic
neurons. These fetal neurons are known to complete their final mitosis between
days 12-14 of gestation in the rat brain (281). The early
presence of 5-HT within these neurons was shown by
histochemical
fluorescence procedures (366a). High levels of 5-HT were detected within the cells before and
during early neurite outgrowth.
This observation raised
the possibility that 5-HT might serve a trophic role during development before assuming its adult functions as
a neurotransmitter.
The 5-HT neurons are located in two separate clusters (Fig. 6) that show a different developmental
pattern during early gestation. The superior group contains
5-HT stores before they migrate from the ependymal
zone toward the DRN, MRN, and B9, but the neurons
comprising
the inferior group appear to produce 5-HT
only after their migration is complete (491). Neurites of
5-HT cells have a robust outgrowth
throughout
the
neuraxis. The pathways used correspond to many of the
main interconnections
established
by nonserotonergic
neurons (33, 304, 491). The ability of the serotonergic
fibers to use preexisiting
fiber pathways
as guides was
termed “epiphytic guidance” (33) and may provide a cellular basis for the many and varied connections formed
by the 5-HT system. Interestingly,
the formation of precise termination
patterns often occurs long after the arrival of the 5-HT fibers. The terminal branching thus
seems to depend on a different set of signals than those
guiding the fibers to the terminal zone. The distinction
between trophic and neurite-extension
factors requires
tissue culture methods. Serotonergic
neurons show accelerated maturation
in culture compared with other
neural systems and respond to many of the same target
cells that they encounter during normal development. A
number of substances
have been shown to function as
serotonergic
growth regulatory
factors.
In this section, these topics are discussed to illustrate the variety of steps required to construct
a complex organization
and how each step is dependent on
both positional and chemical signals.
B. Nuclei
I. Superior
group
In the superior group, the first 5-HT-immunoreactive neurons are seen at 12 days gestation, 10 days before birth in rats (305,491). The cells are small (5-7 pm),
and within 24 h they form long dendrites (up to 135 pm).
These are typical isodendritic
reticular formation developmental patterns. The cells are among the earliest mesencephalic neuroblasts
to differentiate
and the first to
reach the mantle zone. This evidence of precocious neuronal maturation
provides the framework
for its later
expansive network.
At 14 days gestation, the MLF has appeared, and
this tract is surrounded
by 5-HT-immunoreactive
neurons. The neurons have migrated ventrallv from the ven-
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areas. This description
is at odds with the classic view of
serotonergic
fibers in the lower mammals of a diffuse,
highly branched, unmyelinated
midline group of neurons (type I). The possible functional
implications
for
two types of serotonergic
neurons, a slow expansive one
(type I) and a fast precise one (type II), are discussed in
section VIIIC.
Most of the studies on the raphe afferents
have
been performed in rats. The most important
innervation
is probably the interraphe
5-HT connections. Numerous
other afferents
have been described using retrograde
transport,
immunocytochemistry,
and receptor
radioautography.
These studies indicate that important
nonserotonergic
inputs come from the lateral habenula
(glutaminergic)
and the locus coeruleus (noradrenergic). Other neural afferents
reported
include various
neurotransmitters
(e.g., acetylcholine,
GABA,
dopamine, histamine,
and epinephrine)
and neuromodulators (enkephalin,
endorphins,
substance P, CCK, neurotensin, CRF, and ACTH). Nonneuronal
cells also contact
serotonergic
neurons (e.g., oligodendroglial,
ependymal,
and endothelial cells). This convergence of neuronal and
nonneuronal
input provides the raphe cells with a broad
sampling of information
from many brain and endocrine systems that might have neuronotrophic
as well as
physiological
relevance.
An important
topic covered in this section dealt
with the anatomic distribution
of 5-HT receptor subtypes. The raphe nuclei contained mainly the 5-HT,*
receptor. Limbic areas such as the hippocampus,
septum, amygdala, entorhinal,
and pyriform
cortices had
high levels of the 5-HT,*, 5-HT,, and the 5-HT, receptors. The regional distribution
of receptors can be further analyzed at the laminar level. For example, in the
neocortex the 5-HT2 receptors
are highest in primate
layer IV and rat layer VA, whereas the 5-HT, receptor
shows a more diffuse distribution.
It has been suggested
that particular
5-HT fiber types (e.g., fine from DRN)
may be preferentially
associated with particular
5-HT
receptors
(e.g., 5-HT,) (81). Whether
the different receptor subtypes will correspond to separate morphologically identified serotonergic
neurons is a new promising
area of study. Finally, the restricted
localization of serotonergic cell bodies exclusively in the brain stem implies a role in basic brain functions rather than involvement in higher order information
processing. The relative paucity of cell bodies and their highly branched
terminal domain are consistent with a more basic role in
brain functioning
and could imply that these neurons
comprise
a unifiying
system for synchronization
of
functional centers from a restricted
source.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
OF
SEROTONIN
181
mesencephalic reticular formation and may form part
of B9 (305).
At 16 days gestation, the 5-HT cells are more prominent and less compact, forming four paired bilateral
groups. These are the dorsal, medial, lateral, and ventrolateral
(305). The dorsal group is mainly dorsal to
MLF and forms the lateral wings of the DRN. The medial group is in the mantle zone and gives rise to the
MRN. This group may contribute some neurons to B9
and nucleus pontis centralis oralis. The lateral group
gives rise to the scattered neurons in the midbrain reticular formation and part of B9. Finally, the ventrolatera1 group develops into the main body of B9.
Formation
of a fused 5-HT-immunoreactive
nucleus is apparent at 17 days gestation. This occurs because of a joining of the ependymal zone that gives rise
to a thinning septum of nonneuronal cells (135 pm at 1’7
days gestation and 30 pm at 19 days gestation) (305).
Neurons of the lateral wings of the DRN are seen in
contact with the ependymal layer and have direct access
to CSF. These cells probably contribute to the supraependymal layer of 5-HT-immunoreactive
fibers observed in the third and lateral ventricles. The lateral
and ventrolateral
groups give rise to B9, which surrounds the newly formed medial lemniscal fibers.
At 19 days gestation, the decussating fibers of the
superior cerebellar peduncle separates the CLN rostrally, the MRN ventrally and caudally, and the DRN
dorsally. Thus within 1 wk of the first appearance of
5-HT-containing
neurons, all the major nuclei of the superior group can be observed in the fetal brain.
2, Inferior
tricular surface to take up their positions as bilateral
groups near the midline. However, there are a number
of axons and dendrites forming a 5-HT-immunoreactive
commissural
system connecting these bilateral groups
at this early period. Thus 5-HT intercommunication
is
one of the first and most basic forms of neuronal interactions (491).
Major advances are seen by 15 days gestation. The
5-HT-immunoreactive
cells are clustered into two
groups, dorsolateral andventrolateral(491).
In the bilateral group dorsolateral to the MLF, the neurons are seen
in the rostra1 aspect near the mesencephalic flexure and
do not extend very far caudally. These cells are characterized as having thick dendrites still connected to the
ventricular zone and most probably give rise to the lateral wings of the DRN. In the bilateral group located on
the ventrolateral
aspects of the floor plate just beneath
the MLF, the cells form a continuous longitudinal
column that extends to the pontine flexure. The importance of this observation is that later (17 days gestation)
the ventrolateral
group splits to form the MRN and the
interfascicular
portion of the DRN. This provides developmental support for considering these two groups of
5-HT neurons as part of a larger functional grouping, as
previously suggested (45). More laterally displaced cells
become dispersed and give rise to the neurons in the
SYSTEM
group
The inferior group of serotonergic cells forms -2
days after the superior group (305, 491). They are seen
for the first time as a ventral group at the end of 14 days
gestation. The cells are believed to migrate away from
the ependymal zone and to synthesize 5-HT once they
have reached their ventral placement. The caudal group
at 15 days gestation is completely separate from the
superior group with no 5-HT-immunoreactive
cells or
fibers between them. The NRM is the first nucleus to
form in the inferior group, and in whole mount preparations NRM shows more 5-HT immunoreactivity
than
NRO and NRP (15b). It begins in the rostra1 myelencephalon while soon afterward (16 days gestation) NRO
appears in the middle to caudal myelencephalon
at a
slightly more dorsal position. These nuclei have a fair
number of non-5-HT-immunoreactive
cells mixed with
them. At 1’7 days gestation, the three main caudal nuclei
are visible: the NRM, NRO, and NRPa. The cells of NRM
have begun to form as a midline group, and processes
are seen extending across the midline. The neurons are
part of the pontine reticular formation, with some cells
extending fairly laterally. The major change at 19 days
gestation is the appearance of a well-developed group of
5-HT-immunoreactive
neurons in the ventrolateral
medulla near the inferior olive in the ventromedial
aspect
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017
FIG. 6. Sagittal
section
of brain stem of 15-day-gestation
rat
fetus. Note appearance
of superior
(1) and inferior
(2) cell group. IF,
isthmic
fovea. Bar, 100 pm. [Modified
from Lidov and Molliver
(305).]
BRAIN
182
BARRY
L. JACOBS
AND
C. AZMITIA
Volume
?Z?
medullaris to the lateral habenula (17 days gestation),
the external capsule to the claustrum and the internal
capsule to the globus pallidus (17 days gestation) and
the lateral neocortex (18 days gestation), and the ansa
peduncularis and the ventroamygdalofugal pathway to
the pyriform cortex and amygdala.
The ability of 5-HT-immunoreactive
fibers to utilize so many different tracts suggests that these fibers
are using other fibers as mechanical guides to innervate
a target area. This process has been termed epiphytic
guidance (33). The molecule responsible for this form of
guidance is believed to be a general type of pathway
substrate guidance cue that would direct both 5-HT and
non-5-HT fibers (256,304). A likely candidate for such a
substrate factor is laminin, which acts as an effective
adhesive for growing neurites (141, 318, 410). Laminin
immunoreactivity
is observed in the prenatal brain colocalized with 5-HT-immunoreactive fibers (F. C. Zhou,
personal communication). The precocious development
of the 5-HT neurons strategically places them in the
midline of the floor of the brain stem where they can
C. Pathways
and Terminals
contact a large number of laminin-rich pathway primordia. These metabolically active neurons quickly utilize many tracts to reach the entire neuraxis.
I. Subcortical
It is interesting to note that the 5-HT-immunoreactive fibers very early extend into the ventricular system
Almost as soon as the 5-HT-immunoreactive
neuto grow as supraependymal axons. Although the lateral
rons are seen (12 days gestation),
fine processes can be wings of the DRN have thick processes attached to the
followed rostrally
for a distance (50-75 pm), and by 13 ventricular zone as early as 14 days gestation, axonallike projections were not verified on the ependymal surdays gestation,
5-HT-immunoreactive
fibers are seen
crossing the midline (491). These fibers are nonvaricose
face until 17 days gestation. This innervation continues
straight
axons that form tight fascicles as they pass
until the entire ventricular formation is covered by 5HT-immunoreactive
fibers at 6 postnatal days. These
over the mesencephalic
flexure. These fascicles extend
toward the mammillary
complex, possibly following the
fibers have direct access to CSF and can be seen in cerpreviously
formed mammillotegmental
tract [lZ days
tain areas to pass back into brain tissue (43a, 491). The
fibers are most likely not transmitting or receiving elecgestation (304)]. This latter tract is the mesencephalic
component of the MFB (33).
trical information from the ventricle, so the information they process may be trophic in nature. The samThe first connections are those seen decussating the
midline and linking the paired bilateral groups of 5-HT
pling of CSF by the fibers and growth back into neural
tissue would constitute a nonelectrical means of conveyneuronal groups at E-13 days gestation (491). Fibers
ing or receiving information on the metabolic state of
enter the ependymal surface by 17 days gestation. There
is a slow innervation
of the various nuclei as they form
the organism.
The arrival of serotonergic fibers near a terminal
so that by postnatal day I, the cochlear nucleus, the spinal trigeminal,
and the inferior
olivary nucleus are
area does not necessarily mean that they immediately
densely innervated (304). The first fibers are seen in the
will proceed to form a terminal plexus there. For examcerebellum at 21 days gestation, and the innervation
of ple, 5-HT-immunoreactive fibers pass just caudal to the
the internal granular
layer (postnatal
day 1) and the optic chiasm at 16 days gestation (491), yet these fibers
Purkinje cells and molecular layer (10 postnatal days) do not innervate the suprachiasmatic nucleus until 10
postnatal days (304). Interestingly, the optic innervaare reached postnatally.
The signals for terminal
sprouting that occasionally are delayed after the 5-HT
tion of the suprachiasmatic nucleus arrives at 4 postnatal days (149, 448). The neuronal packing in the suprafibers reach a terminal area are not known, but clearly
they represents a timing inherent to the target cells and chiasmatic nucleus declines as the target cells mature
not to the innervating 5-HT afferents themselves.
and grow dendrites. Then at 10 postnatal days the serotonergic fibers innervate the suprachiasmatic nucleus
The 5-HT fibers quickly begin to colonize the forebrain by extending along newly formed pathways (305, itself. This is also true of the mammillary complex,
366a, 491). These include the fasciculus retroflexus to which is not innervated until -3 postnatal days. The
the lateral habenula (15 days gestation), supraoptic de- substantia nigra does not begin to receive 5-HT terminal fibers until after this time. An interesting contrast
cussation into the supraoptic region (16 days gestation),
the same fibers traverse caudally with the optic tract to is seen in the striatum, which is innervated prenatally
by dopaminergic fibers (190,435) but does not receive a
the lateral geniculate body (18 days gestation), the stria
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of the LPGN. These neurons are a lateral migration
from the neurons comprising
the NRM. Medially, the
neurons of the more rostrally
positioned NRM appear to
border the neurons of NRPa at the rostra1 inferior olivary nucleus.
In summary, the various raphe nuclei are phenotypically similar, but during development they show differential
migratory
patterns.
The inferior groups are
parceled into separate nuclei at the same time as the
superior group, although they do not contain 5-HT until
2 days after the superior
group. These differences
in
staining and migratory
pattern between the two major
groups may underlie the other differences
seen in the
adult brain (e.g., eolocalization with peptides). The superior groups give rise to a ventrolateral
cluster that
forms the MRN and the interfascicular
portion of DRN
and the lateral wings of DRN. Finally, it should be emphasized that the fetal neurons begin laterally and move
toward the midline as they mature.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
substantial
5-HT innervation
until 14 postnatal
days
(491). This represents
an interesting
difference between
serotonergic
and dopaminergic
fetal neurons.
2. Hippocampus
3. Neocortex
The neocortical innervation deserves special note.
5-Hydroxytryptamine
fibers reach the frontal cortex by
17 days gestation via a rostra1 route through the septum. They form a bilaminar distribution above and just
below the developing cortical plate (79, 304). Occasionally, some fibers traverse the cortical plate. However,
not until 3 postnatal days is the entire cortex reached.
The adult laminar pattern is not established until at
least 2 wk postnatally. This should be contrasted with
the NA system that innervates frontal cortex at 16 days
gestation and extends over the entire cortex by 20 days
gestation (424, 435). The locus coeruleus-cortical connection reaches the adult pattern by 7 postnatal days
(303,336). This differential sequence between 5-HT and
BRAIN
SEROTONIN
SYSTEM
183
NA immature
neurons suggests the neurons might be
responding
to different signals in the target and may
show different growth rates because of a presynaptic
signal.
Cortical
SHT-immunoreactive
axons
show
a
patchy distribution
in the somatosensory
cortex of neonatal mice (172) where a transient dense patch of SHTimmunoreactive
fibers and [3H]citalopram
(binds to the
reuptake site) labeling was seen in layers IV and VI
(119). This increased patchy labeling was seen from 3 to
17 postnatal days and disappeared by 2 1 postnatal days.
These patches closely overlap the prim .ary sensory area
of cortex . Th e den se patches seen in somatosensory
cortex were not seen in visual (area 17) or auditory cortex
(area 41) where continuous bands of dense axons were
seen i n layers IV and VI. In contrast, motor areas as well
as parietal and visual association
cortex had a sparse
5-HT-immunoreactive
axonal distribution.
In the somatosensory
vibrissae barrel fields, the patches of 5-HTimmunoreactive
axons in layers IV and VI are in vertical register. After 5,7-DHT neurotoxin
treatment,
the
patches
of serotonergic
axons and the density
of
[3H]citalopram
binding is greatly diminished. Recen .tlY 9
it has been observed that removal of the serotonergic
patches in the barrel fields actually suppresses the formation of the barrel fields themselves (80). Thus 5-HT
innervatio In in the cortex appears to serve a trophic influence on the deve lopment of specific sensory receptive
areas. This supports the general hypothesis that the
CNS serotonergic system exerts powerful effects by virtue of its innervation density, a chemical determinant of
the m orphological organization that underlies the physi01WY of the brain
This distinctive
temporal innervation
pattern
serves to underscore th e various steps necessary for development of a 5-HT terminal connection. First, the 5HT neurons migrate from the ventricular surface to assume a paramedian position. This most likely is in response to the presence of tran sient gli al radial fibers
(486). The neu.rons then begin to form neurites in response to a soluble trophic signal (see sect. IV&) and to
be guided to a variety of target areas by laminin tracts.
The final innervation density requires the production of
a local trophic signal that depends on the maturation of
target cells (neurons and glia).
0
Tissue Culture
1. I~~~oduCtio~
Fetal neurons can survive and mature outside their
normal brain environment. The first cultured serotonergic CNS neurons were explants of midbrain raphe
taken from newborn rats (202). These postnatal serotonergic neurons survived for up to 16 days in culture.
The neurons synthesized and metabolized 5-HT. Detectable levels of 5-[3H]hydroxyindole
acetic acid (5[3H]HIAA), the major 5-HT metabolite, were measured
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The hippocampus
illustrates
an example of a structure innervated by separate 5-HT pathways
that arrive
at different times during development
(304, 491). The
5-HT-immunoreactive
fibers that first reach the hippocampus enter from the dorsal supracallosal
route. They
pass through the diagonal band, septum, and in the supracollosal stria in the developing cingulum bundle beof the corpus callosum . These fibers
fore the formation
enter th e retrosplenial
primordial
cortex, pass in to the
subiculum,
and finally into the cornu Ammonis of the
hippocampus by 18 days gestation. The 5-HT-immunoreactive fibers lie above (stratum
oriens) and below (straturn radiatum)
the pyramidal
neurons. The fornix-fimbria route appears to be utilized 1 day later by fibers to
the d.orsal hippocampus.
Finally, the third and final
entry into the hippocampal
formation
via the ventral
amygdalofugal pathways (47) is by laterally coursing
5-HT-immunoreactive
fibers that also project to the
pyriform cortex and the ventrobasal amygdaloid nuclei.
It should be noted that the two dorsal pathways originate in the MRN and interfascicular
part of DRN,
whereas the ventral pathway originates in the lateral
wings of the DRN (530).
The 5-HT-immunoreactive
fibers at 3 postnatal
days are seen in two dense bands in the CA field, a superficial band. in the stratum oriens (over basal dendrites) and a deeper band between the stratum radiuturn and stratum laconosum. This pattern slowly matures into the adult pattern where a dense innervation is
seen over the stratum lacunosum molecular by 10 postnatal days. The innervation of the DG begins at 1 postnatal day with a few fibers in the hilus, and by 2 wk
postnatal it has not yet reached adult patterns. The 5HT innervation of the hippocampus is still increasing 3
mo postnatally in rats (40).
OF
184
BARRY
L. JACOBS
AND
2. Accelerated
maturation
Serotonergic neurons develop more rapidly in culture than neurons in most other neurotransmitter
systems. Process outgrowth occurs within hours in culture
(48), and Ca2+-dependent uptake and release are functional as early as 2-3 days in culture (48,403a). Noradrenergic neurons from fetuses of 18 days gestation show
an immature
phenotype and do not show substantial
uptake for at least 3 wk, while specific uptake into dopaminergic neurons occurs after 5 days in culture (391,
392). Aggregate cultures of dopaminergic
neurons after
4 days in culture (300) and explant cultures of locus
coeruleus neurons after 8-14 days in culture (138, 423)
contain fluorescent cells with fine fibers. Dissociated
mesencephalic cultures do not have fluorescent cell bodies or axonal varicosities for up to 3 wk in culture (403a).
These studies illustrate the variation in morphogenesis
of identified transmitter
systems in culture (391, 392).
In general, serotonergic
neurons appear more precocious than catecholaminergic
neurons. This differential temporal pattern is consistent with developmental
findings in the rodent fetal brain (see sect. IvB). However, the results in culture should be viewed cautiously
since procedures vary dramatically
among laboratories.
C. AZMITIA
Volume
72
3. Target cell interaction
Serotonergic cell growth in culture can be stimulated by a variety of normal target and nontarget cells
(48). Hippocampal
cells, a normal target for the superior
groups, induce the greatest increase of 5-[3H]HT uptake
capacity by mesencephalic
raphe cells. Immunocytochemical staining shows that coculturing with hippocampus did not significantly
change the survival of 5HT-immunoreactive
cells between 48 and 96 h. Thus the
increase in accumulation represents a maturation of the
uptake and storage of the existing cells. A similar result
was achieved in cocultures of dopaminergic
neurons
with striatal cells (35). The stimulation
of 5-HT neurons
by hippocampal
neurons occurs under conditions where
glial cell proliferation
is suppressed (48). Thus the target cell stimulation
is probably due to neuronal-neuronal interactions or to interactions with postmitotic glial
cells. Other target areas, normally innervated by mesencephalic raphe cells (cortex, caudate, and olfactory
bulb), can stimulate the development of the amount of
5-rH]HT accumulated at high plating densities. In addition, serotonergic neurons from the superior group are
stimulated by spinal cord cells that they do not normally
innervate.
The degree of stimulation
observed is not related to
innervation
reached in the adult brain since hippocampal 5-HT levels and fiber density are relatively low (304,
416,450). However, the stimulation
by target tissue may
reflect 5-HT receptor density since hippocampal
5-HT
receptors are among the highest in the CNS (67, 357).
Pharmacological
studies are consistent with this latter
explanation (44.2, 503, 505, 506).
Several neurotransmitter
systems show a tight
coupling between the afferent neurons and their normal
target cells. Dopaminergic
neurons respond to their
normal target cells (corpus striatum) but not to nontarget cells (parietal cortex) by an increased capacity for
uptake and storage for [3H]dopamine
(135, 391, 392).
This stimulation
has been replicated by another laboratory (269). Further evidence for target specificity for
dopaminergic
neurons is shown by the stimulation
produced by the striatal membrane preparations but not by
preparations from the hippocampus, cortex, mesencephalon, or cerebellum, all areas area normally devoid of a
dopaminergic
innervation
(391). These results indicate
that dopaminergic neurons respond only to their normal
target cells in the striatum. Similarly,
rat sympathetic
substance P-containing
neurons are stimulated by their
normal target cells (pineal and salivary gland) but not
by nontarget cells (heart or intestine) (258). These patterns are consistent with a point-to-point
mechanism in
brain development.
In summary,
cultured serotonergic
neurons respond to a wide variety of target neurons within a wide
developmental
window. The combination of diverse target cells and extended time frames could explain the
expansive distribution
of serotonergic fibers in the vertebrate brain. In other words, the serotonergic fibers
have a long period during development in which to lo-
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in the medium after addition of radioactive tryptophan.
In a dissociated embryonic (14-15 days gestation) rat
brain stem raphe, 5-HT neurons were visualized by immunocytochemistry
and by specific uptake of 5-[3H]HT
(522, 282b, 390a). Within 24 h of plating, 3.3% of the
surviving neurons were immunoreactive
to the 5-HT antibody (522). The percent of surviving neurons immunoreactive for 5-HT increased over time, reaching up to
36% of the surviving neurons by 28 days in culture. This
observation emphasizes the hardy nature of serotonergic neurons when compared with other brain stem neurons, including dopaminergic
and noradrenergic. These
cells in culture synthesized, stored, and released 5[3H]HT after exposure to [3H]tryptophan.
It is interesting to note that a large number of GABAergic neurons
were observed in this raphe culture system (up to 40% of
the neurons in 14-day-old cultures). However, these neurons were smaller in somal size and had much shorter
processes than the serotonergic neurons. Furthermore,
GABA was not colocalized with 5-HT in the larger neurons.
Dissociated mesencephalic
raphe cells from fetal
rat (14-18 days gestation) were grown in microcultures
(39,48). The maturation
of serotonergic cells was studied qualitatively
using immunocytochemistry
with a 5HT antibody and quantitatively
by measuring the retention of radioactivity
after incubation in the presence
of a low concentration of 5-PH]HT. The 5-HT-immunoreactive neurons showed specific staining in the perikarya, nuclei, dendrites, axons, and growth cones. These
neurons formed varicose fibers and growth cones within
hours and survived for up to 3 wk in culture. Each serotonergic neuron concentrated ,- 1 fmol5-rH]HT
after 20
min of incubation.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
cate and interact with one of their many target neurons.
The characteristics
of the target cell stimulation
described for 5-HT neurons are uncommon. These findings
hold out the possibility
that a widespread
maturational
factor exists for CNS serotonergic
neurons.
E. Growth
Regulatory
Factors
1. Ions and small molecules
Calcium has a myriad of biologic effects, such as
inhibition of phosphorylation, uncoupling of mitochondria electron transport, activation of phospholipase and
proteases, cell adhesion, and process outgrowth (186,
518, 523). In cultures of mouse cortical neurons or molluscan ganglion neurons, low Ca” results in decreased
process outgrowth (323,518). Conversely, high Ca2+levels intracellularly result in neurite retraction and eventual neuronal death (104, 106). Serotonergic CNS neurons grown in low-Ca2+ medium have fewer 5-HT-immunoreactive cells with smaller somas and fewer
processes after 3 days in culture (49). Conversely, high
levels of Ca2+ result in neuronal death. If a specific Ltype Ca2+ channel antagonist (nimodipine, lo-’ M) is
added to the cultures at the time of plating, then a substantial potentiation in serotonergic survival and process outgrowth is seen over normal conditions (38). This
indicates that regulating the L-type Ca2+ channel can
produce supranormal growth. In vivo studies support
this concept of the L-type Ca2+ channel stimulating serotonergic growth. Treatment of pregnant rats with nimodipine results in accelerated outgrowth of serotonergic fibers to forebrain structures in the offspring (362).
Neurons in culture are assumed to favor supranorma1 glucose concentrations since most of their energy is
derived from the oxidation of glucose (83). Cerebellar
neurons have been shown to survive better in 0.3% (19
mM) glucose than in 0.1% (6 mM) glucose (388). In studies of myelination of central and peripheral neurons, a
glucose concentration of 0.6% (37.5 mM) was found to be
best (350). Likewise, cerebellar explants require 40 mM
BRAIN
SEROTONIN
SYSTEM
185
glucose for optimal development (286). Several reports
on serum-free conditions also advise using glucose concentrations between 25 and 33 mM for optimal neuronal
growth (15, 313, 356). Thus it would be expected, given
their robust outgrowth and high metabolic activity, that
serotonergic neurons would require a similar high-glucose condition. In fact, serotonergic neurons can survive
for up to 2 days, but with reduced outgrowth, when glucose is absent from the medium at the time of plating
(39). As the concentration of glucose is increased to
0.15% (8.3 mM), the growth rate for the cultured serotonergic neurons shows a sharp increase. However,
higher levels of glucose (O&0.5%; 12.5-31 mM) reduce
the development of the serotonergic neurons by as much
as 75%. The decreased outgrowth in high glucose is probably due to increased acidosis from the enhanced production of lactate (see Ref. 361). Interestingly, the glucose
level where serotonergic neurons grow best is similar to
the Michaelis constant (K,) of glucose for uptake into
neurons and glial cells (0.18 and 0.16%, respectively).
Free hydroxyl radicals are believed to be a major
cause of neuronal death (203,206). It has been proposed
that the production of H,O, by monoamine oxidase can
contribute to the generation of free radicals inside neurons (107). Various neurotoxins, such as l-methyl-4
phenyl-l&3,6-tetrahydropyridine
(MPTP) and 6-hydroxydopamine (6-OHDA), appear to act by producing
an excess of free hydroxy radicals (352,463), and it has
been suggested that neurotransmitters themselves can
be converted to potent oxidizing neurotoxins under certain conditions (195,434). In these studies, the addition
of free radical scavengers (e.g., pyruvate, vitamin E) or
antioxidant enzymes (e.g., supraoxide dismutase, catalase, or glutathione peroxidase) prevents the buildup of
oxidants and corresponding cell death (see also Ref.
321). Serotonergic neurons in culture are suppressed by
increased levels of H202 in the medium and are enhanced by addition of catalase and glutathione peroxidase (Azmitia, unpublished observations). These results
indicate that serotonergic neurons are adversely affected by conditions that generate hydroxy radicals or
are stimulated by conditions where free radical buildup
is prevented. Increased levels of free hydroxy radicals in
the aged brain (as evidence by the buildup of lipofuscin)
(440) may offer an explanation for the decrease in longterm survival of transplanted fetal neurons in aged
rats (37).
2. Neurotransmitters
Serotonergic neuronal maturation appears to be
under the influence of its own neurotransmitter during
development. Mesencephalic raphe cells from rats of 14
days gestation cocultured with hippocampal cells from
18 days gestation were grown for up to 4 days in the
presence of various agents known to alter serotonergic
function in the mature brain (505). Pargyline (a nonspecific MAOI) alone and with 5-HT (10-8-10-6 M) inhibited
growth of serotonergic neurons as assessedby uptake of
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Discussion
of development
would be incomplete
without
attention
to the factors
that regulate
the
growth
response of serotonergic
neurons. In this section, we focus on a wide range of factors that have significant effects on the survival,
attachment,
biochemical
maturation,
neurite extension, and death of fetal serotonergic neurons in dissociated
tissue culture. Our selection of factors is broadened to include ions, intermediate metabolites,
antioxidants,
neuropeptides,
gangliosides, neurotransmitters,
and proteins.
A detailed
treatment
of this topic has been published (49) so the
effects of these factors on cultured serotonergic
neurons
are only briefly covered. Furthermore,
the results of
prior depletion of 5-HT on general brain maturation
have been extensively
reviewed (see Refs. 279, 280, 282)
and are not discussed here.
OF
186
BARRY
L. JACOBS
AND
C. AZMITIA
Volume
72
important
functions in regulating the growth of serotonergic neurons. It might be proposed that the vigorous
and precocious innervation of the lateral habenula may
be related to the glutamate stored in these neurons and
the presence of appropriate
excitatory amino acid receptors on the growing serotonergic fibers.
The role of neuropeptides as neuronal growth-regulating factors has been reviewed (49, 125). Neuropeptides comprise the largest group of putative neurotransmitters (neuroregulators)
in the brain and serve as
growth factors that are multifunctional
(446). Their
first appearance in brain during early development is
variable and depends on the peptide and region. For instance, vasoactive intestinal peptide (VIP) and bombesin are not measurable
in the brain before birth,
whereas substance P and somatostatin
occur in all regions examined by 14 days gestation. Somatostatin
and
VIP show large increases in mesencephalon
and forebrain, bombesin shows a similar but less dramatic pattern, whereas substance P displayed its largest developmental increases in diencephalon
and mesencephalon.
Thus brain chemical systems begin to develop early and
increase in complexity as the brain slowly matures.
In dissociated cultures of mesencephalic neurons,
low doses of ACTH (l-10 rig/ml) can stimulate serotonergic development (43). The ACTH family of peptides
[ACTH-(4-IO),
ACTH-(l-23),
ACTH-(l-35),
and Organon 27661 are all effective in culture. However, if hippocampal fetal neurons (target cells) are present, exogenous ACTH is no longer stimulatory
on serotonergic
maturation.
This overriding of the trophic peptide effect
by target neurons supports the idea that positive neuropeptide growth factors are general signals and may
have no direct action on neuronal maintenance after the
neurons reach their target. Enkephalin
was found to be
inhibitory
to these same 5-HT neurons at low concentrations (~1 rig/ml) (124). Interestingly,
the addition of
fetal hippocampal
or spinal cord neurons to the raphe
cultures enhanced the inhibitory
properties
of this
opioid. This result, with a negative neuropeptide growth
factor, indicates that it may have the ability to counteract the trophic effect of the target and halt or repel the
growing fiber. In summary, these studies raise the intriguing possibility that neuropeptides may function as
positive and negative growth regulatory factors in the
brain. Their presence during early development is consistent with this idea. Therefore changes in peptide levels in the brain may contribute to the termination
pattern of the developing 5-HT system.
3. Gangliosides
Gangliosides
are glycosphyngolipids,
which are a
major component of brain membranes. Ganglioside administration
after brain damage has been shown to promote axonal sprouting during nerve regeneration and to
enhance recovery of function. Following partial lesions
of the hippocampus, both aeetylcholinesterase
and 5-HT
uptake are reduced (194). Daily injections of the GM,
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5-[“H]HT. High-affinity
5-HT receptors in fetal brain
stem tissue and in fetal forebrain tissue may regulate
direction and extent of growth. The presence of these
receptors in the fetal brain was confirmed using a direct
binding assay (505). 5-Methoxytryptamine
(5-MT), a 5HT agonist with selectivity
for 5-HT autoreceptors
(322), inhibited growth at nanomolar concentrations.
5Methoxytryptamine
was observed to produce stunted
processes, increase autoinnervation,
and lead to the
death of 5-HT-immunoreactive
neurons. These inhibitory effects on the growth of cultured 5-HT neurons
were reversed at micromolar
concentrations
of 5-MT,
where an actual stimulation
was found. In vivo confirmation of these results used pregnant rats treated with
5-MT. The pups showed suppressed outgrowth of 5-HT
fibers toward the hippocampus
(2 wk to 1 mo) after
treatment with low doses of 5-MT (1 mg/kg SC) (436).
The precise mechanism for these 5-HT receptormediated stimulatory
or inhibitory
effects has received
considerable attention. It is known that glial cells play
an important role since they produce laminin and a host
of trophic factors. High-affinity
serotonergic receptors
exist on brain astroglia (504). The expression of these
receptors varies inversely with the maturational
state
of the astrocyte. Furthermore,
stimulation
of astroglial
cells by specific 5-HT,, agonists produces a conditioned
medium that can stimulate the maturation
of cultured
serotonergic neurons (506). The substance produced by
the astrocytes responsible for this effect is believed to
be the S-100 protein (see sect. 1vE4).
Autoregulation
of fetal serotonergic neuronal development provides a unique way for the CNS 5-HT system
to innervate the brain. This mechanism implies that a
serotonergic neuron will grow until it reaches a brain
region where it produces excess 5-HT because of the
conditions encountered in the target area (e.g., cofactor
availability).
The 5-HT could leak out to interact with
terminal
autoreceptors and stop its own growth. Because serotonergic fibers contain terminal
autoreceptors (191), such a mechanism is feasible as a feedback
brake on neurite extension.
The excitatory amino acid glutamate can produce a
Ca2+-related cell death on neurons containing
an Nmethyl-D-aspartate
(NMDA) receptor (104, 263). The
serotonergic raphe neurons have NMDA receptors in association with the excitatory input derived from the lateral habenula (see sect. IIIC). Addition of 0.5 mM glutamate to serotonergic cultures raised with low glucose
produces evidence of neuropathology
(39). The addition
of MK-801, a specific NMDA antagonist, prevents the
decrease seen in the development of cultured serotonergic neurons. Interestingly,
nimodipine,
the specific Ltype Ca2+ channel antagonist, also attenuated the effects of glutamate
on serotonergic maturation.
These
results indicate that excess Ca2+ entry, via a variety of
channels, is additive in producing neuronal toxicity. In
fact, addition of phencyclidine (PCP) to normal cultures
can enhance serotonergic maturation
in an analogous
fashion as observed with nimodipine. Thus voltage-sensitive and chemically mediated Ca2+ channels may serve
EFRAIN
January
19%
STRUCTURE
AND
FUNCTION
OF
BRAIN
SEROTONIN
SYSTEM
187
HIAA. A similar lack of NGF on fetal mouse brain stem
catecholaminergic
neurons in culture has been reported
(139).
The results with the 5-HT,,-induced
release of astrocytic S-100 have interesting
implications.
For one, it
has been reported that the 5-HT receptors
on astrocytes are reduced when the cells are matured by exposure to adenosine
3’,5’-cyclic
monophosphate
(504).
Thus serotonergic
neurons may determine
their own
innervation
density by stimulating
the release of trophic molecules
when the appropriate
receptors
are
available. When the astrocytes
become mature, the
availability
of receptors
for trophic
stimulation
is
greatly reduced. Interestingly,
damage to the 5-HT
fibers in the adult brain may upregulate
the 5-HT receptors (164, 357) and permit new fibers to stimulate
trophic
factor release. This might account for the
various reports
of hyperinnervation
after damage to
adult 5-HT fibers (46, 168, 169, 528, 529, 533).
4. Proteins
F. Summary
Partial reduction of brain 5-HT fibers by chemical
toxins will eventually result in the collateral sprouting
of undamaged 5-HT fibers (41,531). The signal responsible for this growth
of 5-HT fibers is believed to be a
protein molecule (45b, 49). When a soluble fraction is
prepared from 5,7-DHT-lesioned
hippocampi, a stimulation of fetal serotonergic
neurons is observed in tissue
culture.
The identity of this protein remains unknown. However, recent findings suggest that the molecule may be
S-100 (P. M. Whitaker-Azmitia
and E. C. Azmitia, unpublished observations).
S-100 is found in brain astrocytes, and the P-subunit has been shown to have neurotrophic activity in chick embryo cerebral cortical neurons (261). S-100b is very active
in stimulating
serotonergic
uptake capacity in a microculture
system.
Levels as low as 10 rig/ml of the ,&subunit can increase
the uptake capacity for 5-[“H]HT
by fetal serotonergic
neurons by as much as 100%. In astroglial cultures, the
addition of a specific 5-HT,* receptor agonist, ipsapirone, results in the release of a stimulatory
molecule into
the medium (506). This 5-HT,* neuronotrophic
factor
can increase neurite outgrowth
in cultured serotonergic
neurons. In addition,
all of this trophic
activity
is
blocked by treatment
with a specific S-100 antibody.
The results with S-100 stand in marked contrast to
the lack of serotonergic
trophic activity seen with nerve
growth factor (NGF), a well-recognized
peripheral nervous system (PNS) growth factor (73). Electrolytic
lesions of the serotonergic
input to the hippocampus
resulted in a marked decrease in 5-HT and 5-HIAA.
The
administration
of NGF (10 pg/rat icv twice a week for 2
wk) increased cortical choline acetyltransferase
activity
but had no effect on the lesion-induced
decrease in hippocampal 5-HT and 5-HIAA
(312). Conversely,
injections of GM gangliosides
(30 mg/kg ip for 2 wk) significantly reduced the hippocampal
loss of 5-HT and 5-
Serotonergic
neurons differentiate
very early into
two main clusters of cells, a superior
and an inferior
group (305,491), and begin immediately
to produce neurites that extend anteriorly from the superior group and
caudally from the inferior group (305). These fibers use
other nonserotonergic
pathways,
blood vessels, and the
ependymal cells as guides for innervating
their target
areas. This ability to utilize a variety of substrates
for
the growing processes has been termed epiphytic guidance (33).
Serotonergic
fibers reach their target very early in
development. They reach the hypothalamus
by 16 days
gestation and the cortex by 17 days gestation (304). However, the final termination
density is not reached in
these structures
until the second week of postnatal development. The reason for this delayed maturation
may
involve production of some local growth regulatory
substance (49). It has been demonstrated
by tissue culture
studies that serotonergic
neurons respond to a variety
of substances
produced by neurons, glia, or endocrine
tissue. The timing of the availability
of these factors
may act as the signal for the completion of innervation.
There is evidence that this might occur following
enhanced terminal activity (see Ref. 304). The studies indicating that 5-HT can function as a trophic factor indicate that a shift in the timing of serotonergic
ingrowth
may have important
consequences on the maturation
of
the target neurons. The delayed onset of barrel fields in
the cortex produced by 5,7-DHT injections supports this
idea (80).
A number of different factors have been described
that can function as negative or positive serotonergic
growth factors. The factors can often be stimulatory
at
one and inhibitory
at another concentration
or condition. Calcium, for instance, can produce accelerated
growth at 0.5 mM but actually suppress growth at 2.0
mM. The same is true for glucose, 5-MT, gangliosides,
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ganglioside resulted in enhancement of recovery in both
neuronal systems at 6 and 21 days postlesion. In another
paradigm, electrolytic
lesions in the midbrain caused a
significant decrease in hippocampal
5-HT and its major
metabolite 5-HIAA
(312). Daily systemic injections of
the GM, ganglioside significantly
reduced the injury-induced decrease in hippocampal
5-HT and 5-HIAA
at
6-14 days. Similar results have been seen with the dopaminergic neurons (464). Low doses of GM,, gangliosides
(pg/ml) were stimulatory
on both serotonergic
and dopaminergic neurons in low-density
cultures (E. C. Azmitia
and E. Yavin, unpublished
observations).
However,
in
high-density
cultures, a higher dose of gangliosides (pg/
ml) was inhibitory.
The effects of GD1 gangliosides,
which have two sialic acid residues, are much more potent than GM1 on serotonergic
neurons. These results
imply that gangliosides can be beneficial when a neuron
has been stressed but may be inhibitory
when a neuron
is growing under optimal conditions.
188
BARRY
L. JACOBS
AND
and neuropeptides.
It appears that the conditions
of
growth
are finely tuned so that imbalances on either
side of the ideal condition can suppress the maturational rate. The manipulation
of these factors during
development
to produce a change in the final innervation density and behavior holds promise for correcting
developmental
disorders
(436).
V.
ADULT
PLASTICITY
A. Introduction
B. Regeneration
The ability of injured neurons to send their axons
back into the same area they vacated following axotomy
C. AZMITIA
Volume
72
is termed regeneration.
This process had been well
characterized
in the PNS by a variety of researchers,
but in the CNS many scientists still adhere to the words
of Ramon y Cajal (397): “once the development
was
ended, the founts of growth
and regeneration
of the
axons and dendrites
dried up irrevocably.
In adult
centers, the nerve paths are something fixed, ended, and
immutable. Everything
may die, nothing may be regenerated.” Early studies of 5-HT regeneration
in spinal
cord and brain stem have been previously reviewed (33,
72). A case of serotonergic
regeneration
is discussed: the
morphological
and functional
recovery after 5,7-DHT
lesions in the hypothalamus,
where the regrowing
fibers
not only reestablish
their normal synaptic contacts but
can also hyperinnervate
the target.
Unilateral
microinjection
of 5,7-DHT into the MFB
of adult female rats results in the destruction
of the
serotonergic
fibers traveling in this pathway. Within 1
wk, the hypothalamus
is largely devoid of 5-HT-immunoreactive fibers (166), and levels of 5-HT and 5-HIAA
are greatly reduced (e.g., 90% decrease in the dorsomedial nucleus) (169). Behaviorally,
progesterone-primed
ovariectomized
rats show a facilitated lordosis response
to injected estrogen after bilateral 5,7-DHT injection
(314). Between 14 and 19 days after the neurotoxin
injection, 5-HT-immunoreactive
axons in the caudal hypothalamus have smooth bulbous tips that begin to emit
fine wispy processes. The 5-HT-immunoreactive
fibers
extend back into the previously
denervated
hypothalamic nuclei by 1 mo, with the levels of 5-HT increasing
to 50% of control. The facilitated
lordosis begins to be
attenuated at this time. The results at 8 wk after lesion
show that 5-HT-immunoreactive
fibers have hyperinnervated the hypothalamus
and 5-HT and 5-HIAA have
returned to control levels in many areas. The facilitated
lordosis response to estrogen is once again normal after
9 wk.
This series of studies indicates that damaged serotonergic fibers can survive an injury and with time send
back their axons to reinnervate
the denervated region.
The hyperinnervation
observed with immunocytochemistry is confirmed by light- and electron-microscopic
radioautography
(167,168). At the ultrastructural
level, 50
days after 5,7-DHT neurotoxin
injection, there was no
significant difference in the types of appositions
or synaptic frequency
between 5-[3H]HT-labeled
axons and
the postsynaptic
neurons compared with sham injected.
Thus the regenerating
serotonergic
axons grow back to
some of the same nuclei (dorsomedial
hypothalamus
and zona incerta), reestablish
synaptic and neurotransmitter function, and restore the behavior to normal levels. The ability to achieve this complicated sequence of
events in an adult brain emphasizes a target-directed
regrowth
signal for serotonergic
fibers should exist.
Successful regeneration
in the adult CNS, because
it must occur in an environment
deprived of signals
available to developing fibers, requires
months. The
time required for the fibers to reach their target is much
longer than that needed by the growing fetal neurons,
which can innervate
the hypothalamus
within
a few
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The adult nervous system loses many of the properties associated
with the developing brain. There is a
more complex and varied collection of neuronal and glial
cells in the adult. Many of the radial glial cells clearly
visible during early development
are gone. Substrate
molecules like laminin seen throughout
the immature
brain are no longer present (307). Myelinated pathways
not seen prenatally
are abundant in every region of the
adult brain. The number of synaptic contacts observed
in the mature neuropil illustrates
how extensively
interconnected
the adult brain has become. Thus it is not
surprising
that the dynamic plastic nature of adult neurons has been difficult to study.
Serotonergic
neurons are exceptional in their regenerative capacity in the adult brain. Damage to fine 5-HT
fibers produces only temporary
decreases in the levels
of 5-HT in target areas. Given sufficient time (several
months) the damaged neurons will reestablish
both a
structural
and a functional presence. Adult target cells
can provide many of the crucial molecules to promote
sprouting,
although key guidance cues (e.g., laminin)
may be absent. This may explain why regenerating
fibers require more time to innervate an area than do
the fetal cells during development.
Undamaged
serotonergic
fibers in the brain can
also expand when neighboring
5-HT fibers are removed.
This process of homotypic collateral sprouting
was established for the first time with 5-HT innervation
of the
hippocampus
by utilizing a specific neurotoxin for 5-HT
fibers (41). The characteristics
of this form of adult plasticity are discussed in section vC.
Neuronal transplantation
enables fetal neurons to
be inserted into the mature environment
of the adult
brain. This interaction
of neurons of different ages provides unique insights into the process of adult plasticity.
This procedure underscores
the tremendous
capacity of
the adult brain for reorganization,
both in structure
and
function. Locally placed fetal serotonergic
neurons can
substitute
for the loss of normal afferent input arriving
from the brain stem raphe nuclei. Furthermore,
the response of fetal neurons to the adult neuropil may suggest which factors are needed to facilitate regrowth
of
damaged adult neurons.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
days. The temporal discrepancy might be due to the lack
of a proper substrate in the adult for the growing axonal
sprouts. For example, the substrate molecule laminin is
not detected in the adult neuropil (205, 307). However,
injections of laminin can accelerate this process (182).
The fibers perform properly when they arrive at the
target site as indicated by the ability to restore morphological, biochemical, and functional connections. The
problem is getting to the target, and laminin may be an
important chemical substrate to direct the growing
sprouts.
OF
BRAIN
SEROTONIN
A
5,7-DHT
C
Symmetry
Asymmetry
Supracallosal
5,7-DHT
C
(14 days)
LTCB
(42 days)
+STFI
(14 days)
FIG. 7. Schematic
representation
of 5-HT innervation
of hippocampus by supracallosal
fibers traveling
in cingulum
bundle (CB) and
by infracallosal
fibers traveling
in fimbria
(FI). These fibers originate
from different
neurons
in median
raphe (MR) nucleus and ascend to
forebrain
in medial forebrain
bundle (MFB).
A: degenerating
5-HT
axons
(dashed
line) after
unilateral
short-term
cingulum
bundle
(STCB) microinjection
of 5,7-dihydroxytryptamine
(5,VDHT)
and resultant
anatomic
asymmetry
produced.
B: collateral
sprouting
of infracallosal5-HT
axons into dorsal hippocampus,
which restores
symmetry
[after
long-term
cingulum
bundle
(LTCB)
injection
of 5,7DHT]. C: degenerating
5-HT axons (dashed line) produced
by midline
microinjection
of 5,7-DHT
into fimbria
(STFI)
of LTCB rats and resultant
asymmetry
produced.
Midline
microinjection
into fimbria
of
normal rats (not shown) produced
no such asymmetry.
[From Azmitia
et al. (41).]
are densely distributed in the infragranular
layer of
DG, in the stratum lacunosum-molecular of the cornu
Ammonis, and in the area fasciola cinerea. The density
of the 5-HT-immunoreactive fibers in the dorsal hippocampus is greatly decreased but maintains a similar
laminar pattern 3 days after lesioning by 5,7-DHT into
the CB. An apparently normal density and distribution
pattern of 5-HT-immunoreactive
fibers is seen by 42
days postlesion. The fasciola cinerea in the hippocampus is among the first regions reinnervated by 5-HT-immunoreactive fibers with very dense and large varicosities. Restitution of 5-HT-immunoreactive
fibers in the
dorsal hippocampus after the 5,7-DHT lesion in the CB
is accompanied by a marked increase in the number and
intensity of 5-HT-immunoreactive fibers in the FF. No
evidence of a regeneration of 5-HT-immunoreactive
fibers is seen distal to the injection site. These observations provide direct evidence for serotonergic collateral
sprouting (SCS) in the CNS induced by removal of 5-HT
fibers.
Collateral sprouting of cholinergic and noradrenergic fibers after partial deafferentation of the hippocampus by lesions has also been demonstrated (137, 176,
184). In general, these studies show a time course of
sprouting similar to the more specific chemical lesions.
The phenomenon of homotypic collateral sprouting is
slower than seen by reactive synaptogenesis (RS) where
the sprouting begins within days and is essentially complete by 2 wk (see Ref. 110). Several other differences
between SCS and RS have been noted (50). One is that
the chemical specificity of the lesioned fiber is critical
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Homotypic collateral sprouting is the ability of an
undamaged neuron to expand its terminal innervation
density in response to removal of fibers containing the
same neurotransmitter.
This plastic response was first
shown in the CNS of adult rats by 5,7-DHT-selective
destruction of the serotonergic hippocampal afferents
traveling in the cingulum bundle (CB) and by monitoring the induced expansion of the undamaged serotonergic fibers traveling in the fornix-fimbria
(FF) (Fig. 7).
Serotonergic fibers were traced by anterograde transport after injections of [3H]proline in the midbrain
raphe nuclei by the radioautographic technique. After a
Z-wk delay, the labeled fibers in the FF began to grow
into the space left by the lesioned CB fibers, and within
42 days the innervation appeared normal. Evidence for a
hyperinnervation after 90 days was noted. These unilateral CB-lesioned animals also showed an asymmetrical
turning response to a 5-HT agonist that returned to
normal after 42 days. To demonstrate that the anatomic
and functional recoveries were due to sprouting fibers
from the FF pathway, the midline injection of 5,7-DHT
into the FF at 4.2 days after a previous unilateral CB
lesion reinstated both the functional and anatomic deficits.
Further evidence in support of this phenomenon involved HRP-retrograde transport studies (531). With
this procedure a significant increase was seen in HRPlabeled neurons in the MRN at 21 and 42 days compared
with 3 days after CB lesion, with the number of cells at
4.2 days actually being supranormal (121% of sham injected). Neurons in the interfascicular subnucleus of the
DRN that normally travel in the CB to innervate the
dorsal hippocampus were not labeled at any time after
the injection of 5,7-DHT in the CB. In addition, there
was no increase in cell labeling for 2 mo after combined
CB-FF 5,7-DHT lesions. These results indicate that the
CB midbrain raphe fibers do not regenerate into the
dorsal hippocampus, but FF fibers can increase their
territory by collateral sprouting in response to homotypic denervation.
The availability of sensitive antibodies against 5HT enables these neurons to be observed directly without the use of transported material (533). The 5-HT
fibers are distributed in a laminar pattern in the hippocampus (Fig. 8). These fibers have large varicosities and
8
Asymmetry
STCB
C Collateral Sprouting
189
SYSTEM
190
BARRY
L. JACOBS
AND
for SCS but not for RS. In RS the proximity
of the fiber
to the site of lesion is critical, whereas in SCS the collateral sprouts can travel long distances (up to a centimeter).
Further
evidence that SCS is a separate process
from RS is their differential
response to adrenal steafferents after a lesion of
roids. In RS of hippocampal
C. AZMITIA
Volume
7.2
the entorhinal
cortex, the removal of circulating
adrenal steroids enhances the appearance of new sprouts
(421), whereas in SCS the adrenal steroids are necessary
for sprouting
(532). The opposite actions of growth modulatory substances are an important
principle for understanding
recovery of function. Furthermore,
the innervation
density of a particular
system should be
viewed as being in a dynamic equilibrium,
and increasing the amount of target area (partial deafferentation)
or of the afferent input (see next section) can change the
level of innervation.
Established
contacts
are fairly
stable to changes in growth regulatory factors (e.g., glucocorticoid).
These factors are effective when the system is in flux. Thus the maintenance
of neuronal connections is resistant
to daily changes in the environment, but when these connections
are severed a new
balance can be easily created.
A final word should be mentioned concerning the
signal for SCS. When 5-HT fibers are damaged, there is
a period of time (2-3 wk) before new collateral sprouts
are formed (50). This appears to be due to the formation
of a soluble trophic factor by the target cells. The hippocampus can be removed at various times after 5,7-DHT
lesions and a soluble extract can be studied in culture.
No significant
enhancement
of trophic activity is seen
until 2 wk postlesion (45b). This is in marked contrast to
the production
of NGF, which increases within a few
days and peaks in the hippocampus
at 2 wk postlesion
(116). Furthermore,
when 5-HT fibers begin to grow
there is evidence that they can overshoot their normal
levels and hyperinnervate
their target neurons.
D. Transplantation
Integration
of fetal cells with adult neurons is probably the most extreme form of brain plasticity.
Therefore this topic is reviewed in some detail because it illustrates several important
anatomic and physiological
aspects of both the immature
and mature serotonergic
system.
I. Introduction
The first transplantation
of serotonergic
neurons
in the brain was made into a cavity in the retrosplenial
cortex of rats (71). Serotonergic
neurons from fetal embryonic pontine raphe region (15 days gestation)
grew
along the lesioned temporoammonic
perforant
path to
innervate the partly denervated
hippocampus.
At l-3
mo, 5-HT fibers were seen to conform to the normal
distribution
of terminals of the perforant
path. The 5,7DHT-lesioned
and nontreated
hosts were reported to
support the same outgrowth
pattern.
Transplantation
of serotonergic
neurons into the
spinal cord was first performed
by Nygren et al. (364).
The fetal neurons (17-19 days gestation; crown-rump
length 19-26 mm) were inserted into the lumbar spinal
cord with a glass pipette. With the use of fluorescence
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FIG. 8. Schematic
drawings
of 5-HT fiber distribution
in coronal
sections
of dorsal hippocampi
of normal
(A), 3 days after 5,7-DHT
lesion
lesion in cingulum
bundle (CB) (B), and 42 days after 5,7-DHT
in CB (C). A: dorsal hippocampus
receives 5-HT innervation
via supracallosal pathway
[medial CB-indesium
griseum
(IG)] and infracallosal
pathway
[fornix-fimbria
(FF)]. These afferent
5-HT fibers mainly terminate
in stratum
lacunosum-moleculare
of cornu Ammonis
(CA),
infragranular
layer of dentate
gyrus (DG), and area fasciola
cinerea
(FC). Less dense 5-HT fibers are distributed
in stratum
oriens and
stratum
radiatum
of CA areas. Density
of 5-HT fibers in CA areas
appears
in sequence of CAl, CA4, and CA3. B: 3 days after 5,7-DHT
lesion in CB, degenerated
fibers are observed
in CB, IG, and CA areas
and in DG. Density
of 5-HT-immunoreactive
fibers are significantly
decreased
in CB, IG, infragranular
layer of dentate,
stratum
lacunosum-moleculare
of CA, and FC. 5-HT-immunoreactive
fibers in FF
appear
normal.
C: 42 days after lesion, 5-HT-immunoreactive
fibers
are restored
to normal
density
and pattern
except for a greater
than
normal
fiber density
in FC. On lesion side, 5-HT fibers regenerate
from degenerated
stumps but do not extend far from lesion site. Reactive growth
of compensatory
5-HT fibers is observed
in FF, which
results in abnormally
high density
of 5-HT-immunoreactive
fibers in
ipsilateral
FF. ALV, alveus; CC, corpus callosum;
Fi, fimbria;
G, granular cell layer; LM, stratum
lacunosum-moleculare;
LV, lateral ventricle; M, stratum
moleculare;
0, stratum
oriens; P, stratum
pyramidalis;
PI, stratum
polymorphic;
Pm, pyramidal
cell layer; R, stratum
radiaturn. [From Zhou and Azmitia
(533).]
EFRAIN
January
STRUCTURE
19%
AND
FUNCTION
histochemistry,
the fate of the donor neurons was studied. Only 25% of the transplants
were successful. Serotonergic neurons survived for up to 3 mo, and fluorescent fibers could be traced 10 mm cranially or caudally
from the cell bodies. The reason for the low survival was
attributed
to the graft being extruded from the spinal
cord because of pressure and bleeding.
2. Descriptive
studies
OF
BRAIN
SEROTONIN
RAPHE
SYSTEM
& LC
TRANSPLANTS
IN
5,7-DHT
HIPPOCAMPUS
and hyperinnervation
N
DHT
DYT
DkjT
R-TP
LC-TP
Ml
N
DHT
DYT
DYT
R-TP
LC-TP
FIG. 9. 5-HT and norepinephrine
(NE) levels in control
hippocampus
(N), hippocampus
with 5,7-DHT
lesions (DHT),
and after
raphe (R-TP)
or locus coeruleus
transplantation
(LC-TP)
in hippocampus with lesion. When hippocampus
(Hipp)
was deprived
of 5-HT
innervation
by 5,‘7-DHT
lesion, growth
of transplanted
fetal 5-HTraphe neurons
is stimulated,
but growth
of transplanted
fetal NE-LC
neurons
was not facilitated
(see drawing).
A: 5-HT levels. R-TP had
greater
increase
in level of 5-HT when placed in hippocampus
with
lesion than when placed in control
hippocampus.
LC-TP did not increase 5-HT level in hippocampus
with 5,7-DHT
lesion. B: NE levels.
There was no difference
in NE levels between
control
hippocampus
and hippocampus
with 5,7-DHT
lesion nor LC-TP or R-TP in hippocampus with 5,7-DHT
lesion. NE levels were not higher
in LC-TP in
5,7-DHT-lesioned
hippocampus
compared
with LC-TP in control hippocampus.
[From
Zhou et al. (528).]
fetal serotonergic
neurons. Fetal raphe neurons (16-18
days gestation)
were transp lanted into the entorhinal
cortex of 6-day-old neonatal rat recipients that had received a fimbria and entorhinal
cortex ablation 3 days
earlier (219). One week after transplant,
some 5-HT-immunoreactive
fibers were seen in the host cortex but
none in the hippocampus.
Between 14-21 days, some 5HT-immunoreactive
fibers were seen in CAZ-CA3 and
some in infragranular
DG. After 3 wk, a hyperinnervation was noted in the infra- and supragranular
layers of
the DG, especially in the hilus area. The general lamination throughout
the hippocampus was not clear, but this
was attributed
to the extensive denervation
by the fornix and entorhinal lesions. The authors noted that their
implants decreased in size between 30 and 60 days.
The 5-HT fetal transplants
(E-14 days gestation;
crown-rump
length 8-12 mm) were placed into adult
hippocampus and examined with immunocytochemistry
after 5,7-DHT lesions. The authors observed that the
number of surviving fetal neurons was stable for -7 wk
and then decreased at 5 mo to 37% of the number of cells
seen at 3 wk. Nevertheless.
at the longest nostonerative
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017
The first microinjections
of fetal serotonergic
neurons were made into the hippocampus
of adult and aged
animals (46). Isogeneic mice (16 days gestation)
were
used, and the transplanted
fetal raphe area was examined with an antibody raised against 5-HT. The 5-HTimmunoreactive
fibers hyperinnervated
both cornu
Ammonis
(stratum
lacunosum-molecular)
and the DG
(both the polymorphic
and the molecular layers). This
ability to supercede the normal levels of innervation
is
unique. If fetal hippocampus was transplanted,
then the
adult host serotonergic
fibers grew into the donor target
tissue. These studies demonstrated
that the innervation
density of the brain by serotonergic
neurons can exceed
normal levels when fetal neurons are supplied. Furthermore, fetal hippocampus
can induce an expansive response from the adult neurons. It can be concluded that
neuronotrophic
interactions
are not governed solely by
the ages of the cells. This is especially noteworthy
in the
aged brain where the endogenous serotonergic
fibers
are degenerating
(40,47).
Unlike most neuronal systems, hyperinnervation
of
target areas by fetal serotonergic
neurons is a well-confirmed event by anatomic and biochemical measures of
5-[3H]HT uptake and by 5-HT and 5-HIAA
levels. If
transplants
were made into unlesioned
brains, then
high-affinity
uptake of 5-[3H]HT increased by 53% over
normal levels, 5-HT levels increased by 50%) and 5-HTimmunoreactive
cells and fiber density of 5-HT-immunoreactive fibers were supranormal
after 1 mo (32). A
prior 5,7-DHT lesion reduced both uptake of 5-[3H]HT
and 5-HT levels to 36 and 30% of normal values, respectively (Fig. 9). If transplants
were made into these animals, then a dramatic increase of high-affinity
uptake
(345% over control and 946% over 5,7-DHT-primed
hippocampus)
and 5-HT levels (225% over control and
850% over 5,7-DHT-primed
hippocampus)
was found.
Similar results were seen 2 wk after intracisternal
injection of 5,7-DHT (472). Levels were 205 and 234%
above normal levels and 3,383 and 1,504% above the 5,7DHT-treated
levels for hippocampal5-HT
and 5-HIAA,
respectively.
There was also evidence for a change in
rotational
behavior
after ipsilateral
transplantation,
which was significantly
correlated with the asymmetry
in 5-HT levels. These results demonstrate
that transplanted fetal neurons can replace adult hippocampal5HT in a motor response. Correlation
between hippocamI pal 5-HT levels and locomotor activity has been previously demonstrated
in normal and lesioned rats (515).
Other workers
have noted a hvnerinnervation
bv
192
BARRY
L. JACOBS
AND
3. Role of target sites
With the use of a transplantation
procedure with
cell suspensions,
fetal serotonergic
neurons (14 days
gestation)
from the forebrain
projecting
raphe nuclei
were transplanted
into the spinal cord (389,390), which
normally
is innervated
by the descending medullary
raphe nuclei. The spinal cord was previously transected
at the lower thoracic level, and after laminectomy
a lmm thick segment was excised. After 1 wk, the raphe
cell suspension was injected into the distal cord. Within
10 days, 5-HT-immunoreactive
fibers were seen 15 mm
from the transplant
site, and by 60 days, fibers were
seen 20 mm from the site. The fibers appeared to travel
near the central canal and heavily innervated
the nucleus intermediolateralis.
Recent work suggests that 5HT transplants
in damaged spinal cord preparations
in
primates may result in the return of some of the lost
function (A. Privat, unpublished
observations).
It is interesting
to note that the fetal serotonergic
cells can
innervate
and restore functions
lost by transection
of
the spinal cord, even if those 5-HT cells would normally
have projected to the forebrain.
Similar evidence for
neuronotrophic
interactions
between the rostra1 raphe
nuclei and the spinal cord was provided by coculturing
methods (48). The developmental
maturation
of the superior group of serotonergic
neurons was stimulated by
the presence of spinal cord neurons when grown together for 3 days in culture.
An extensive series of papers dealt with 5-HT-, subneurons transstance P-, and TRH-immunoreactive
planted from medullary or mesencephalic (rostra1 brain
C. AZMITIA
Volume
7.2
stem) raphe nuclei into the spinal cord, hippocampus,
or
corpus striatum
(160-163). Adult rats were pretreated
with 5,7-DHT, and fetal raphe cells (13-14 days gestation) injected into target sites. In the spinal cord, mesencephalic cells grew out ~15 mm after 2-12 mo and did
not innervate
motoneurons.
Some small substance
P
cells with intratransplant
fibers were seen. No TRH
fibers were seen. In the hippocampus,
there was a substantial outgrowth
of 5-HT-immunoreactive
fibers but
no detectable substance P or TRH. Finally, in the striaturn, the mesencephalic
5-HT-immunoreactive
neurons
grew better than in the spinal cord. Colocalization
of
5-HT/substance
P was found in this area for the first
time. Very few fibers for substance P were seen in the
graft, and no TRH fibers were seen. The situation with
medullary transplants
was different. In the spinal cord,
the 5-HT-immunoreactive
neurons innervated motoneurons. Colocalizations
of 5-HT with substance P and TRH
were seen. The 5-HT-immunoreactive
fibers extended
up to 15 mm. Some outgrowth
of substance P and TRH
fibers was seen. In hippocampus,
the medullary 5-HTimmunoreactive
neurons behaved like the mesencephalic raphe group where no substance P or TRH fibers
were seen outside the graft. Finally, in the striatum,
5-HT, substance P, and TRH fibers were restricted
to
the graft.
This series of studies emphasizes the importance of
considering
not only the source and age of the donor
fetal neurons but also trying to match the fetal cell with
its proper target area to achieve the most satisfactory
result. Neuronotrophic
interactions
can be very specific
but not always logical. For instance, medullary
5-HT
neurons normally
innervate
spinal cord, but if transplanted into hippocampus,
they grow out, whereas
if
transplanted
into striatum,
they will not grow out. The
role of neuropeptides
as neuronal growth
regulating
factors is discussed in section IVE.
4. Host trophic factor
Partial removal of 5-HT fibers from the adult hippocampus by injections of 5,7-DHT can induce homotypie collateral sprouting
of the undamaged 5-HT fiber
(41, 50, 533). These results indicate the presence of an
endogenous trophic factor for 5-HT fibers that can induce adult 5-HT sprouting.
Can fetal 5-HT neurons be
similarly
affected?
This question of a lesion-induced
neuronotrophic
response was studied in greater detail. Transplants
of
raphe neurons into an intact adult hippocampus showed
increases in high-affinity
uptake of 5-[“H]HT, 5-HT levels, and 5-HT-immunoreactive
fiber density above normal but not as high as that seen if the transplants
were
made into brains previously injected with 5,7-DHT (32,
528). Although
the extent of fiber outgrowth
of the 5HT-immunoreactive
neurons was increased, the number of surviving
5-HT-immunoreactive
cells was not
changed significantly.
The neuronotrophic
effect of the
5,7-DHT lesion was attributed
to a soluble factor since
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times (7 wk and 5 mo), some of the animals exhibited
extensive
hyperinnervation
patterns
throughout
the
dorsal part of Ammon’s horn and the DG.
In ultrastructural
studies of raphe transplants,
15% of 5-HT-immunoreactive
fibers in the hippocampus formed asymmetrical
contacts in normal rats. This
percentage
was not changed after fetal raphe transplants, even though the number of fibers was greatly
increased (22). These results concerning hyperinnervation of denervated adult tissue by transplanted
fetal 5HT neurons illustrate
the robust trophic and dynamic
interactions
of serotonergic
neurons and their targets.
The ability of sprouts to reach the proper target sites
(and even make appropriate
synaptic contacts)
in the
absence of the developmental
guidance cues is remarkable. What trophic factors are these neurons seeking?
There are many trophic factors that stimulate 5-HT neurons. It is interesting
that the trophic signals must be
present in great supply despite the age of the animal
(see sect. W4).
How can a fetal neuron substitute
for the
adult neuron equipped with its normal complement
of
afferents obtained from the brain stem raphe? The ability of transplanted
fetal 5-HT neurons to restore normal
behavior lost by deafferentation
of the adult 5-HT neuron emphasizes the basic information
conveyed by these
neurons.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
5. Electrophysiological
maturation
The development
of mature electrophysiological
properties
by transplanted
serotonergic
cells was studied in a hippocampal
slice preparation
(430, 431). The
serotonergic
neurons from 15- to 19-day-gestation
rat
embryos showed few of the adult characteristics.
After
transplantation,
5-HT neurons develop all the normal
characteristics
associated with the raphe neurons that
BRAIN
SEROTONIN
SYSTEM
193
develop in situ. In this work, it was also demonstrated
that electrical stimulation
of the transplant
caused a
marked hyperpolarization
of hippocampal
pyramidal
neurons. This established
the existence of a connection
between host and donor tissue and verified that the electrophysiological
characteristics
are similar
to those
seen for the normal raphe-hippocampal
serotonergic
projection. Again, evidence is obtained that an ectopitally situated fetal serotonergic
neuron can substitute
functionally
for adult brain stem raphe neurons.
Electrophysiological
studies have also been carried
out on a cotransplant
of fetal raphe and spinal tissue
into the anterior chamber of the eye (210). Dorsal raphe
neurons from the midbrain
formed cellular plexuses
around large spinal cord neurons. Firing rates of both
5-HT and spinal cord neurons were obtained. In the spinal cord, glutamate triggered cell firing and 5-HT (3 x
10e4 M) accelerated the rate. Metergoline, a 5-HT receptor antagonist, blocked the response to exogenous 5-HT.
In certain cotransplants,
5-HT terminals
hyperinnervated the spinal cord and slowed spontaneous cell firing.
If the dorsal raphe grafts were stimulated,
the spinal
cord neurons’ firing rates were increased over the elevated glutamate-induced
rate. Thus a mini-brain
circuit
can be established in the eye, and the physiology of 5-HT
on motoneurons
can be studied.
6. Functional
recovery
In the most complete study of functional recovery
by transplanted fetal serotonergic neurons, it was first
shown that neurotoxin-induced destruction of serotonergic terminals in the medial hypothalamus facilitated
lordosis, decreased 5-HT-immunoreactive fiber density,
and reduced 5-HT and 5-HIAA levels (314). Transplantation of serotonergic neurons then restored 5-HT and
5-HIAA levels 50 days later and completely restored the
change in lordosis to control levels or below. The 5-HTimmunoreactive fibers were also seen at normal or hyperinnervated levels in the medial hypothalamus. These
studies demonstrate that not only motor behavior but
complex hormonal-dependent sex-linked behaviors require the presence of 5-HT, whether derived from normal midbrain raphe neurons or from transplanted fetal
neurons.
Several other complex behaviors can function when
fetal 5-HT neurons are substituted for those of the
adult. Transplantation
of fetal serotonergic neurons
into the fourth ventricle may correct sleep disturbances
(181, 189, 329). The 5,7-DHT lesions in neonates significantly decrease paradoxical sleep in adult rats. Three
months after intracisternal 5,7-DHT, 16-day-gestation
raphe tissue was transplanted into the fourth ventricle.
Paradoxical sleep in these animals was normal. A few
5-HT-immunoreactive
cells and fibers were seen near
the transplant site. In another study, 5,7-DHT microinjection into the FF resulted in a facilitation of complex
maze learning by reducing the mean number of errors
and trials to criterion in rats (17). The transplantation
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an extract from a lesioned-primed
hippocampus
could
stimulate
the development
of transplanted
5-HT neurons when transplanted
to an intact hippocampus
(528).
Hyperinnervation
of the transplanted
neurons did not
appear to displace the normal afferent inputs to the hippocampus from the midbrain raphe neurons (529). Thus
fetal and adult afferents can coexist in an adult target
area and establish a much higher degree of innervation.
It is important
to note that the enhanced development
of fetal serotonergic
neurons after lesions with 5,7-DHT
did not enhance the development of fetal locus coeruleus
neurons (528). In fact, the noradrenergic
neurons grew
more poorly into the 5-HT-denervated
than into the
normal hippocampus
(Fig. 9). This provides evidence
that the damaged adult brain can produce specific neuronotrophic
or neuronotoxic
factors that differentiate
between 5-HT- and norepinephrine-producing
neurons.
One of the problems with fetal axonal growth into
adult tissue may be the lack of a suitable substrate
for
the axons to attach to when growing.
To test whether
laminin has a similar role in the adult brain, sequentially injected laminin and transplanted
fetal neurons
were made into various brain regions to determine if the
fetal neurons would preferentially
grow along a laminin
injection tract. Serotonergic
neurons were transplanted
to the motor cortex, neostriatum,
or hippocampus
of
adult animals (534). The tract used for microinjection
of
cell suspension was then immediately filled with suspension medium or laminin.
In other animals, laminin
or control solution was injected in a separate needle
tract displaced 0.3-l mm from the transplant
injection
tract. Straight
and thick 5-HT-immunoreactive
fibers
(stained with anti-5-HT
antiserum)
were predominant
within the laminin-treated
tracts or were directed toward the laminin-treated
parallel tracts when it was
positioned ~0.5 mm from the transplant
site. The density of 5-HT-immunoreactive
fibers in the injection
tracts in all three brain areas was much higher for
laminin than that seen with control medium. Thin axonal fibers of fetal 5-HT
neurons
were observed
surrounding
the laminin-treated
tracts but not in vehicle-treated tracts. Finally, laminin injection to the hippocampus, motor cortex, or neostriatum
of the adult
brain did not stimulate sprouting
of undamaged adult
5-HT fibers. These results suggest that purified laminin
can facilitate and guide process outgrowth
of 5-HT neurons during an early developmental
stage but does not
induce sprouting on these same fiber types in the adult
brain.
OF
194
BARRY
L. JACOBS
AND
VI.
C. AZMITIA
CELLULAR
Volume
72
NEUROPHYSIOLOGY
A. Unit Activity
in Anesthetized
Animals
or Brain Slices
I. Introduction
Neurophysiological
experiments carried out in anesthetized animals or in tissue slices maintained in vitro
are of limited value in elucidating questions regarding
organismic
physiology
or behavior.
Nonetheless,
the
simplicity
and/or stability
afforded by these preparations have proven invaluable for examining the synaptic
physiology
and membrane properties
of neurons. Several recent papers have been devoted exclusively to this
aspect of serotonergic
neurons (5,10, g&115,429,
516).
E. Summary
2. Intrinsic
activity
Serotonergic
neurons, because they have extensive
projections
and can regulate a wide range of biological
functions,
are an excellent neuronal system to use for
the study of adult plasticity.
Undamaged
serotonergic
neurons will respond to the loss of neighboring
5-HT
fibers by collateral
sprouting
(41). This demonstrates
that the adult serotonergic
system has not reached its
growth potential but is fact limited by the environment
of the target structure.
What does determine the innervation density? This particular
question has intrigued
investigators
for many years, and transplantation
studies have provided some clues.
Serotonergic
neurons are a good source of cells for
transplantation
work. The cells can be easily dissected,
accurately
measured
by biochemical
methods, sensitively stained by immunocytochemistry,
specifically destroyed by neurotoxins,
and reliably transplanted
into
cerebral tissue. A major conclusion from this work is
that the age of the host or donor tissue does not provide
a complete barrier to communication
between serotonergic neurons and billions of its target neurons. For example, they can quickly contact the hippocampal
pyramidal cells and spinal cord motoneurons
and establish
electrical and chemical connections.
This colonization
can regulate complex functions such as sexual behavior
and cognitive processing. These transplantation
studies
indicate that the innervation
density of 5-HT in the
adult brain can be experimentally
manipulated
by increasing either the number of 5-HT neurons or by enlarging the target area. The lack of a suitable substrate
for fiber attachment
in the adult brain can hinder the
rate of fetal neurite growth (534). Recent evidence indicates that astrocytes
that produce a number of serotonergic trophic molecules, such as S-100, undergo aging
and may lose the receptors necessary for proper trophic
responses (504,506). Interestingly,
the 5,7-DHT lesion of
the adult hippocampus
appears to reactivate
the glial
cells to once again produce S-loop in response to a 5-HT
signal (45a).
In the late 196Os,Aghajanian et al. (6,7) descri.bed a
distinctive type of neuronal activity recorded in the area
of the DRN or MRN of rats anesthetized with chloral
hydrate. They consistently recorded cells with a slow
(1-Z spikes/s) and highly regular discharge pattern
(Fig. lo), whose activity was completely suppressed by
systemic administration of LSD. On the basis of these
initial results, they hypothesized that these slow and
regular firing cells were serotonergic, since this type of
activity was found only in brain areas known to contain
serotonergic cells and since only this type of neuron
showed a consistent depressant response to low dosesof
LSD (an effect predicted on the basis of previous neurochemical studies examining the effects of LSD on brain
5-HT). The assumption that these were serotonergic
neurons has now been supported by a large body of experimental evidence (for review see Ref. 238).
The characteristic slow and regular discharge of
serotonergic neurons led to the hypothesis that their
activity might be endogenous, driven by an intrinsic
pacemaker mechanism. In the first attempt to explore
this issue, rats were prepared with a complete transection of the neuraxis, immediately rostra1 to the DRN,
thus isolating it from the influence of the entire forebrain (347). The activity of DRN neurons in these brain
stem-transected animals was virtually
indistinguishable from that found in intact animals. Although their
discharge rate was ~30% higher than that of a corresponding group of neurons in intact animals (possibly
representing removal of a tonic forebrain inhibitory influence), these cells still displayed the slow rhythmic
discharge pattern that characterizes serotonergic neurons. In an attempt to explore this issue more directly,
the activity of serotonergic neurons in the rat DRN was
examined in vitro (346). By cutting a 400-pm thick section through the rat brain stem, the serotonergic neurons in this tissue slice were isolated from neurons in
the remaining forebrain and brain stem. In vitro, most
DRN neurons displayed discharge patterns indistin-
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017
of these animals with fetal serotonergic
neurons reversed this facilitation
(395). Finally, it has recently
been shown that cognitive functions associated with the
cortex and hippocampus
are dependent on both the serotonergic and cholinergic
systems (358, 483). Rats injected with 5,7-DHT or administered
atropine (a cholinergic antagonist)
show only slight dysfunction
in spatial
memory, but when both treatments
are given there is a
severe loss of spatial memory (432). If these 5,7-DHT-lesioned animals receive a serotonergic
fetal neuronal
transplant
into the dorsal hippocampus,
then the atropine injection is no longer effective at disrupting
memory. Thus in cortically
based cognitive behaviors, fetal
serotonergic
neurons provide the input for normal responses. The only requirement
is a trophic one: the
growth of the fetal 5-HT neurons into the proper target
zone. Once the 5-HT fibers are in place, the brain appears to function normally once again.
EFRAIN
January 1992
STRUCTURE
AND
FUNCTION
OF
BRAIN
SEROTONIN
SYSTEM
195
pacemaker cells studied in both vertebrate and invertebrate preparations (310).
3. Development
from
typical
rat. Each
guishable from those observed in vivo. They uniformly
fired with a regular pattern; some fired at a rate substantially higher than that observed in vivo, but over
60% of the cells had a mean discharge rate between 0.8
and 2.5 spikes/s (Fig. 11).
The most direct and compelling evidence for the intrinsic activity of brain serotonergic neurons came in a
study employing intracellular recordings in vivo. This
analysis revealed that these cells display a large afterhyperpolarization (AHP) followed by a gradual depolarization, without evidence of excitatory postsynaptic potentials (EPSPs), eventuating in the succeeding spike
(lla). This same pattern was also found in subsequent
intracellular analyses of serotonergic unit activity recorded in DRN tissue slices (115, 481). These neurons
also had very high membrane input resistances and very
long time constants (both -3-4 times that of neighboring neurons). Further studies in tissue slices led to the
following proposed sequence of membrane events generating this slow rhythmic activity (5, 92, 429). During
an action potential, calcium enters serotonergic neurons
through a high-threshold calcium channel. This is followed by a large (15-20 mV) AHP generated by a calcium-activated potassium conductance. This AHP results in a long relative refractory period, thus preventing discharges in bursts and insuring slow rates of
firing. As the AHP decreases (due to sequestration and/
or extrusion of calcium), it deinactivates a low-threshold calcium current and an early transient outward potassium current (IA). The currents generated by the activation of these two voltage-dependent channels are
opposed, with IA tending to slow the rate of depolarization and the low-threshold calcium conductance increasing it. Under normal conditions, the calcium conductance is stronger, thus leading to a shallow ramp
which ultimately
reaches threshold,
depolarization,
fires, and, as the calcium enters the cell, reinitiates the
sequence of events. The slope of this ramp is what determines the rate of discharge of serotonergic neurons.
This sequence of events is similar to that seen for other
FIG. 11. Spontaneous
single-unit
activity
of rat DRN neurons
recorded
in vitro. Single oscilloscope
sweep traces of 3 different
units
are shown at left (bar, 1 s). Interspike
interval
histograms
corresponding to same 3 units are shown at right (bars, 1 s and 25 spikes). [From
Mosko and Jacobs (346).]
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017
FIG. 10. Oscilloscope
trace of single-unit
recording
serotonergic
neuron in DRN of chloral hydrate-anesthetized
trace is 10 s long. [From Jacobs et al. (238a).]
Developmental analyses of the single-unit activity
of DRN serotonergic neurons indicate that they take on
adult characteristics very early postnatally or even prenatally. Embryonic rat brain serotonergic neurons have
been studied in slices (431). No activity was observed at
embryonic days 14-16, but the characteristic activity of
brain serotonergic neurons was seen on embryonic days
17-18 (i.e., 3-4 days prenatal). The slow and regular discharge that characterizes adult DRN serotonergic neurons was also seen in anesthetized 3-day-old rat pups
(278,177). Additionally, these cells displayed a suppression of activity in response to a 5-HT autoreceptor agonist drug, a response characteristic of DRN serotonergic
neurons in adults. Finally, as early as the first postnatal
week in kittens, DRN cells displayed the slow and regular activity and pharmacological response characteristic of adults. These cells also showed the adultlike pattern of activity across the sleep-wake cycle (2). Thus this
group of neurons is among the first in the CNS to take
on adultlike activity both spontaneously as well as dur-
196
BARRY
L. JACOBS
AND
ing a physiologically
driven change. This is consistent
with the aforementioned
precocial morphological
development of neurons in the superior group of serotonergic
cells.
4. Feedback regulation
C. AZMITIA
Volume
72
gic neurons in the same, as well as other, raphe nuclei, it
was proposed that these cells are under local, rather
than long-loop, feedback control (343). Third, Aghajanian has further
refined and elaborated this latter concept by providing physiological
evidence that serotonergic neurons are under the influence of axon collateral
inhibition (493) and anatomic evidence that neighboring
serotonergic
neurons make dendrodendritic
and dendrosomatic
connections (4; see sect. IIIF).
Apparently,
this feedback exerts a tonic influence
since administration
of 5-HT autoreceptor
antagonist
drugs increases the firing rate, and regularizes
the discharge pattern, of serotonergic
neurons in behaving animals (159; Fig. 12). Interestingly,
when this same type
of experiment
was conducted in anesthetized
animals,
no increase in discharge rate was seen (74, 315). Additionally, as would be predicted on theoretical
grounds,
the feedback becomes increasingly
manifested
as the
basic neuronal activity increases. Thus under conditions
where serotonergic
neurons are known to be relatively
inactive, for example, during sleep, autoreceptor
blockade has little, if any, effect on neuronal activity. However, under conditions where serotonergic
neurons are
activated and 5-HT release would be expected to exert
feedback inhibition, for example, during arousal, autoreceptor blockade is manifested as a significantly
larger
increase in the activity of serotonergic
neurons (159).
These results provide evidence for the physiological
significance of this feedback inhibition.
Finally, there is
indirect evidence that the density of cell body autoreceptors on individual neurons may be a factor in determining both the level of spontaneous
activity of serotonergic neurons as well as the degree to which they
respond to 5-HT agonist drugs (212,237). Thus neurons
with few autoreceptors
would be faster firing, due to
less feedback inhibition, and less responsive to 5-HT agonist drugs, due to fewer sites for drug-receptor
interactions.
Neighboring
serotonergic
neurons in the DRN (two
cells were recorded simultaneously
with the same electrode) display complex patterns
of interactions
(496).
Baseline
8-OH-DPAT
(5pg/kg,i.v.)
Spiperone
(1 mg/kg,i.v.)
8-OH-DPAT
(5yg/kg,i.v.)
FIG. 12. Polygraph
traces displaying single-unit
activity
of serotonergic
neuron
in DRN of cat, following
systemic administration
of 5-HT,,
agonist
drug [Ghydroxy-2-(di-n-propylamino)tetralin
@-OH-DPAT)]
and a 5-HT,, antagonist
drug (spiperone).
Note rapid
and potent effect of agonist.
Note also
that antagonist
not only blocks effect of
agonist
(bottom
trace),
but it significantly increases baseline level of neuronal activity
(middle
trace).
III I I
+
~
4
,,,, ,,,,, ,,,,. .. .,,. .. . ... .. . .. . .... .... .... .. . .. . . .. . .. ... ... . . ....
.
10 set
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017
A major variable that controls the activity of brain
serotonergic
neurons
is negative neuronal feedback.
Thus as the level of brain 5-HT increases, for example,
following
administration
of its precursors,
L-tryptophan or L-5hydroxytryptophan,
the activity of brain
serotonergic
neurons correspondingly
decreases (3,467).
It is presumed that this neuronal decrease is a homeostatic response that acts to compensate for increases in
synaptic levels of 5-HT. The activity of serotonergic
neurons is also strongly inhibited by 5-HT autoreceptor
agonist drugs such as 8-OH-DPAT
(Fig. 12).
During the past several years, a good deal of evidence has been accumulated to indicate that the nature
of this feedback is a short loop, or local, rather than a
long loop, involving the response of postsynaptic
target
neurons. Increased
levels of synaptic 5-HT appear to
inhibit serotonergic
neurons by a direct action at somatodendritic
5-HT receptors (the 5-HT,* autoreceptors).
The following
evidence indicates that this is a result of
5-HT being released from axons or dendrites of neighboring serotonergic
neurons or via axon collateral feedback. First, studies employing iontophoresis
show that
serotonergic
neurons are directly responsive
to the application of 5-HT. For example, the ejection of 5-HT
onto serotonergic
neurons in the DRN strongly
depresses their rate of firing (8). Second, drugs that release the endogenous stores of 5-HT, or block its reuptake, depress serotonergic
unit activity in the DRN independent of any feedback from the forebrain,
since the
effectiveness
of these compounds was undiminished
in
animals whose neuraxes were transected
immediately
rostra1 to the nucleus (347). When these neurophysiological findings were coupled with anatomic evidence that
serotonergic
neurons make connections with serotoner-
EFRAIN
January
19%
STRUCTURE
AND
FUNCTION
5. Aflerent
control
Physiological
analysis of the control of serotonergic
neurons by afferent inputs follows from anatomic studies. There is evidence for afferents from GABAergic
interneurons
in the area of the DRN (64,192), from catecholaminergic
neurons (52,105,173,252),
from neurons
utilizing excitatory
amino acids as their neurotransmitter (250), and from histaminergic
neurons (462). A large
number of other afferent inputs to raphe nuclei have
been demonstrated
through the use of retrograde
tracing techniques and were discussed in section IIIC. These
additional inputs are not discussed here because their
lack of neurochemical
identity precludes their manipulation by pharmacological
means. Furthermore,
in many
cases, neurochemically
identified afferents have simply
not been studied electrophysiologically.
The studies on
afferent control have focused primarily
on the issue of
whether the input increases or decreases the activity of
serotonergic
neurons and the strength and/or temporal
characteristics
of the effects. In some cases, the ionic
bases of these effects have begun to be investigated;
however, this is not described in detail here because it is not
of primary importance
to our central themes (see Refs.
10, 516).
Stimulation
of the pontine reticular formation has
been found to produce a short-latency
suppression
of
serotonergic
unit activity in the DRN (497). The effects
were blocked by systemic administration
of the GABA
antagonist picrotoxin but not by the glycine antagonist
strychnine.
There was some specificity
to this action,
since stimulation
of the neighboring locus coeruleus did
not produce these suppressive
effects (20,497). The lateral habenula constitutes
the densest input to the DRN,
thus providing a pathway
for extrapyramidal
and limbit systems to exert control over the brain stem serotonergic system. This led to the examination
of the effects
of electrical stimulation
of this region in anesthetized
rats (455, 495). Once again a strong suppressive
effect
(latency of 15 ms and duration of 50-400 ms) on serotonergic neurons was seen. This effect was also blocked by
picrotoxin
but not by strychnine
(495). On the basis of
these data, the authors hypothesized
that the habenu-
BRAIN
SEROTONIN
SYSTEM
197
loraphe pathway was GABAergic
in nature. Other evidence, however, indicates that the GABAergic
cells involved in this response are not long-axoned neurons located in the habenula but are small interneurons
found
in the area of the DRN (64, 192). Habenula lesions increase choline acetyltransferase
activity in the DRN,
thus suggesting that the habenuloraphe
pathway
must
be cholinergic (180). Intracellular
recording of DRN neurons in anesthetized
rats indicates that stimulation
of
the habenula produces inhibitory
postsynaptic
potentials mediated by an increased chloride conductance
(375). Somewhat complicating
the issue, these latter results also indicate that these effects are both mono- and
polysynaptically
mediated. It has been proposed, on the
basis of anatomic and neurochemical
studies, that the
lateral habenula also exerts a direct excitatory
influence on the DRN, which is mediated by an excitatory
amino acid (250, 254). These results regarding
GABA
and excitatory
amino acids are consistent
with recent
intracellular
analyses of rat DRN neurons studied in
vitro. Electrical stimulation
just below the cerebral aqueduct evoked a short-latency
(0.5-5.0 ms) and longduration (20-200 ms) GABA-mediated
inhibition and excitatory amino acid-mediated
excitation (370).
The issue of neurochemical
identity of afferent inputs to serotonergic
neurons in the DRN has also been
addressed by means of studies employing microiontophoresis. Application
of either GABA or glycine suppressed the activity of serotonergic
neurons, and these
effects were blocked by their respective antagonists,
picrotoxin and strychnine
(179). More recently, iontophoretie application of histamine has been found to exert a
depressant
effect on serotonergic
unit activity in the
DRN (275). Iontophoretic
application of norepinephrine
had no effect on serotonergic
unit activity (178, 457);
however, the application of noradrenergic
antagonists
strongly
suppressed
the activity of these neurons, an
effect that could be reversed by the iontophoretic
application of norepinephrine
(51). These data imply that noradrenergic
neurons provide a tonic excitatory
input to
serotonergic
neurons in the DRN. [Voltage-clamp
studies indicate that this is mediated by a suppression
of two
potassium
currents,
a resting conductance and a voltage-dependent
transient
conductance (5, lo).] Because
systemic or iontophoretic
application of a variety of cyadrenoceptor
antagonist
drugs (also reserpine and low
doses of clonidine) can completely suppress the activity
of serotonergic
neurons in the DRN of chloral hydrateanesthetized
rats, it was proposed that the activity of
serotonergic
neurons
is dependent
on a continued
adrenergic input (51,53,178,457).
Similar experiments
have also been conducted in freely moving cats (214).
When the change in behavioral state produced by systemic administration
of adrenergic
drugs was taken
into account, however, little or no net change in activity
of serotonergic
neurons was seen. On the basis of these
latter results, it appears that an excitatory
adrenergic
input to the DRN influences the activity of serotonergic
neurons under physiological
conditions, but their con-
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They exhibit synchronization
of activity, inhibition,
or
mutual inhibition of each other. These data are consistent with the aforementioned
concept that serotonergic
neurons influence the activity of other serotonergic
neurons. The fact that neighboring
neurons rarely fire simultaneously
or on a one-to-one basis (496) indicates
that they are not electrotonically
coupled (there is also
no ultrastructural
evidence to support this type of interaction).
The release of 5-HT is also believed to be modulated
by local feedback inhibition operating at the axon terminal (for review see Refs. 100,449). However, there is no
evidence to indicate that this action at an axon terminal
autoreceptor
(5-HT&
(145,224) is not related to neuronal activity or release in a linear manner.
OF
198
BARRY
L. JACOBS
AND
6. Environmental/physiological
manipulations
Serotonergic unit activity in anesthetized animals
(primarily rats) has been studied in conjunction with
the manipulation of several different physiological systems (e.g., body temperature and blood pressure), several pituitary-adrenal
hormones (e.g., ACTH and corticosterone), and with stimuli from several sensory modalities (e.g., light flash and electrical shocks to peripheral
nerves). In general, the studies have revealed that these
manipulations produce either no effect on serotonergic
unit activity or the results of several studies on the same
issue have been contradictory.
Experiments utilizing a variety of physiological
and pharmacological approaches have implicated brain
5-HT in the regulation of body temperature (351). Therefore it is not surprising that a number of studies have
explored the relationship between elevation of body temperature and raphe unit activity. The first experiment
to do so reported that the single-unit activity of midbrain raphe neurons in chloral hydrate-anesthetized
rats was significantly increased in response to elevated
body temperature induced by radiant heat (500). However, a subsequent study, utilizing the identical procedure, failed to find any evidence for such a relationship,
despite elevations of body temperature as great as 4OC
(468). This latter result is consistent with another study
that failed to find an increase in DRN unit activity desnite a 12°C increase in bode temperature (88). Addi-
C. AZMITIA
Volume
72
tional studies on this issue have been inconsistent.
Heat-sensitive neurons were found in the MRN; however, no attempt was made to identify these neurons as
serotonergic (239). Indeed, in all likelihood, they were
nonserotonergic cells since they fired at high rates (~6
spikes/s) and were not regular in their discharge pattern. Another study in urethan-anesthetized
rats reported that slow and regular firing neurons in the DRN,
MRN, NRM, and nucleus raphe pontis were unaffected
by changes in body temperature (134). However, some
cells, especially in the NRM were found to be responsive
to increases or decreases in skin temperature independent of changes in core temperature. Finally, slow and
regular firing midbrain raphe neurons in chloral hydrate-anesthetized cats were reported to increase their
activity in response to locally induced elevation of brain
temperature (112).
Although not examined extensively, two studies
have reported a lack of relationship between DRN unit
activity and variations in blood pressure in anesthetized
rats. Intravenous administration of l-norepinephrine
had no effect on unit activity, despite an increase in
blood pressure (154). The other study reported that no
relationship existed between spontaneous discharge
rate of DRN neurons and basal fluctuations in blood
pressure (88).
A large literature exists relating 5-HT to pituitaryadrenal function. Two studies have investigated the effect of pituitary-adrenal
hormones on the unit activity
of serotonergic neurons in the mesencephalon of chloral
hydrate-anesthetized rats. Intravenous administration
of hydrocortisone was reported to produce a decrease in
the activity of these neurons (153). In an attempt to follow up these results, a similar study reported no effect
on serotonergic unit activity of various intravenous
doses of corticosterone, hydrocortisone, and ACTH
(345). No ready explanation is available to explain these
differences.
Finally, the activity of midbrain raphe neurons in
anesthetized rats has been examined in response to sensory afferent input. Repetitive presentation of light
flashes had no effect on neuronal activity (344), whereas
direct stimulation of a peripheral nerve did elicit activation of DRN serotonergic neurons (14).
7. Summary
Most salient is the fact that serotonergic neurons
have endogenous biological mechanisms for generating
their slow and highly rhythmic activity. This characteristic pattern of neuronal activity is seen not only in the
adult but is manifested early in development. This basic
activity is then available for modification by the variety
of afferent inputs received by serotonergic neurons.
B. Unit Activity
in Behaving Animals
I. Introduction
Because of the general lack of response of serotonergic neurons in anesthetized animals to a varietv of
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017
tinued activity does not seem to be dependent on this
tonic input.
To summarize,
5-HT, GABA, and histamine
exert
depressive
effects on serotonergic
neurons. Evidence
also indicates that glycine may be an inhibitory
neurotransmitter
on these neurons. Norepinephrine
is the
transmitter
best known to exert an excitatory
influence
on serotonergic
neurons; however, a glutaminergic
input has been described, and this is also likely to exert an
excitatory
effect. The noradrenergic
input seems to be a
tonic one operating at a near maximal level, since the
iontophoretic
application of norepinephrine
on spontaneously active cells has little further effect. These data
are of obvious relevance to pharmacological
studies,
since it can reasonably be assumed that drugs known to
affect serotonergic,
GABAergic,
histaminergic,
glutaminergic,
and noradrenergic
neurotransmission
will
also engage or disengage the activity of brain serotonergic neurons.
These results set the stage for a set of related questions, for example, when is the powerful influence of the
habenula exerted, under what conditions
is the GABAergic inhibitory
influence exerted or lifted, and when
is the excitatory
noradrenergic
input engaged or disengaged? As described in section VI& we know that serotonergic neurons in behaving animals can be inhibited
(e.g., during sleep) or excited (e.g., in response to phasic
sensory input), but studies are just beginning to explore
the CNS sites from which these influences originate and
the neurochemical
systems that mediate them.
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
2. Neurochemical
identi$cation
Because single-unit
studies of serotonergic
neurons
in behaving animals examine extracellularly
recorded
action potentials, the identification
of neurons as “serotonergic”
is necessarily
indirect. The criteria used in
this identification
are derived largely from studies
carried out in anesthetized
rats. As discussed in section
VIA& a population of neurons found within the midbrain raphe nuclei consistently
displayed the following
characteristics:
discharging
in a slow (-1-Z
spikes/s)
and highly regular manner, having biphasic action potentials of long duration (>2 ms), and having activity
that is selectively
and completely
suppressed
by low
doses of serotonergic
agonist drugs (6,10,127). Neurons
displaying similar characteristics
have been observed in
all the brain areas where serotonergic
neurons are localized and are not found in regions devoid of serotonergic
neurons. These initial results were strengthened
by subsequent studies providing direct evidence for the serotonergic identity of these neurons. With the use of a
double-labeling
technique,
experiments
were c.arried
out in which the slow and regular firing DRN cells were
injected intracellularly
with fluorescent dye, then localized, and subsequently
examined for the presence of 5HT by histochemical
fluorescence
(11). All cells that
displayed this characteristic
activity were found to contain 5-HT. Additional indirect evidence in support of the
serotonergic
identity
of these neurons has been reviewed in detail (238).
Studies in behaving animals capitalize on this foregoing body of evidence. Serotonergic
neurons are initially identified on-line by their slow and regular activity and their characteristic
action potential waveform
BRAIN
9
SEROTONIN
199
SYSTEM
C
200
>
=I
0
v)
1TE
B
200
0.5
400
600
set
INTER-SPIKE
INTERVAL
(met)
FIG. 13. A: oscilloscope
trace of extracellular
action potential
recorded
from serotonergic
NRM neuron
recorded
in behaving
cat
(positive
is down). B: oscilloscope
trace of slow and regular
discharge
of serotonergic
NRM neuron.
C: interspike
interval
histogram
generated from activity
of same serotonergic
NRM
neuron
during
quiet
waking.
Histogram
was constructed
from 1,024 intervals
using bin
width of 10 ms. [From Fornal
et al. (155).]
and duration (Fig. 13). Further confirmation
of the serotonergic identity of these neurons derives from evidence that neuronal activity is completely suppressed
by systemic
administration
of 5-HT somatodendritic
autoreceptor
agonists, such as 5-methoxy-N,N-dimethyltryptamine
or GOH-DPAT.
After completion of an
experiment,
the localization
of presumed serotonergic
neurons
is examined
histologically
to determine
whether they were in areas known to contain dense concentrations
of serotonergic
neurons.
3. Basic characteristics
I) SLEEP-WAKE-AROUSAL
CYCLE.
The first study to
examine the activity of serotonergic
neurons in behaving animals was carried out in the DRN of cats (328).
During quiet waking behavior these cells displayed the
same slow (ml-3 spikes/s)
and highly regular activity
found in earlier studies of the DRN in anesthetized
rats
or in tissue slices (328, 469).
This stable pattern of slow and highly regular activity during quiet waking progressively
changes across
the sleep-wake-arousal
cycle (469). As the animals become drowsy and then enter slow-wave
sleep, neuronal
activity displays a corresponding
decrease in rate and
loss of regularity.
This culminates
during rapid-eyemovement (REM) sleep, where a complete cessation of
DRN neuronal activity is seen (Fig. 14). Because it is
well known that a primary feature of REM sleep is centrally induced atonia, this decreased neuronal activity
might, in some way, be related to this loss of muscle
tone. This issue has been examined from several perspectives, and the results provide general support for
the existence of a relationship
between tonic level of
motor activity and DRN serotonergic
unit activity in
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017
manipulations,
and/or the equivocal nature of these
findings, investigators
began studying
the activity of
serotonergic
neurons in unanesthetized
freely moving
animals. To date, such studies have been confined exclusively to one species, domestic cats. In cats, the vast
majority of serotonergic
neurons are found in four main
clusters that correspond,
roughly, to the midline DRN,
MRN, NRM, and NRO/NRPa
(236,511). Single-unit
activity has been examined, at least to some extent, in all
four of these groups. In addition, however,
~25% of
brain serotonergic
neurons are located at some distance
from these midline groups (236, 511). These “pararaphe” serotonergic
neurons have not been studied in
terms of their single-unit
activity.
The preponderance
of these single-unit
studies
have examined the activity of serotonergic
neurons in
the DRN. Therefore most of our discussion focuses on
this group. However,
some attention has also been devoted to the NRM because of its involvement in nociception and analgesia. Finally, because the MRN and NRO/
NRPa groups have been the subject of very few experiments, these data are discussed
primarily
for the
purpose of comparison
and contrast
with the DRN
and NRM.
OF
Volume
X?
EOG
EMG
UNIT
111
‘1I
iI’ I
-11
REM
SLEEP
r
I
I
SLEEP
I
dim
EEG
11
5 SEC
FIG. 14. Polygraph
traces displaying
single-unit
activity
of DRN serotonergic
neuron recorded
across sleep-wake-arousal
cycle in cat. Note
how slow and regular
activity
is maintained
during active and quiet waking
but then falls off during slow-wave
sleep. Cell became totally silent
during rapid-eye-movement
(REM)
sleep. EEG, electroencephalogram;
EOG, electrooculogram;
EMG, electromyogram.
behaving cats. Destruction
of a discrete area of the dorsomedial pontine reticular
formation
in cats produces
REM sleep without
atonia (209, 248). An animal with
such a lesion has epochs that, by all criteria, appear to
be REM sleep, except that antigravity
muscle tonus is
present and the animals are therefore capable of moving
and even engaging in complex motor acts, including locomotion. When the activity of DRN serotonergic
neurons was examined during REM sleep in these animals,
it was found to be increased well above that of normal
animals in REM sleep (471). In fact, the level of neuronal
activity was directly related to the degree of restored
motor activity during REM sleep. During other phases
of the sleep-wake-arousal
cycle in these animals, the
activity of DRN neurons was similar to that observed in
normal animals.
A second series of experiments
examined this issue
in a somewhat
reciprocal manner. When cholinomimetic agents such as carbachol are injected directly into
the dorsomedial
pontine reticular
formation,
they can
produce atonia in an otherwise
awake cat. Dorsal raphe
nucleus serotonergic
unit activity during this centrally
triggered atonia was found to be completely suppressed
(454). Carbachol
injections
into nearby areas, which
failed to induce atonia, had no effect on DRN unit activity. Several additional experiments
in this series led to
the conclusion that the critical variable was centrally
induced atonia rather than muscle paralysis per se. Systemic administration
of dantrolene, a peripherally
act-
ing muscle relaxant, or succinylcholine,
an antagonist at
the neuromuscular
junction,
produced no change in
DRN unit activity
despite a profound
loss of muscle
tonus (the animals given succinylcholine
had to be artificially respirated).
However,
systemic administration
of mephenesin, a centrally acting muscle relaxant, once
again produced a large decrease in DRN unit activity.
Several points should be made regarding
these
data. First, it is clear that this relationship
is not one
where a decrease in serotonergic
neuronal
activity
causes atonia. When serotonergic
neurotransmission
is
blocked by any of a variety of pharmacological
means
(inhibition
of 5-HT synthesis,
blockade of 5-HT receptors, or central application
of a serotonergic
neurotoxin), muscle paralysis
or atonia does not ensue.
Similarly, the decreased neuronal activity cannot be attributed to diminished proprioceptive
input since succinylcholine produces muscle flaccidity but no change in
DRN unit activity.
Thus it appears that some CNS
group(s)
of neurons that produces atonia also causes
these serotonergic
neurons to decrease their activity.
Second, if a functional relation does exist between central motor systems and serotonergic
unit activity, it is
not a simple linear relation, since small changes in muscle tone are frequently
unaccompanied
by changes in
serotonergic
unit activity (316). Thus the relationship
appears to be at a more gross level, perhaps one reflective of the general behavioral state of the organism. Although serotonergic
neurons in the other raphe groups
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SLOW-WAVE
January
19%
STRUCTURE
AND
FUNCTION
OF
BRAIN
SEROTONIN
UNIT
FIG. 15. Polygraph
record showing
activity
of mesencephalic
5-HT neuron
during
transition
from
REM sleep to
wakefulness.
Note resumption
of neuronal activity
before arousal
from REM
sleep [as indicated
by return
of neck
electromyogram
(EMG)
activity]
and
high level of this activity.
Pontogeniculate occipital
waves can be seen in latera1 geniculate
(LGN)
trace. EEG, electroencephalogram;
EOG,
electrooculogram.
EEG
201
SYSTEM
lrn
,
,I .‘-
I’
.
’ ‘IW
10 set
have not been studied under these conditions, the fact
that they are all REM-off leads to the prediction that
they would also be atonia-off.
A final feature of DRN neuronal activity during
sleep is seen in a resumption
of the activity of these
neurons signaling the end of a REM sleep epoch. Over
50% of DRN cells examined displayed a dramatic increase in neuronal activity as much as 10 s before the
end of a REM period (Fig. 15), as defined by behavioral
and/or polygraphic criteria (the mean lead time was 3.2
s). Most interesting was the fact that the level of this
resumed activity was approximately
twice that observed during a quiet waking state and was approximately equivalent to that seen during phasic arousal.
This high level of neuronal activity in anticipation
of
awakening may serve to prepare the various CNS target
systems of the organism for responding to external
stimuli.
Reciprocal to the decrease in activity that DRN
cells display during sleep, an increase in activity is seen
as the animal becomes behaviorally
activated (469). Increases above the level of neuronal activity during quiet
waking are seen during an alert or active waking state
(-20%) and during an aroused state (-40%).
In response to an arousing stimulus, such as a click that
elicits an orienting response, DRN neuronal activity
displays a phasic activation (l-2 s in duration) to a level
approximately
twice that of the quiet waking level. As
the ability of a stimulus to elicit an orienting response
wanes with repeated presentations, so does the concomitant phasic activation of DRN cells.
In summary, a dramatic, almost linear, positive relationship exists between the level of tonic motor activity/behavioral
activation of the organism that is seen
spontaneously, across the sleep-wake-arousal
cycle, and
the discharge rate of DRN serotonergic neurons. These
results have been confirmed by several laboratories (93,
316,437). In section VI, B5 and B6, we review how these
neurons respond to more extreme changes in behavioral
state, such as those induced by painful stimuli or a varlety of stressors and during specific complex behaviors.
When the same type of analysis was extended to
serotonergic neurons in the MRN, NRM, and NRO/
NRPa of behaving cats, a generally similar pattern of
state-related
neuronal activity was observed (93, 155,
211,401, 418; Fig. 16). However, there were several important differences, especially between the superior
group of serotonergic neurons (DRN and MRN) and the
inferior group of serotonergic neurons (NRM and NRO/
NRPa). For example, medullary
serotonergic neurons
generally display a higher (30-60%) spontaneous firing
rate during comparable behavioral states. In addition,
mesencephalic serotonergic neuronal activity exhibits a
strong inverse relationship
to the occurrence of sleep
spindles and pontogeniculate-occipital
cortex (PGO)
waves, two events associated with slow-wave sleep and
REM sleep, respectively.2 In contrast, the activity of
medullary
serotonergic neurons is unrelated to these
events. The activity of NRO/NRPa
neurons, as well as a
subset of MRN neurons, is not as strongly related to
behavioral state as DRN, NRM, and the majority
of
MRN cells. Thus they did not display as great a decline
in activity across the sleep-wake cycle. Their activity,
although significantly
reduced during REM sleep, was
not completely suppressed. They were generally unresponsive to arousing stimuli, whereas DRN, NRM, and
the majority of MRN cells were often strongly activated
by such stimuli.
A subgroup of DRN neurons whose activity often
deviates from the highly regular discharge pattern that
2 This is consistent
with neuropharmacological
evidence
that
PGO waves are held under inhibitory
serotonergic
control
(413). In
addition,
these data are consistent
with the concept that decreased
serotonergic
(and noradrenergic)
tone, in conjunction
with increased
brain
stem cholinergic
activity,
influence
thalamocortical
mechanisms responsible
for important
physiological
and behavioral
aspects
of sleep (454a).
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EOG
202
BARRY
6
L. JACOBS
AND
EFRAIN
C. AZMITIA
DRN 3 6
4
i::iii~i~~-~~~iiiiii~.
QW
DRO
SWS,
SWS2
SWS3
PREREM
REM
AW
QW
DRO
SWS,
SW!39
SWS2
SWS3
PREREM
REM
PREREM
REM
NRPa
QW
DRO
generally characterizes
serotonergic
neurons has been
described. Accordingly,
these cells have been described
as “nonclocklike”
(353,437). As with other serotonergic
neurons, the activity of these cells changes dramatically
across the sleep-wake
cycle. They become silent during
REM sleep; however, they often display little change in
activity in going from quiet waking to slow-wave
sleep.
Additionally,
as with other serotonergic
neurons, their
activity is strongly suppressed by the systemic administration of 5-HT,* agonist drugs. They do not appear to
be localized to any particular
portions of the DRN but
instead are scattered throughout
the nucleus (437). As
described in section VIB~, many neurons in this subgroup have a specific relationship to particular behaviors in cats (406).
Finally, an attempt to understand the neurochemical mechanisms l mediating the suppression of DRN neuronal activity during sleep has recently been initiated.
With the use of single-unit recordings in conjunction
with multibarrel microiontophoresis in the conscious
but head-restrained cat, preliminary evidence suggests
that a GABA-mediated inhibition may be responsible
(289a). When the GABA antagonist bicuculline was applied onto DRN serotonergic neurons during sleep, in
some cases their activity was restored to waking levels.
However, when bicuculline was applied during waking,
there was no change in neuronal activity, suggesting
that this GABAergic influence was selectively activated
during sleep.
II)CIRCADIANVARIATION.
A basic feature of many
biological systems is variation in a circadian manner.
Because brain 5-HT metabolism is strongly influenced
by the light-dark cycle (for review see Ref. 470) (true
circadian rhythmicity
has not been examined directly),
SWS,
SWS2
SWS3
PREREM
FIG.16.Meandischargeratesof
serotonergic
neurons in 2 mesencephalic
[dorsalis
(DRN)
and medianus
(MRN)]
and 2 medullary
[magnus
(NRM)
and
pallidus
(NRPa)]
raphe
nuclei
across
sleep-waking
continuum
in cat. Each
bar represents
mean * SE; n = 18-47
cells/nucleus.
AW, active waking;
QW,
quiet
waking;
DRO,
drowsy;
SWS,,
SWS,,
SWS,, beginning,
middle,
and
end of slow-wave
sleep epoch, respectively;
PRE-REM,
minute
before REM
onset. [From Fornal
and Jacobs (156).]
REM
it is of interest to ask whether the activity of serotonergic neurons varies as a function of environmental lighting. If behavioral state is held constant, i.e., when the
activity of a given neuron is studied repetitively during
REM sleep or quiet waking at various times across a
12:12-h light-dark cycle, the activity of DRN serotonergic neurons is invariant (470). Thus the activity of these
cells is not influenced by the light-dark cycle nor, by
inference, the circadian cycle. How then can we account
for the numerous reports that 5-HT metabolism does
vary as a function of environmental lighting? There are
two obvious, non-mutually exclusive, possibilities. First,
because neurotransmitter metabolism occurs predominantly at the axon terminal, local conditions at these
sites -may modulate metabo llism somewhat independently of constant neuronal activ ity at the cell body.
Such -modulatory influences are known to exist (1,W
Second, the variation in 5-HT metabol ism across the
light-dark cycle may simply be secondary to the wellknown distribution of sleep and waking across this cycle. For example, rats are known to spend most of their
time in the dark, awake and active. During this time
brain serotonergic neuron s would also be active and metabolism would be increased. In contrast, rats in the
light spend most of their time sleeping. During this period the activity of serotonergic neurons would be decreased, as would metabolism. In all likelihood, a combination of these two factors accounts for the observed
variation in brain 5-HT metabolism across the lightdark cycle.
III)SENSORY
STIMULATION.
As discussed in section
VIkt6, ‘in anesthetized animals serotonergic neurons
show no response to ph asic environmental stim .uli, such
as light flashes. In contrast, in conscious animals, repeti1.
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SWS,
SWS3
-
NRM liw6
c35
DRO
72
MRN
5
it
3;
z
Volume
January
199.2
STRUCTURE
AND
FUNCTION
OF
SEROTONIN
SYSTEM
203
Finally,
confirming
the speculation
that the response of DRN neurons in conscious animals might be
blunted by anesthesia,
it has been shown experimentally that chloral hydrate anesthesia completely blocks
the response of these neurons to either clicks or flashes
(213). An important
aspect of this study was the fact
that the responses of the same neuron were examined
under identical conditions
while the animal was initially unanesthetized
and then anesthetized.
4. Physiological
manipulations
I) INTRODUCTION. The next two sections review the
data on the response of serotonergic
neurons to a variety of physiological
manipulations
and behavioral/environmental stressors. Many of these physiological
manipulations can also be considered to be stressors,
as indicated by their ability to produce increased
plasma
catecholamine
levels and increased tonic heart rate.
There are two reasons for carrying
out these studies.
First, it enables us to explore, at the single cell level,
issues in which 5-HT previously
has been implicated,
e.g., thermoregulation
or nociception. Second, the possibility of noting a change in serotonergic
neuronal activity would be enhanced by exposing the animal to strong
stimuli from a variety of domains. Thus the neuronal
responses to these strong stimuli might provide clues to
other conditions that could influence serotonergic
activity in a more subtle manner.
II) THERMOREGULATORY
CHALLENGES.
Historically, brain 5-HT has been most closely tied to the central mechanisms
underlying
thermoregulation
(351).
The primary focus of this research has been on the anterior hypothalamus/preoptic
area, which receives its
serotonergic
input from neurons lying within the mesencephalic groups of serotonergic
neurons (47,82,476). Hypothalamic 5-HT has been implicated in both heat loss
responses (those occurring in reaction to a heated environment, e.g., panting and vasodilation)
and heat gain
responses (such as those occurring
during the febrile
response of disease states, e.g., shivering
and vasoconstriction).
A comparison
of these two situations
is of
interest because they both involve increased body temperature but are produced as opposite thermoregulatory
mechanisms
are engaged.
The previous research in this area, employing primarily pharmacological
approaches, does not provide a
clear-cut consistent
picture (for review see Refs. 156,
157). This may be attributable,
in part, to the nonphysiological nature of such experiments.
Until recently,
single-cell studies of serotonergic
neurons and thermoregulation
were carried out primarily
in anesthetized
animals, and the results were contradictory
or, at best,
equivocal.
Experiments
conducted in behaving animals have
examined the response of DRN neurons to both increased ambient
temperature
and pyrogen-induced
fever (157). For environmental
heating, the temperature
in the experimental
chamber was maintained
at 43 t
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tive presentation
(once every 2 s) of clicks or flashes
produces an excitation of DRN neurons that is followed
by a period of inhibition (215,402). The excitation has a
latency of ~40-50 ms and a duration of ~60-80 ms. The
inhibitory
period that follows the excitation has a mean
duration of -250 ms and closely corresponds
to the interspike interval that normally follows the discharge of
these highly regular firing neurons. For this reason, it
has been proposed that this does not constitute
a true
excitation
of these neurons but simply a resetting
of
their discharge pattern. Thus these phasic sensory inputs may cause the entire group of DRN serotonergic
neurons to fire and then pause in unison. A similar type
of response to sensory stimuli was observed in most
MRN serotonergic
neurons (401). Interestingly,
a subgroup of MRN serotonergic
neurons displayed the opposite response, i.e., an inhibition
followed by a modest
excitation. In contrast to DRN and MRN cells, the activity of caudal medullary NRO/NRPa
cells was either unaffected or only weakly responsive
to either clicks or
flashes (211). Finally, the response of rostra1 medullary
NRM cells was intermediate
between the strong excitation of DRN and MRN cells and the weak excitatory
response of NRO/NRPa
cells (155). Thus a gross caudalto-rostra1
brain stem gradient of increasing excitation
of serotonergic
neurons by sensory stimuli appears to
exist. This may be related to the predominantly
ascending projections
of the rostra1 groups and their resulting
influence on forebrain target sites, which may be more
closely involved in processing exteroceptive
sensory inputs.
The fact that the response of DRN serotonergic
neurons to sensory stimuli does not habituate, even over the
course of hundreds
of repetitions,
may be important
(215, 402). It is well known that many nonserotonergic
mesencephalic
neurons also respond to simple sensory
stimuli, but this response invariably
displays habituation (65, 422, 433). Thus the maintained responsiveness
of DRN serotonergic
neurons to repeated sensory stimuli is rare among brain stem neurons. The possible functional significance of the maintained responsiveness
of
these neurons, in the face of a declining response of virtually all other brain stem neurons, is discussed in section VIIIA.
Recently, an attempt to understand the neurochemical mechanisms
mediating the sensory-induced
excitation of DRN serotonergic
neurons has been initiated.
With the use of single-unit
recordings
in conjunction
with multibarrel
microiontophoresis
in the conscious
but head-restrained
cat, preliminary
evidence suggests
that an excitatory
amino acid mediated effect may be
responsible
(E. S. Levine and B. L. Jacobs, unpublished
observations).
When the excitatory
amino acid antagonist kynurenine
was applied onto DRN neurons during
exposure to a series of clicks, the typically observed excitation was significantly
diminished.
However,
this
same treatment
had no effect on the spontaneous
activity of these cells, suggesting that this excitatory
amino
acid influence was at least somewhat
selective to this
sensorv-induced
activation.
BRAIN
BARRY L. JACOBS AND EFRAIN
:
:
0’
8’
:
i
:
:
8’
8’
,’
a
0 !
6
3
’
0 !
FIG. 17. Example of heating trial showing effects of acute heat
stress on brain temperature, respiratory rate, and activity of single
serotonergic DRN neuron in awake cat. Firing rate of this neuron was
net significantly different from baseline at any time during heating
trial, although respiratory rate was increased 2- to 12-fold, and brain
temperature was elevated by as much as 13°C. [From Fornal et al.
Wm.1
Volume
72
Body/brain temperature began to increase within 30
min, reached peak at I-2 h, and returned to predrug
level by 6 h. The peak elevation of body temperature
that was attained was typically 1.5~25°C. Once again,
no change in DRN single-unit activity was observed
during any phase of the pyrogen-induced febrile response when compared with the level of activity measured during a comparable control behavioral state.
These data indicate that at the single-unit level of
analysis, DRN serotonergic neurons are not involved in
the mechanisms for either heat gain or heat loss. Additionally, they do not appear to be heat sensitive, i.e.,
responding to elevations in skin, body, or brain temperature. Overall, these results do not support a specific role
for these neurons in thermoregulation. However, they
may be involved in the well-known state-dependent
changes in thermoregulation, since it is clear that serotonergic neuronal activity
does vary dramatically
across the sleep-wake-arousal cycle. Furthermore, these
data do not rule out the possibility that the release of
5-HT in the hypothalamus may be controlled locally by
circulating neurohormones, neurotransmitters,
or by
changes in brain temperature. Exploration of the possibility that, under certain conditions, there may be a dissociation between single-unit activity and axon terminal release of 5-HT is described in section VIB7.
III) CARDIOVASCULAR
CHALLENGES.
Both the ascending serotonergic system, in its projections to cortex,
limbic system, and hypothalamus, and the descending
serotonergic system, in its projections to autonomic regulatory regions of the brain stem and spinal cord, have
been implicated in cardiovascular control (24,272,517).
As with any complex system, such as the cardiovascular,
attempts to relate a central neurotransmitter to its control or modulation are difficult to do with any degree of
precision or certainty. This is attributable both to the
inherent nonspecificity
in some experimental
approaches, such as electrical stimulation of the brain or
systemic drug treatment, and to the multiple sites of
possible interaction between serotonergic neurons and
the cardiovascular system. As discussed, previous studies examining DRN unit activity and blood pressure in
anesthetized rats failed to find any relationship
(88,154).
l°C until each cat displayed continuous panting (respiratory rate --150/min). The activity of DRN neurons
remained unaffected during the interval when ambient
temperature was increased from 25 to 43°C (Fig. 17).
During this initial phase of heating, no appreciable behavioral or physiological responses were seen. However,
following prolonged heat exposure, cats displayed intense panting, relaxation of posture, and a progressive
rise in body/brain temperature (range: 0.520°C). Once
again, however, no change in DRN single-unit activity
was observed in comparison to the level of activity measured during a comparable behavioral state in the absence of heat.
In studies of DRN neuronal activity during the febrile response, the synthetic pyrogen muramyl dipeptide was administered svstemicallv (50 ue/ke iv) (157).
The first study to examine the relationship between
the cardiovascular system and DRN and NRM unit activity in behaving animals found that, under resting conditions, the discharge of these neurons was unrelated to
the cardiac cycle (338). This finding becomes more
meaningful when one considers that a neighboring
group of neurochemically identified neurons, the noradrenergic cells of the locus coeruleus, did display a cardiac-related periodicity (338). These data indicate that
these serotonergic neurons do not receive direct afferent
input from the cardiovascular system, at least under
resting conditions.
The response of DRN neurons and a small group of
NRM neurons has also been studied in relation to
changes in blood pressure induced by peripherally acting drugs. A iueular catheter was used to administer
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38.5
C. AZMITIA
January
1992
STRUCTURE
AND FUNCTION
OF BRAIN SEROTONIN
205
V) DISCUSSION. These data indicate that, at least at
the single-unit level of analysis, brain serotonergic neurons are not changed from their basal level of activity by
manipulations of several important physiological regulatory systems. Several things militate against simply
dismissing these data as trivial or uninteresting. First,
this lack of neuronal change from that seen during a
comparable baseline behavioral state occurs in the face
of significant physiological responses of the organism to
defend against these challenges (339,341). Second, these
studies manipulated a number of different physiological
regulatory systems. Finally, in a parallel series of studies, a neighboring group of neurochemically identifiable
neurons, the noradrenergic cells of the locus coeruleus,
were found to be highly responsive to the same thermoregulatory (339), cardiovascular (340), and glucoregulatory (341) challenges. The implications of the differential responsiveness of these two widely projecting brain
stem neurochemical systems is discussed in section
VIIIA.
5. Environmental
stressors
I) INTRODUCTION.
Paralleling the experiments just
described, the studies in this section explore, at the single-cell level, behavioral and environmental issues in
which 5-HT has been historically implicated, e.g., aggression and nociception. These studies also utilized
strong stimuli from several domains in the hope that
this would optimize the possibility of finding a change.
II) STRESSORS. This series of studies examined the
responses of DRN serotonergic neurons to three environmental challenges from differing domains: noise, restraint, and exposure to a natural enemy.
Exposure of various species, including humans, to
loud noise for a sustained period of time is known to be
stressful. When cats are exposed to loud white noise (100
dB) for 15 min, the stimulus elicits strong sympathetic
activation, as indicated by a 30-50% increase in tonic
heart rate and a lOO-150% increase in plasma norepinephrine. It also evokes a stereotyped behavioral response of crouching, with ears flattened (I, 514). Despite
this strong organismic activation, the activity of a group
of DRN neurons was not significantly different from
that observed during an undisturbed active waking baseline (514).
Because serotonergic neurons are unaffected by a
stressor that impinges on the organism via its telereceptors (i.e., white noise), it was of interest to examine
the effects of a proximal stressor, such as restraint.
When cats are restrained for 15 min, a strong sympathetic activation is once again seen. Struggling and vocalizations during the restraint provide additional behavioral evidence for the stressful nature of the stimulus (1, 514). Despite this behavioral and physiological
activation, the activity of a group of DRN neurons was
not significantly different from that observed during an
undisturbed active waking baseline (166).
Confronting an animal with one of its natural ene-
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phenylephrine, to induce increases in blood pressure; sodium nitroprusside, to phasically decrease blood pressure; and hydralazine, to produce prolonged hypotension. The drugs were administered while the animals
were in a quiet waking state. Over a range of ZO- to
70-mmHg increases and IO- to 50-mmHg decreases in
mean arterial pressure, there was no change in the discharge rate of DRN or NRM serotonergic neurons (158,
158a). This is despite the fact that these blood pressure
changes were of sufficient magnitude to produce significant reflexive changes in heart rate and plasma catecholamines. These data are another indication that serotonergic neurons are unresponsive to afferent input deriving from the cardiovascular system. This lack of
change in activity of serotonergic neurons to manipulations of the cardiovascular system is also consistent
with the results of a recent study of medullary serotonergic neurons in anesthetized cats (326).
As discussed with regard to thermoregulation,
these data do not preclude the possibility that changes
in serotonergic neuronal activity across the sleep-wake
cycle may be involved in the well-established state-dependent changes in the cardiovascular system (368,525).
This conceptualization of the functional role of the serotonergic system is more fully developed in section VIII. It
should also be noted that the serotonergic neurons in
the ventrolateral medulla, an area known to be important in cardiovascular regulation, have not been studied
under these conditions.
IV)GLUCOREGULATORYCHALLENGE.
Glucoserepresents the main source of energy for the brain. Therefore
it is of interest to manipulate blood glucose levels in
both directions and to examine the effects on DRN single-unit activity in behaving animals. This issue is of
interest for two reasons. The central serotonergic system is thought to be a broad neuromodulatory system,
and therefore its neuronal activity might be responsive
to this fundamental biochemical variable. In addition,
brain 5-HT has been implicated specifically in the control of food intake.
The activity of serotonergic DRN neurons in behaving cats was not significantly altered by bolus injections
of glucose (500 mg/kg iv) that elevated blood glucose
levels three- to fivefold (158). Likewise, the activity of
these neurons was not significantly affected by the administration of a dose of insulin (2-4 IU/kg iv) that lowered blood glucose by 250% or after the rapid reversal
of this hypoglycemia by subsequent glucose administration.
These results indicate that the activity of serotonergic DRN neurons is uninfluenced by alterations in
blood glucose, nor is it sensitive to elevations of endogenous circulating insulin levels or to the administration
of exogenous insulin. Furthermore, changes in the activity of these neurons do not appear to be a component of
glucoregulatory mechanisms evoked by either hyper- or
hypoglycemia. This is despite strong pituitary and adrenal activation by insulin-induced hypoglycemia and is
therefore unexpected since brain 5-HT has often been
implicated in the neuroendocrine response to stress.
SYSTEM
206
BARRY
L. JACOBS
AND
C. AZMITIA
Volume
72
stimulation (Fig. 18, top). In contrast, some neighboring
nonserotonergic neurons were dramatically activated
when the stimulus reached a noxious level but showed a
minimal response at nonnoxious levels (Fig. 18, bottom).
The response of these latter neurons to noxious stimuli
was blocked by a systemic injection of morphine (Fig.
18, bottom right).
The response of NRM serotonergic neurons has also
been studied during the application of a more prolonged
painful stimulus. A subcutaneous injection of dilute
Formalin solution produces a series of stereotyped behavioral responses in cats: initially it elicits licking, biting, and shaking of the injected paw; with time this
abates and the paw is simply held aloft; then it is placed
on the floor but no weight is applied; finally, after -30
min, use of the paw returns to normal. Throughout this
30-min episode the activity of NRM serotonergic neurons was no different from that seen during a comparison undisturbed active waking state (30).
Because a critical link in opioid-induced analgesia
may be the release of 5-HT in the spinal cord, the response of NRM neurons to systemic administration of
morphine has been examined. Across a dose range of
0.5-4.0 mg/kg ip, the response of NRM neurons in behaving cats was unchanged from that observed during a
control waking condition, despite clear-cut behavioral
evidence of drug-induced analgesia (30). These results
are consistent with those seen in similar studies of NRM
neurons conducted in anesthetized rats (103). These results do not exclude the possibility that opioid drugs
may exert an action directly on serotonergic nerve terminals in the spinal cord (69). However, it is also worth
noting that several studies have questioned the involvement of descending serotonergic neurons in opioid-induced analgesia (242, 393, 394).
To summarize, morphine-induced analgesia does
not appear to be dependent on the activation of serotonergic NRM neurons and, at the single-unit level, serotonergic NRM neurons do not appear to be specifically
involved in pain modulation, since they respond to
noxious and nonnoxious stimuli to approximately the
same degree. This suggests that they are not an essential component of the analgesic system. However, the
activation of these neurons in response to arousing stimuli could be part of a more general sensory modulatory
system operating during periods of increased motor activity and behavioral arousal. This is consistent with
one of the central themes of this review, i.e., that serotonergic neurons exert modulatory control over a large
variety of sensory, motor, and endocrine functions. Another possibility is that painful stimuli can directly increase the release of 5-HT at the nerve terminal, thus
bypassing an action on the cell body, leaving single-unit
activity unchanged. This issue is examined below in experiments described in section VIB7.
6. Behavior
One major domain remains to be discussed with respect to its relationshiD to serotonergic neuronal activ-
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mies represents a powerful stimulus of particular biologic relevance. In this experiment, cats were exposed to
a dog while physiological measures were taken and DRN
single-unit activity was recorded. When a dog was
brought into view of the cat for 5 min, it evoked the
typical stereotyped feline defense reaction of arched
back, facing broadside, piloerection, and often growling
and hissing. Sympathetic activation, as indicated by an
increase in tonic heart rate and an increase in plasma
norepinephrine level, was once again seen. Despite this
behavioral and physiological activation, the discharge
rate of a group of DRN serotonergic neurons was unchanged from that observed during an undisturbed, active waking baseline condition (514).
These negative results with DRN neurons are somewhat surprising in light of the literature implicating
5-HT in aggression and/or defense reactions in a variety
of mammalian species. Additionally, as with physiological challenges, when the activity of locus coeruleus noradrenergic neurons was examined in response to these
same behavioral/environmental
stressors, large increases in activity were consistently observed (1, 289).
III) NOCICEPTION/ANALGESIA.
Much attention has
been devoted to the role of central 5-HT in nociception
and analgesia, especially because of the important social and clinical relevance of this subject. The bulk of the
data in support of an important role for 5-HT in analgesia derive from anatomic, pharmacological, and brain
stimulation studies (reviewed in Refs. 30,156,283).
Very
few studies have addressed this question at the singlecell level, and only one has done so in behaving animals.
Most of the experimental attention regarding the role of
5-HT in nociception and analgesia has been focused on
the medullary serotonergic neurons of the NRM.
It has been proposed that painful stimuli activate
brain stem opioid-containing neurons, which in turn activate spinal cord-projecting NRM serotonergic neurons, thus suppressing pain (56). If opioid- and nociception-induced analgesia are dependent on activation of
these neurons, then both painful stimuli and opioid
drugs should increase their activity. As the series of
studies described in this section shows, however, the experimental data do not provide support for this hypothesis.
If radiant heat is applied to an animal’s tail, the
animal will move its tail to escape this stimulus as it
becomes painful. When the activity of cat NRM serotonergic neurons was studied during such a procedure, it
was found that there was no consistent increase in discharge during the application of the heat (30). NRM neurons were equally activated by arousing but nonpainful
stimuli, such as tapping on the experimental chamber or
entering the experimental room.
In an attempt to provide a quantification of the response of these neurons to painful stimuli, the inferior
alveolar (dental) nerve was electrically
stimulated
while the neuronal response was analyzed (30). The data
indicated that NRM serotonergic neurons show no
greater response to high-intensity noxious stimulation
of the alveolar nerve than to low-intensitv nonnoxious
EFRAIN
January 1992
STRUCTURE
AND FUNCTION
OF BRAIN
SEROTONERGIC
30
z
z
\
SYSTEM
207
NEURON
1
NOCIRESPONSIVE
NEURON
II
100
( NON-NOXIOUS
UA
400
1
ity. Do serotonergic neurons display changes in activity
in association with any specific behaviors? Recently, a
group of DRN and MRN 5-HT neurons have been found
to undergo dramatic alterations in neuronal activity in
relation to two types of behavior, oral-buccal movements and orienting responses.
A substantial number (as many as 3050%) of serotonergic neurons in the DRN and MRN of cats are tonically activated (Z- to &fold) during repetitive motor activities involving the oral-buccal region: chewing/biting
(Fig. 19, top and middle), lapping, and grooming the body
surface with the tongue (406). During these periods of
activation the discharge pattern of these neurons frequently becomes extremely regular (i.e., clocklike). Interestingly, these neurons do not appear to be activated
during any other types of behavior; in fact, their activity
typically decreases during many active behaviors (Fig.
19, bottom), such as locomotion and orientation.
Individual neurons within this subpopulation
may display
characteristics
not shared by the others within this
group. Some may increase their activity in anticipation
of initiating
the behavior, for example, in response to
seeing or smelling food. Others may increase their activity in phase with the licking or biting responses (most,
however, appear to be tonically activated during these
behaviors). Finally, many of these neurons can also be
activated by somatosensory stimuli, especially those applied to the head and neck area (C. A. Fornal and B. L.
Jacobs, unpublished observations).
Many (perhaps all) of the aforementioned
serotonergic neurons that increase their activity during oral-
(
NON-NO
lJA
XIOUS )
800
( NOXIOUS
A
uA
f
800
1
POS
-
200
msec
UA
T MORP
HINE
buccal movements display the opposite response during
orientation to an external stimulus (Fornal and Jacobs,
unpublished
observations).
Phasic auditory or visual
stimuli often elicit the typical feline orienting response,
which is to foveate the source of the stimulus by moving
the head and eyes toward it. This is usually accompanied
by a suppression of all other overt movement. During
this brief orientation,
and for several seconds following
it, the activity of some DRN and MRN neurons is suppressed, often completely. This may be related to the
previously known fact that 5-HT neuronal activity is
suppressed during PGO waves during deep slow-wave
sleep and REM sleep (156). Morrison and colleagues
(341b) have shown that PGO waves during sleep are a
sign of internal alerting. Of more importance to the present discussion, they also demonstrated that PG 0 waves
could be evoked by phasic stimuli during the waking
state, where they were frequently associated with startle or orienting responses (external alerting). Completing this picture, the suppression of 5-HT unit activity
during orientation
is frequently accompanied by PGO
waves.
7. In vivo release
Up to this point, our discussion of functional analyses of serotonergic neurons has focused on studies of
single-unit activity. A basic assumption inherent to the
usefulness of single-unit activity is that such activity is
tightly coupled to the axon terminal release of neuro-
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18. Computer-generated
peristimulus time histograms of simultaneously recorded responses of serotonergic neuron of NRM (top) and neurochemitally unidentified nociresponsive neuron
(bottom) to repetitive stimulation (constant current, monophasic pulses of 1.4ms
duration once every 2 s) of alveolar nerve.
Serotonergic neuron and nociresponsive
neuron were recorded on adjacent leads of
same microelectrode bundle in behaving
cat. Current intensity of 100 PA was
threshold for eliciting jaw-opening reflex,
and 400 PA elicited a strong jaw-opening
reflex without any behavioral signs of
pain. Current intensity of 800 PA elicited
signs of pain. Current intensity of 800 PA
was repeated 30 min after administration
of morphine (2 mg/kg ip), a dose that eliminated behavioral signs of pain. Occurrence
of stimulation is marked by arrow. Each
histogram was constructed from 64 trials
using bin width of 10 ms. See text for discussion. [From Auerbach et al. (30).]
FIG.
SEROTONIN
208
BARRY
QUIET
L. JACOBS
AND
EFRAIN
C. AZMITIA
Volume
72
WAKING
The most serious is the tissue damage that results from
having fluid pushed into the brain with a high flow rate
ti II, I I I I ,Ir\
and under positive pressure and then withdrawn under
negative pressure.
EM More recently, two other approaches to this problem have been developed, voltammetry and intracereEOO G
---+
bra1 microdialysis. In vivo voltammetry is based on the
principle that electrooxidizable substances, such as 5HT, can be oxidized by applying a specific voltage between an electrode in the brain area of interest and
FEEDING
some neutral point (262, 447). The resulting current is
assumed to be proportional to the concentration of the
substances oxidizing at the electrode surface. At present, the major impediment to studying 5-HT with this
approach is that both 5-HT and its major metabolite,
SHIAA, are oxidized at the same potential. However, a
number of laboratories are currently attempting to develop a treated electrode that would not oxidize the metabolite, thus leaving 5-HT to be measured. A recent
ACTIVE
WAKING
study reports some success in this endeavor (111).
In vivo brain dialysis can be likened to a push-pull
IIIB,l\llii\l\Illlli\lllI\\\ !I II lllrn II !lIl!mmmI!!l III I \I III!\ Imu II e II lnrrirmrrrr!
cannula approach conducted inside of a dialysis membrane (for review see Refs. 262,475,501). Thus the brain
tissue is not directly exposed to moving fluids under differing pressure. Instead, substances released in brain
diffuse across the dialysis membrane (typically with a
mol wt cutoff of 5,000) at a rate proportional to their
concentration gradient. They are then collected and an10 SEC
alyzed by radioimmunoassay or sensitive high-pressure
FIG. 19. Polygraph
traces displaying
single-unit
activity
of seroliquid chromatography coupled to a variety of detection
tonergic
neuron
in DRN of cat during
quiet waking,
feeding,
and actechniques, including electrochemical, coulometric, or
tive waking.
Note dramatic
increase
in activity
during
feeding
and
fluorimetric techniques.
pauses that occur during active waking,
often in association
with eye
movements.
EMG, electromyogram;
EOG, electrooculogram.
To date, most studies employing in vivo dialysis
have used pharmacological and basic biochemical manipulations to validate that the method does, in fact,
transmitter. If that is untrue to any significant degree, measure neuronally released 5-HT that diffuses into the
it undermines the value of single-unit activity as a re- extracellular space (31, 253, 501). More importantly,
flection of the functioning of brain neurochemical sys- from the perspective of this review, a few studies have
used this approach to examine release of 5-HT under
tems.
Theoretically, there are a number of different mech- physiological conditions. The data are consistent with
anisms that could allow release to become uncoupled what has been found with single-unit analyses. Thus
from neuronal activity: as neuronal activity increases, 5-HT release in the anterior hypothalamus of cats inrelease may be unable to follow in a linear fashion; di- creases from sleep to waking to arousal by ~50-100%
rect axoaxonal connections may modulate release (pre- (513,513a). Perhaps more significantly, when cats were
synaptic inhibition or facilitation); factors, such as pep- exposed to two different challenges (synthetic pyrogen,
tides or small ions, circulating in the extracellular space which significantly increased body temperature, and inaround the axon terminal may influence release; and tense white noise) there was no change in 5-HT release
mechanisms intrinsic to the axon terminal may vary in beyond that attributable to a change in behavioral state
produced by the manipulation, i.e., the 50-100% ina manner unrelated to neuronal activity (e.g., circadian
crease seen in going from sleep to waking to arousal
variation in reuptake).
One of the few ways to explore this issue is to exam- (513, 513a). These data directly parallel the single-unit
ine release in vivo, especially under conditions similar to results from studies employing the same manipulations.
Similar results have also been reported in studies using
those in which single-unit activity has been studied. Historically, measurement of neurotransmitter
release in in vivo dialysis in rats. From a basal level of hippocamvivo was approached through the use of a push-pull per- pal 5-HT release during sleep (defined by behavioral crifusion technique, where artificial CSF is pumped into a teria), there was a 36% increase during a drowsy state
target site and then the extracellular fluid that it has and a 45% increase during an alert state (251). In another part of this same study, handling and tail pinch
mixed with is pulled out. There are, however, significant
drawbacks to this approach (for review see Ref. 262). both increased hippocampal 5-HT release by ~50%
UNIT
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January
1992
STRUCTURE
AND
FUNCTION
from an unspecified baseline period in the light phase of
the light-dark
cycle.
Thus studies in both cats and rats indicate that activating a sleeping or quiescent animal increases the
release of 5-HT by -5O-100%)
closely paralleling
the
electrophysiological
data on serotonergic
neurons.
8. Summary
BRAIN
SEROTONIN
SYSTEM
209
nergic neurons
are coactivated
in association
with
rhythmic central pattern generator-mediated
behaviors
and by the afferent inputs that normally influence such
behaviors. The motoneurons
that would mediate at least
some of these oral-buccal behaviors in mammals receive
very dense 5-HT inputs (e.g., MoV and MoVII). It is also
interesting
to note that 5-HT plays an important
role in
feeding (287, 412) and in other rhythmic
behaviors (for
review see Ref. 376a) in several invertebrate
species. Finally, a further
elaboration
of this hypothesis
is that
serotonergic
neurons in more caudal groups (e.g., NRM
or NRO/NRPa)
might display activation in association
with other central pattern generators,
such as locomotion, scratching,
or respiration,
that are controlled by
more caudal portions of the neuraxis.
Because this increase in serotonergic
neuronal activity has been seen exclusively in association with oralbuccal movements,
it raises a number of interesting
questions. Is this relation peculiar to cats; would rats or
monkeys manifest a different type of movement-related
activity? Is this an emergent property of more complex
vertebrate species or a remnant of serotonergic
neurons
related to feeding in invertebrates
(412). Will other
movement-related
subgroups
of serotonergic
neurons
be found in the DRN and MRN? Will all such neuronal
increases be related exclusively to central pattern generator-mediated
responses? Finally, what are the efferent
projections
of these serotonergic
neurons
and from
what sources do their afferents derive?
VII.
POSTSYNAPTIC
ACTIONS
A. Introduction
The early literature
on the postsynaptic
effects of
5-HT in the CNS is frequently
contradictory.
Investigators initially
were expecting to find a consistent
and
unitary action, i.e., that 5-HT would universally
be excitatory (increasing the activity of postsynaptic
target neurons) or inhibitory
(decreasing target neuron activity).
It is now clear that this was simplistic.
A given neurotransmitter
can exert multiple, sometimes opposing, actions, and this is determined
by the characteristics
of
the receptor with which it interacts and the membrane
and intracellular
biochemical
mechanisms
coupled to
the receptor. During the past 10 years it has become
evident that there are multiple 5-HT receptors
in the
mammalian
CNS (at least three families of receptors
with six different membrane binding sites3 have now
been reported
for 5-HT). In this section we describe
some of the more salient and well-established
postsynaptic actions of 5-HT and the particular
receptors
3 Establishing
the existence
of a true neurotransmitter
receptor
is much more conservative
than the demonstration
of a binding site. A
receptor
implies
a demonstrable
physiological
function
mediated
by
it, whereas
a binding site merely implies attachment
of the ligand to a
membrane.
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A large literature
implicates
brain serotonergic
neurons in the control of physiological
processes (such
as cardiovascular
function and thermoregulation)
and
the response to environmental
conditions (such as noise,
restraint,
and pain). However,
the experiments
described here imply that at least the raphe groups of serotonergic
neurons are not primarily
involved in these
activities, since no effect on serotonergic
single-unit
activity in behaving animals was seen independent
of
changes in behavioral arousal. This was true despite the
fact that many of these studies employed stimuli chosen
explicitly for the strength of their impact on the organism. The tonic activity of serotonergic
neurons appears
in general to vary in a stereotypical
manner in association with motoric activity and behavioral state and not
specifically
in association
with any process. The discharge of these neurons appears to reach a “ceiling”
during active waking beyond which they cannot be activated, regardless
of the impact of the evoking stimulus
on the organism. Therefore
the primary
role of these
neurons in physiology
and behavior may be to coordinate the activity of target structures
(or set the tone) in
conjunction with the organism’s
level of motor activity/
behavioral arousal. This is a major task for the nervous
system because not only behavior but the output of all
physiological
systems in the organism is strongly modulated across the sleep-wake-arousal
cycle (368, 525).
Consistent with this view is the fact that serotonergic neurons discharge with an extraordinary
tonic regularity, suggesting
that little neuronal information
is
conveyed by this steady-state
activity. Thus the message to target structures
from serotonergic
neurons
may be an analog signal that indicates that the organism is in REM sleep (-0.0-0.3
spikes/s),
in slow-wave
sleep (-0.3-1.5
spikes/s),
drowsy (- 1.5-2.0 spikes/s),
in
quiet waking (-2.0-3.0
spikes/s),
active waking (-3.O5.0 spikes/s),
or physically aroused (-4.0-7.0
spikes/s).
In this schema, within a given behavioral state, the degree of influence that these neurons exert on their target sites is determined primarily
by the density of their
innervation
of particular
CNS regions and whether
there is conjunctive neuronal activity in that target site.
The necessity for coordinated neuronal activity is what
transforms
the serotonergic
system from one exerting
an indiscriminate
or ubiquitous influence on the CNS to
one that confers a specificity of action.
Such specificity can also be imparted by the activation of at least some DRN and MRN neurons in association with particular
complex behaviors. One hypothesis
to account for these data is the DRN and MRN seroto-
OF
210
BARRY
L. JACOBS
AND
thought to mediate these effects (for review see Refs. 12,
78, 387, 479). The molecular biology of 5-HT receptors
and the intracellular
second-messenger
systems and ion
channels
to which 5-HT receptors
are coupled are
beyond the scope of this paper (for review see Refs. 169a,
357a, 507).
B. Inhibition/Modulation
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72
these data is that they are derived from several species.
Unfortunately,
little definitive information
is available
regarding
the receptor subtype involved in these actions. However,
a recent study indicates that the 5-HT,
receptors may be more effective than the 5-HT, receptor
in regulating nociception. When 5-HT was iontophoretitally applied to dorsal horn neurons in the spinal cord of
anesthetized
rats, 5-HTlB agonists mimicked the antinociceptive effects of 5-HT, while a 5-HT, agonist was
without
effect (5-HT,*
agonists
had nonspecific
actions) (143).
As described, 5-HT also exerts a strong inhibitory
action on serotonergic
neurons themselves (autoinhibition). This is the most studied and well-understood
effect of 5-HT in the mammalian
CNS and is attributable
to an action at the 5-HT,* receptor (for review see Ref.
10). These receptors
are found on the somatodendritic
regions of serotonergic
neurons (16), and the action of
5-HT here produces an increased conductance through
the opening of potassium channels (9).
Finally, we come to a postsynaptic
action of 5-HT
that cannot be described with unidimensional
terms
such as inhibition or excitation. If 5-HT is applied iontophoretically
onto neurons in the somatosensory
or visual cortex of anesthetized rats, it exerts little effect on
the spontaneous
activity of these cells (497a, 499). However, when 5-HT is applied in conjunction with afferentinduced excitation of these cells, it preferentially
suppresses this activity. The same effect of 5-HT was observed in conjunction
with afferent-induced
inhibition
of these neurons. (Unfortunately,
no attempt was made
in these studies to determine the 5-HT receptor subtype
where these actions were mediated.) Recently, 5-HT has
been shown to decrease glutamateand quisqualate-induced excitation
of cerebellar neurons in tissue slices
from young rats (215a). These actions have been
variously
referred
to as a reduction
in the signal-tonoise ratio, a disenabling effect, or synaptic modulation
(76, 136, 519). A similar modulatory
action of 5-HT in
the brain stem is described in the next section.
C. Excitation/Modulation
The predominant
postsynaptic
action of 5-HT in the
brain stem and spinal cord (other than the aforementioned inhibitory
action associated with nociception)
is
direct excitation or potentiation
of a heterosynaptic
excitatory effect. These data derive primarily
from studies
employing extracellular
recordings in conjunction with
microiontophoresis
of 5-HT or electrical stimulation
of
serotonergic
neurons (54, 89, 183, 221, 223, 273a, 324,
508a, 509). Most of this work is directed at actions on
motoneurons,
e.g., in the rat facial nucleus or in the ventral horn of the spinal cord.
In these studies, iontophoretic
application of 5-HT
alone produced little or no change in single-unit
activity. However,
when this is interacted
with excitatory
influences on motoneurons,
produced either by direct
anolication
of excitatorv
amino acids or bv electrical
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The predominant
postsynaptic
action of 5-HT in the
forebrain,
especially in the neocortex, is inhibition
(27,
78,1.21,170,201,245,246,257a,
366,403,409,420,427,428,
494). However,
it is clear that excitation
is also frequently seen (87, 244,245, 376,405,408).
These data derive primarily
from studies employing extracellular
recordings in conjunction
with either microiontophoresis
of 5-HT or electrical stimulation
of serotonergic
neurons. In some cases, intracellular
analyses have been
carried out, and the inhibition is attributable
to membrane hyperpolarization
(23,428). There are several reasons why these data often seem confusing. First, they
are frequently
derived from studies employing different
methodologies,
e.g., in vivo versus in vitro preparations
or iontophoretic
drug application versus bath application versus electrical stimulation.
Second, many of these
studies were carried out before the introduction
of antagonists specific to particular
5-HT receptor subtypes.
These latter pharmacological
tools now frequently
permit the systematic
parceling out of apparently
complex
groups of data. Finally, these studies may be comparing
data gathered from different sets of neurons within
a
particular
structure,
e.g., specific regions of hippocampus or particular
layers of cortex.
Nonetheless,
despite these limitations,
a relatively
consistent picture is beginning to emerge. At least some
of the inhibitory
effects of 5-HT seen in the dorsal hippocampus and the neocortex, the two areas most extensively examined, appear to be attributable
to the activation of a 5-HT,* receptor. This conclusion is based on
electrophysiological
studies employing agonists and/or
antagonists
selective for particular
5-HT receptor subtypes (23,108,520). Whether this action accounts for all
of the reports of inhibition by 5-HT in the forebrain is
not known. The ionic basis or the hyperpolarization
produced by 5-HT in the hippocampus
appears to be an
increased resting conductance for potassium ions that is
not calcium dependent (23,108,428). When the postsynaptic effects of the 5-HT,* receptor are examined in combination with the activation of the 5-HT, receptor, their
interaction
is subtractive,
i.e., a reduction in the degree
of inhibition (87, 123, 274).
Another site in the CNS where extensive evidence
shows that 5-HT produces an inhibitory
action is on somatosensory
neurons in the spinal cord, especially dorsal horn cells involved in nociception (for review see Ref.
94). These data are based on both electrical stimulation
of the cell bodies of descending serotonergic
neurons
(140, 185, 196, 249, 333, 407) and on the iontophoretic
abnlication
of 5-HT (63. 198. 247. 398). A strength
of
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January
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STRUCTURE
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OF
stimulation of the dorsal root or motor cortex, 5-HT
produced a strong facilitation of neuronal activity (324,
509). These effects can also be characterized as enabling,
increased signal-to-noise ratio, or modulation. Such an
action of 5-HT would have the effect of greatly potentiating the responsiveness of target cells to incoming
excitatory stimuli. Intracellular analyses indicate that
this increased excitability is attributable to a depolarization, probably mediated by a decreased resting membrane conductance to potassium ions (480). Furthermore, pharmacological analyses indicate that an action at a 5-HT, receptor appears to mediate this effect
BRAIN
SEROTONIN
SYSTEM
211
(508). In contrast, when applied in the absence of the
excitatory amino acid effects, these three substances
had little effect of their own. This implies that all three
may exert a modulatory influence on motoneurons. Contrary to these results, in the dorsal horn, 5-HT and substance P appear to exert opposing synaptic effects, inhibition and excitation, respectively (122, 240). No data
are available on the postsynaptic actions of the simultaneous administration of two or three of these substances. Neither are any data available regarding
whether the substances are coreleased and, if so, under
what conditions.
(324, 510).
D. Cotransmission
As described in section III, 5-HT and other neurotransmitters are frequently found to coexist in axon terminals along with hormones or neuropeptides. This
raises the interesting questions of whether and how cotransmission differs from more traditional, single neurotransmitter synaptic actions in the CNS.
Medullary neurons that project to the ventral horn
of rats have been shown to contain 5-HT and substance
P, or 5-HT and TRH, or all three substance (243; for
review see Ref. 508). On a gross physiological level, 5HT, substance P, and TRH appear to interact synergistically (for review see Ref. 466). Their effects are additive or even multiplicative in facilitating motor output.
Unfortunately, only limited neurophysiological data are
available regarding the direct postsynaptic actions of
these substances. Nonetheless, they support the gross
behavioral and physiological data. When applied iontophoretically onto motoneurons in the ventral horn of
rats, all three substances augment or facilitate the increased motoneuron excitability produced by glutamate
E. Latency and Duration of Action
An important dimension of the postsynaptic actions of 5-HT is the temporal characteristics of its actions, i.e., latency and duration. This not only describes
the window in which activation or inhibition of serotonergic neurons is likely to influence its target cells but
may also bear on the nature of important physiological
properties of serotonergic neurons, such as nonsynaptic
release and receptor-coupled messenger systems.
Phasic electrical stimulation of serotonergic cell
bodies or axons is one of the only ways of addressing
these issues. It is assumed that this causes an action
potential(s) to invade the axon terminal to release physiological stores of the neurotransmitter. Because electrical stimuli are discrete short-lived events, their sequelae can be timed with precision. In contrast, several
difficulties are inherent with employing iontophoretic
application of 5-HT to answer these questions: inability
of knowing whether the amount released is of the same
order of magnitude as physiological release, the possibility that the amount released may swamp the ability of
the presynaptic neuron for removal by a reuptake mechanism, lack of knowledge regarding the somatodendritic
domain of the target cell where 5-HT is acting, and inability to precisely time events relative to the time of
release.
Almost the entire literature on latency and duration of the postsynaptic actions of 5-HT derives from
studies employing anesthetized rats. Stimulation of the
DRN or MRN consistently produces a relatively longduration (100-400 ms) inhibition of target neurons in
the cerebral cortex, hippocampus, thalamus, septal
area, and amygdala (27,244,257a, 420,427,494). A major
discrepancy arises in the literature regarding latency of
action. Several studies report that the inhibitory effects
have a relatively short latency of lo-20 ms (27,244,494),
whereas most of the other studies cited report a considerably longer latency of ~100 ms. The factor(s) responsible for this difference has not been determined, although differential activation of small- and large-caliber axons remains a possibility.
Keeping in mind the aforementioned caveats regarding the iontophoretic application of neurotransmitter, it is interesting to note that effects on motoneurons
lasting up to two orders of magnitude longer (i.e., tens or
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An interesting corollary of these motoneuron results is the finding that 5-HT also exerts an excitatory
action on neurons in the caudate/putamen of the extrapyramidal motor system. This conclusion is based on
studies in anesthetized rats employing intracellular recordings in conjunction with electrical stimulation of
the DRN (376,482).
It is also important to place these motoric effects of
5-HT in a broader context. First, if animals are given
any of a variety of drugs known to increase synaptic
5-HT, they display a constellation of active motor signs
that has been called the “5-HT syndrome” (231). This is
believed to be a direct reflection of 5-HT-induced motoneuron excitation. Also, recall the relation of serotonergic neuronal activity to oral-buccal activity and their
general relationship to motor tone. The motor effects of
5-HT are, at least in part, mediated by its ability to
move motoneurons from a stable hyperpolarized state,
with little or no neuronal activity, to a stable depolarized “plateau” state, with tonic neuronal activity (22233,
223). All of this is consistent with our theme that a
strong tie exists between serotonergic neuronal activity
and various aspects of motor function.
21.2
BARRY
L. JACOBS
AND
hundreds of seconds) than those found with electrical
stimulation
have been reported
(324, 480, 508a).
Whether such effects, which typically depend on continuous application of 5-HT for tens of seconds, are physiologically relevant (and involve engaging more complex
membrane
and/or
intracellular
mechanisms)
is not
known.
F. Summary
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72
hibitory,
or modulatory)
and temporal characteristics
of these actions are provided by the specific type of 5-HT
receptor subtype affected and the membrane and intracellular biochemical
mechanisms
that are coupled to
them. The long latency and long duration of these synaptic actions are compatible with the modulatory
nature of the brain 5-HT system that is a focus of this
review.4 It is also interesting
to note that a similar conclusion can be reached regarding the temporal aspects
of the synaptic actions of norepinephrine.
The effect on
target neurons of activating
brain noradrenergic
neurons is generally reported to be a long-latency,
longduration
inhibition
of activity
(75, 152). Thus monoamine neurotransmitters
may be fundamentally
different than faster acting excitatory
and inhibitory
amino
acid neurotransmitters,
which act almost exclusively by
means of simple receptor-mediated
ionic conductance
changes (439). The implications
of such an arrangement
are discussed next.
VIII.
DISCUSSION
AND
SPECULATION
Serotonergic
neurons comprise the most expansive
neurochemical
network
in the vertebrate
CNS. These
midline brain stem neurons innervate virtually
all portions of the neuraxis. It is also a system that appears to
be conserved across phylogeny. Developmentally,
they
are among the first neurons to appear, and the fact that
they synthesize 5-HT before making contact with their
target neurons is consistent
with their serving important trophic functions. In turn, they also respond vigorously during development to a variety of growth regulatory factors. Similarly,
when these neurons are damaged in the adult, they demonstrate
a robust sprouting
response that is also responsive
to many of these factors.
This vast anatomy is complemented by a basic physiological role for this system. Functionally,
serotonergic
neurons exert a gain control primarily
through a modulatory action on behavioral and physiological
processes
by interacting
with six or more 5-HT receptor subtypes.
This influence on target neurons is expressed in direct
association with the motor activity and behavioral state
of the organism, i.e., highest during arousal and active
waking, intermediate
during quiet waking, and off during REM sleep. (Serotonergic
neuronal activity is unperturbed by a variety of environmental
and physiological stressors.)
Finally, there is evidence from anatomic studies for
an evolutionary
trend toward greater specificity in the
serotonergic
system. Thus in higher mammals, serotonergic neurons
are more likely to have myelinated
4 A recent
paper
provides
a challenge
to this generalization
(129). In the peripheral
nervous
system
of the guinea pig there is
evidence that the excitatory
effect of 5-HT at a 5-HT, receptor
may be
a “fast”
one (short latency
and short duration)
mediated
by a direct
action at a ligand-gated
cation channel.
This remains
to be demonstrated
for the CNS.
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It is becoming increasingly
clear that neurotransmitters such as 5-HT exert multiple postsynaptic
effects
in the CNS and that these differential
effects are often
directly attributable
to actions at specific receptor subtypes. An area of intense current interest is the identification of the heterogeneous
membrane and intracellular biochemical
mechanisms
coupled to each receptor
subtype, e.g., G proteins that either activate or inhibit
formation
of CAMP or that activate phospholipase
C,
those that act on a specific simple ion channel, and those
that may influence a calcium-activated
potassium
conductance (10, 419). The issue regarding the duration of
the postsynaptic
effects of 5-HT will also certainly
be
shown to be dependent on which of these aforementioned mechanisms
is activated.
Recent evidence in rats also indicates that different
receptor subtypes, 5-HT, and 5-HT,, may be related to
different types of 5-HT axons, thin and thick, deriving
from different nuclei, DRN and MRN, respectively
(for
review see Ref. 81; see sect. III@.
It will be interesting
to
see whether such a relationship
also holds for other species. The different caliber of axon, the specific receptor,
and the type of release site, synaptic or nonsynaptic,
are
the major determinants
of the latency of the postsynaptic action of 5-HT.
One of the limiting factors in this research on 5-HT
is the lack of cross-species
data. For example, whereas
most of the physiological
studies of the postsynaptic
actions of 5-HT have been carried out in rats (typically
under anesthesia),
all of the studies of the activity of
serotonergic
neurons
under physiological
conditions
have been carried out in behaving cats. There are almost
no data from primates on either of these issues.
The finding that different 5-HT receptors in a given
brain region may mediate reciprocal or antagonistic
actions is deserving of further
investigation.
This may
imply that the postsynaptic
actions of 5-HT under normal physiological
conditions
are in a somewhat
balanced state. This concept of dynamic balance may also
apply to control at a more molar level. For example,
there is increasing evidence that 5-HT, by its actions at
two or more receptor sites, may exert opposing effects
on the control of blood pressure (187). It may also explain why hallucinogenic
drugs, acting preferentially
at
a 5-HT, receptor subtype, and thus creating an imbalance, can exert such strong organismic effects (232,234).
In summary, 5-HT generally seems to exert a longlatency and long-duration
action on its postsynaptic
target neurons. Diversitv
in the nature (excitatorv.
in-
EFRAIN
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STRUCTURE
1992
AND
FUNCTION
axons, more discrete projection
domains, and a higher
percentage of synaptic release sites. We speculate that
this anatomic trend may be associated with a similar
degree of physiological
specialization.
In this final section, we elaborate on these major
themes in this review.
A. Activity
and Actions
of Serotonergic
Neurons
BRAIN
SEROTONIN
SYSTEM
213
of activity in association
with the sleep-wake-arousal
cycle, going from total inactivity
during REM sleep to
their highest discharge (3-5 spikes/s) during an aroused
or active waking state. We believe that this reflects a
primary role of serotonergic
neurons in the mammalian
CNS: to send information
to their target neurons restricted to that regarding the level of motor activity/behavioral state of the organism. (Their slow and highly
regular discharge pattern dictates that their activity
can convey little in the way of complex information.)
Then, in accordance with their conjunctive relation with
the particular
target structure,
they may facilitate
or
suppress its activity.
Aghajanian
and colleagues (12,
479) have speculated that serotonergic
neurons may act
to facilitate
motor systems while suppressing
sensory
systems. Thus when an organism is aroused or stressed,
serotonergic
neurons will facilitate
motor output and
inhibit afferent input. Reciprocally,
as the organism becomes drowsy or falls asleep, motor output will be disfacilitated, and afferent sensory input will be disinhibited.
Serotonergic
neurons also modulate the activity of the
autonomic nervous system so that its output is appropriate to the level of motor activity being displayed by
the organism. Several points are worth recalling in this
context. First, the activity of serotonergic
neurons anticipates the end of REM sleep, seemingly preparing the
organism for responding to the external world. Second,
serotonergic
neurons do not become inactive during
REM sleep in brain-lesioned
animals displaying
REM
sleep without
atonia. Finally, a group of serotonergic
neurons in the DRN and MRN of cats are strongly activated during central pattern generator-mediated
oralbuccal movements, such as feeding, lapping, and grooming. These latter results are consistent with a large body
of pharmacological
and physiological
data showing that
5-HT facilitates
a number of vertebrate rhythmic motor
activities (207, 231, 341a, 488a, 492).
One of the major factors determining
how serotonergic neurons influence their target neurons is their
duration of action. If the influence of 5-HT is a modulatory one, then the coincidence of its action with other
afferent inputs is the critical event. If a serotonergic
neuron is discharging
at 2 spikes/s during waking and
its postsynaptic
action lasts for 500 ms, then it will exert
an almost continuous
influence on its target neurons
during this behavioral
state. On the other hand, if it
exerts a “fast” action, lasting for only IO ms, then any
heterosynaptic
afferent inputs may have to be precisely
timed to be coincident with (and thus modulated by) this
serotonergic
effect.
Throughout
this review we described much of the
CNS serotonergic
system as broad and expansive. It
should be clear, however, that this concept should not be
extended to imply that its influence is diffuse and nonselective. This point can best be made, perhaps, by examining the serotonergic
input to the primary visual cortex
(area 17) in monkeys. As discussed in section III, not only
do serotonergic
neurons display a preferential
innervation of particular
laminae in this cortical area, but this
pattern implies a relationship
to specific visual func-
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Activity
of brain serotonergic
neurons is slow and
often highly regular. There is evidence that this characteristic pattern of activity is generated by mechanisms
intrinsic to individual serotonergic
neurons. Some serotonergic neurons have also been described as having
nonclocklike
activity; however, this activity can become
highly regular under specific conditions,
such as their
activation during oral-buccal movements and following
pharmacological
blockade
of autoreceptor-mediated
feedback inhibition.
Thus all serotonergic
neurons appear to have an intrinsic
clocklike activity that can become nonclocklike
when any of a variety of extrinsic
factors act on them.
The slow discharge
of serotonergic
neurons may
also exert a significant
influence on the “state”
of the
postsynaptic
receptors on their target neurons. Leysen
and Pauwels (302) have speculated that this may account for the ease with which many treatments
can
downregulate
5-HT receptors
(decrease the apparent
number of available receptor sites) but few, if any, can
upregulate them. This would imply that any plasticity
manifested
in this system under physiological
conditions is much more likely to be seen when serotonergic
neurons are activated rather than deactivated.
The slow and highly regular neuronal activity of
serotonergic
neurons appears to be necessary
for the
normal function of a number of complex processes. The
ability of transplanted,
isolated fetal serotonergic
neurons to restore the lost adult serotonergic
connection
lends strong support to the concept that these neurons
must convey a basic message to their postsynaptic
target neurons. Several points are especially pertinent.
First,
the fetal serotonergic
neurons can find their
proper target sites and reestablish
lost synaptic contacts. Trophic signals play a dominant role in achieving
the appropriate
innervation
density that provides the
source of 5-HT. Second, the neurons release 5-HT to the
target cells in a physiologically
relevant manner. The
studies showing that raphe neurons have an intrinsic
firing pattern provide the mechanism for release. The
loss of the normal afferents
to serotonergic
neurons
does not appear to be critical, possibly because some of
these afferents may be reestablished
at the transplant
site. Finally, the information
conveyed by serotonergic
neurons is communicated
by a slow and rhythmic
firing
rate. It is not known whether information
about state
will still be supplied by the transplanted
neurons. This
obviously depends on whether these neurons receive signals providing state-dependent
information.
Serotonergic
neurons displav their complete range
OF
214
BARRY
L. JACOBS
AND
C. AZMITIA
Volume
7.2
ergic system should dominate, whereas the serotonergic
system should prevail under conditions where a stimulus is repeatedly presented on a continuing basis. Both
systems are thought to exert their strongest
and most
specific influence when their activity is coordinate with
that in particular
target sites in the CNS.
B. Growth/Plasticity
and Physiology
An implicit assumption
throughout
this review has
been that the growth and plasticity of serotonergic
neurons is linked to their physiology and function. In this
section, we make explicit our ideas of how and why this
synthesis
might occur.
The initial growth and development of serotonergic
neurons in the fetus is under genetic control. Beyond
that, both genetic and epigenetic factors serve to guide
and sculpt serotonergic
neurons until they reach their
adult form. A number of the neurotrophic
factors controlling that growth are described in this review. *4dditionally, we believe that the characteristic
neuronal
activity of serotonergic
neurons contributes
to their expansive axon terminal domain. Recall that an endogenously generated, slow, and highly regular discharge
pattern of neuronal activity is first observed in the late
fetal stage or in the early postnatal period. Thus during
development,
serotonergic
neurons are physiologically
either silent or highly rhythmic
in their activity. This
general lack of episodic or pulsatile activity increases
the likelihood that serotonergic
neurons will be active in
phase with their target neurons. This may contribute to
their ability to establish
connections
with neurons
throughout
the neuraxis. It has further
been proposed
that inactive neurons may make more indiscriminate
connections (95). Thus the final adultlike anatomic pattern may be attained when this slow and regular activity is concordant with the pattern of activity of postsynaptic target neurons. This idea is supported by the developmental studies of Changeux and Danchin (94) and
Constantine-Paton
(log), who showed, both physiologically and morphologically,
that synaptic formation
is
made between neurons that are simultaneously
active
and is eliminated
for neurons
whose
activity
is
asynchronous.
Thus this basic rhythmic
activity stabilizes the extensive
connections
made by the rapidly
growing
and branching
fetal serotonergic
neurons.
Once formed, these morphological
connections
can be
strengthened
by physiological
activity (208). Consistent
with this general line of thought regarding the interaction between anatomy and physiology, we also described
evidence that serotonergic
neurons may grow to the periphery of a particular
CNS target site and then await
some signal before the final infiltration
takes place.
The type of neuronal activity
displayed by these
neurons may also be relevant to their regrowth
following damage to serotonergic
neurons in the adult. The
regenerating
sprouts in the adult brain will most likely
encounter
many neurons displaying
a variety of discharge patterns.
The serotonergic
fibers, by virtue of
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tion. Thus 5-HT more heavily innervates
the magnocellular stream over the parvocellular
stream, which implies that 5-HT may be more importantly
involved in
the processing
of visual information
regarding
movement over that regarding form (485).
Finally, the topic of the role of CNS 5-HT frequently includes a discussion of CNS norepinephrine
as
a parallel or interacting
system. In fact, these two neurotransmitter
systems are often seen as being strongly
interrelated.
Recall our initial discussion of Brodie and
Shore’s (90) hypothesis
that they might serve coordinated functions as the central representation
of the two
branches of the autonomic nervous system.
From an electrophysiological
perspective it is interesting to note that the norepinephrine
neurons of the
locus coeruleus are the only other group of neurons in
the CNS (other than cu-motoneurons)
that display the
same pattern of activity across the sleep-wake-arousal
cycle as do serotonergic
neurons. They are silent in REM
sleep, display a low level of activity during slow-wave
sleep, become moderately
active during waking, and increase their activity still further
during active waking
or arousal (152, 234a). There are, however, several important differences
in the response properties
of serotonergic and noradrenergic
neurons. First, whereas serotonergic
neurons display no habituation
in their response to sensory stimuli, the response of noradrenergic
neurons rapidly habituates
so that they often become
unresponsive
within
a few repeated trials. Second, we
described a wide variety of situations that evoked physiological activation of the organism (e.g., the sympathoadrenal system) but that failed to activate serotonergic
neuronal activity above an undisturbed
active waking
baseline level. However,
when the discharge of noradrenergic neurons in the area of the locus coeruleus was
examined under the same conditions, it invariably
displayed a doubling or tripling of activity above an active
waking baseline (1,339-341). Thus we may characterize
the serotonergic
system as a stable or conservative
system whose activity is tied strongly to the tonic level of
motor activity/sleep-wake-arousal
state of the organism. Beyond that, it is essentially
unperturbed
by challenging or stressful
conditions.
In contrast,
the brain
noradrenergic
system is seen as dynamic and as a system whose full range of activity goes beyond a linear
relationship
to the behavioral
state of the organism.
This system responds strongly
to phasically
arousing
stimuli and to tonic stimuli that represent a real or apparent danger to the organism. Third, a group of serotonergic neurons are strongly
activated during rhythmic vegetative activities,
such as feeding, licking, and
grooming, and are inhibited during orientation.
Reciprocally, locus coeruleus neurons are phasically
activated
during orientation
and either display a decrease or no
change in discharge during the aforementioned
vegetative activities
(27a). Shifts in the predominance
of the
activity of one or the other of these two neurochemical
systems will have profound implications
for their target
neurons. For example, in emergency situations,
such as
hemorrhage
or restraint,
the influence of the noradren-
EFRAIN
January
1992
STRUCTURE
AND
FUNCTION
BRAIN
SEROTONIN
SYSTEM
215
liminary evidence indicates that albino rats reared from
birth for 30 days in either constant light or constant
dark display serotonergic
innervation
of the nucleus suprachiasmaticus
of the hypothalamus
that is significantly different from that of rats reared under standard
light-dark
laboratory
conditions (259a). Animals reared
under these constant conditions have a sparser innervation of the nucleus suprachiasmaticus
of the hypothalamus, and, in addition, those reared in constant light display innervation
that is more restricted
to the ventral
portion of the nucleus. Nearby nonvisual regions of the
preoptic area of the hypothalamus
did not display these
differences.
C. Two Serotonergic
Systems
One of the most interesting
developments
in this
field in recent years has been the emergence of the concept that there are two types of serotonergic
systems.
The strongest
evidence for this comes from anatomic
studies; however, this appears to be paralleled by evidence from physiological
studies.
Anatomically,
there is strong evidence for two
types of nuclear organization
of serotonergic
neurons.
In the rat brain stem these cells are densely packed in
midline nuclei (type I), whereas in higher mammals the
cells are often seen in smaller, less densely packed clusters located off the midline (type II). The morphological
description
of the serotonergic
neurons can also be
grouped in two general types. In rats, the neurons are
fine in caliber, highly branched, largely unmyelinated,
and with large nonsynaptic
terminal
fields (type I),
whereas in higher mammals the axons are thicker, have
fewer branches, are often myelinated,
and have more
restricted
synaptic terminal fields (type II) (see Ref. 83a
for a discussion of the phylogenetic
trend toward less
collateralization
of descending serotonergic
neurons).
Although
this classification
into two types of nuclear
organizations
and two types of neuronal morphology
has not been firmly correlated, it has been proposed that
laterally placed serotonergic
neurons have fewer collaterals (148).
Physiologically,
in behaving animals, the typical
serotonergic
neuron displays a slow and regular activity
that is relatively unperturbable
across a wide variety of
behavioral,
environmental,
and physiological
conditions. On the other hand, we have also seen evidence for
a group of serotonergic
neurons whose activity is significantly altered in association
with specific conditions:
increased during repetitive
oral-buccal
related movements and decreased during orientation.
These anatomic and physiological
data raise the interesting
question of whether
these two data sets are
related, i.e., whether functionally
specific serotonergic
neurons are of the type II class. If they are, then a phylogenetic trend toward type II neurons would have profound implications
for serotonergic
function in the human brain.
Downloaded from http://physrev.physiology.org/ by 10.220.33.6 on May 12, 2017
their continuous rhythmic
activity, would be able to interact more easily with this ongoing activity. Thus their
growth would not be arrested, as would be expected if
they showed episodic activity out of phase with the neurons encountered.
Another
aspect of this issue is related to the evidence that functionally
related nuclei can be innervated
by the same group of serotonergic
neurons and in some
cases by the same serotonergic
neuron (33,229,235,334,
489,498). For example, the MRN innervates
the cingulate cortex, septal nuclei, and hippocampus
while the
DRN innervates
the substantia
nigra, corpus striatum,
amygdala, and nucleus accumbens. Individual
neurons
in the DRN and MRN of rats may project to sensorimotor portions of both cerebral and cerebellar cortex or to
visual portions of both, but they do not seem to project
to two different functional
systems in these structures
(498). Similar examples can be found in the inferior
groups where the NRO innervates
the motor cranial nuclei of X and XII and the ventral horn of the spinal cord
while the NRM innervates
the substantia
gelatinosum
of the spinal cord. As discussed, the connections made
by a synaptically
linked area could be stabilized by the
collaterals
of a single serotonergic
neuron or groups of
serotonergic
neurons. This suggests that when serotonergic neurons are active (e.g., during motor activity or
arousal),
the interactions
of preformed
connections
would become stabilized, but when the neurons are silent (e.g., during sleep), the interconnections
would become labile. Consistent with this line of thought, during
development 5-HT terminals
may be one of the last extrinsic afferents
to complete their innervation
of the
cortex (304). It was proposed that the 5-HT innervation
signals the completion of various neuronal circuits and
is involved in verification
and consolidation
of interneuronal contacts (304). This extends the idea that functionally
interconnected
nuclei are innervated
by the
same group of serotonergic
nuclei (33, 36).
The final point in this section concerns the relationship
between
developmental
growth
factors
and
adult function. The responsiveness
of the sprouting
serotonergic
neurons to a variety of growth signals contrasts with the known physiology
of the adult system.
Attracted
by a number of neuronotrophic
and guidance
factors, the fibers actively send out processes to innervate the CNS. Many of the same factors that produce
substantial
changes in growth rate (e.g., steroids, peptides, and glucose) produce no change in the firing rate
of the neurons in the adult animal. We believe that the
role of these trophic factors is to determine the serotonergic innervation
density during periods of growth.
Thus perturbations
occurring
during normal development may influence the ability of serotonergic
neurons
to reach and innervate
their appropriate
targets and
may have long-lasting
effects on the physiology
of the
system.
In an attempt to explore this issue directly, we recently have shown that manipulating
environmental
conditions
during development
can alter that pattern
and density of serotonergic
terminal innervation.
Pre-
OF
216
BARRY
L. JACOBS
AND
We wish to thank Dr. Patricia Whitaker-Azmitia
for assistance in writing
certain sections of this manuscript
and
Arlene Kronewitter
for preparing
the manuscript.
This work was supported by National Institute of Mental
Health Grant MH-23433 and Air Force Office of Scientific Research Grant 87-301 (to B. L. Jacobs) and by National Science
Foundation
Grant BNS-8812892 (to E. C. Azmitia).
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