Download Brainstem Afferents of the Cholinoceptive Pontine Wave Generation

Sleep Research Online 2(3): 79-82, 1999
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Brainstem Afferents of the Cholinoceptive
Pontine Wave Generation Sites in the Rat
Subimal Datta, Elissa H. Patterson and Donald F. Siwek
Sleep Research Laboratory, Department of Psychiatry, Boston University School of Medicine,
Boston, Massachusetts 02118, USA,
The present study was designed to investigate the distribution of brainstem neurons projecting to the pontine wave (Pwave)-generating sites in the rat. In six rats, biotinylated dextran amine (BDA) was microinjected into the physiologically
identified cholinoceptive P-wave generation site. In all cases, microinjections of BDA in the cholinoceptive P-wave
generating site resulted in retrograde labeling of cell bodies in many parts of the brainstem. The majority of those retrogradely
labeled cells were in the pedunculopontine tegmentum, pontine reticular nucleus oralis, parabrachial nucleus, vestibular
nucleus, and gigantocellular reticular nucleus. The results presented in this study provide anatomical evidence that the
cholinoceptive P-wave generation site in the rat receives anatomical projections from other parts of the brainstem known to
be involved in the REM sleep-generation mechanism.
CURRENT CLAIM: Distribution of brainstem neurons projecting to the P-wave generator in the rat.
A group of neurons in the pontine tegmentum generates a
prominent field potential just prior to the onset of and
throughout REM sleep (Datta and Hobson, 1994, 1995; Datta
et al., 1998). These field potentials have been recorded from
the pons, lateral geniculate body, and occipital cortex. Since, in
the cat, these potentials originate in the pons (P) and propagate
to the lateral geniculate body (G) and occipital cortex (O), they
are called ponto-geniculo-occipital (PGO) waves (Brooks and
Bizzi, 1963; Jeannerod et al., 1965). These field potentials
have also been recorded from many other parts of the brain
which receive excitatory inputs from the P-wave-generation
site (Datta, 1997). The P-wave in the rat is equivalent to the
pontine component of the PGO waves in the cat (Marks et al.,
1980; Sanford et al., 1995; Datta et al., 1998). In the rat, these
field potentials are absent in the lateral geniculate body (LGB)
due to the lack of afferent inputs from P-wave-generating cells
(Stern et al., 1974; Datta et al., 1998). Since these field
potentials are absent in the LGB of the rat, in this report we call
these field potentials P-waves rather than pontine PGO waves.
In addition to REM sleep induction, PGO waves have been
implicated in several other important brain functions such as
sensorimotor integration, learning, memory, cognition,
dreaming, self-organization, development of the visual system,
visual hallucination and startle responses (reviewed in Datta,
1997). In our earlier studies we localized cholinoceptive Pwave generation sites both in the cat and rat (Datta et al., 1992,
1998). More recently we have identified brain regions
receiving efferent projections from the physiologically
identified P-wave generation sites in the rat (Datta et al., 1998).
On the basis of this efferent mapping study, we have produced
anatomical evidence indicating that the P-wave generating
cells are involved in sleep-dependent memory consolidation.
In the present study, we investigated the distribution of
brainstem neurons projecting to the P-wave generating sites in
the rat. Identification of afferent inputs potentially may identify
other brain regions that are capable of modulating P-wave
generating cells and P-wave production. Although PGO-wave
generating sites in the cat have been mapped in previous
studies (Quattrochi et al., 1998), this is the first study to our
knowledge that maps afferents of functionally identified Pwave generating sites in the rat.
METHODS
Experiments were performed on 6 male Sprague-Dawley
rats (Charles River, Wilmington, MA) weighing between
200 and 300 gm. Animals were deeply anesthetized (chloral
hydrate, 400 mg/kg, i.p.) and placed in a stereotaxic
apparatus (Kopf, model 1730). A scalp incision was made,
the skin retracted, and a hole was drilled in the skull
overlying the cholinoceptive P-wave generation site
(stereotaxic, AP: -0.50; L: 1.25; H: 2.0) as identified earlier
(Datta et al., 1998).
A single carbachol microinjection (50 ng in 50 nl saline
solution) was made within the cholinoceptive pontine P-wave
generation zone as described earlier (Datta et al., 1998). These
microinjections were made to positively identify the
cholinoceptive pontine P-wave generation site. Following
positive identification, using the same chemitrode, these
cholinoceptive pontine P-wave generation sites were
microinjected with 50 nl of 4% Biotinylated Dextran Amine in
saline (BDA, Molecular Probes, Inc., Eugene, OR). The scalp
skin incisions were then sutured and the animals were allowed
to recover from anesthesia.
Correspondence: Subimal Datta, Ph.D., Sleep Research Laboratory, Department of Psychiatry, Boston University School of Medicine,
M 934, 715 Albany St., Boston, MA 02118, USA, Tel: 617-638-5863, Fax: 617-638-5862, E-mail: [email protected].
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DATTA ET AL.
from a Vectastain kit (Vectastain, ABC Elite Kit, Vector Labs.,
Inc., Burlingame, CA). On the following day, the sections
were rinsed well in 5 changes of PBS and then reacted in a
DAB reagent kit (DAB-Plus reagent set; Zymed Laboratories,
San Fancisco, CA) for approximately 25 minutes. The
progress of this reaction was monitored under the microscope
to prevent excess non-specific staining. Sections were rinsed
well in several changes of PBS, mounted onto chrom-alum
subbed glass slides and allowed to air dry. Slides were then
sequenced through distilled water for 4 minutes, 70% ethyl
alcohol for 3 minutes, two washes of 95% ethyl alcohol for 3
minutes each, two two-minute washes of 100% ethyl alcohol,
a four-minute Histoclear wash, and a final Histoclear wash.
From the final Histoclear wash, the slides were coverslipped
using Permount. All consecutive sections were processed for
quantitative analysis, except in two cases, in which one in
every four sections was counterstained with cresyl violet to
demonstrate nuclear boundaries.
All sections were examined microscopically under light
field illumination. Using camera lucida tracings of these
sections and with the aid of a rat brain atlas (Paxinos and
Watson, 1986), brainstem nuclear divisions were identified.
Cells retrogradely labeled with BDA are identified by the dark
brown color in the surroundings of pale unstained cells (see
Fig. 1). These boundaries and the position of labeled cells and
injection sites were plotted on the drawings. Following
microscopic examination, retrogradely labelled cell counts
were obtained from the plots for well-known groups of
neurons in the brainstem. Counts from the two sides of the
brain were combined. Combined mean and standard
deviations were calculated from the six individual cases.
RESULTS
Figure 1. Digitized brightfield photomicrographs (Optimas, Bioscane)
showing examples of neurons that are retrogradely labeled after
injection of BDA into the cholinoceptive P-wave generation site. (A)
Labeled neurons in the laterodorsal tegmentum. (B) Labeled neurons in
the vestibular nucleus. (C) Labeled neurons in the gigantocellular
reticular nucleus. Scale bar = 100 micrometer.
After ten days, rats were deeply re-anesthetized with
chloral hydrate, and their brains were fixed by transcardial
perfusion of approximately 60 ml 0.9% saline containing 1%
sodium nitrite followed by 200 ml 4% paraformaldehyde and
0.25% gluteraldehyde in 0.1M phosphate buffer (pH of 7.4).
The brains were then removed and cut in serial sections of 50
micrometer thickness with a vibratome. These sections were
treated to reveal the presence of BDA as follows. Sections
were first rinsed in three changes of phosphate buffered saline
(PBS), followed by immersion in 0.3% hydrogen peroxide for
15 minutes to remove endogenous peroxidases and to lyse red
blood cells. Sections were then carried through four rinses in
PBS or until small bubbles were no longer present. Sections
were then incubated overnight at 4°C in a solution of 10 ml
Triton-X, 20 ml PBS, and 8 drops each of solution A and B
All injections were placed within the stereotaxic
coordinates of antero-posterior -0.30 to -0.80, lateral 1.1 to
1.4, and dorsoventral 1.9 to 2.3 (Paxinos and Watson, 1986).
Histological identification showed that microinjections were
made in the dorsal part of the nucleus subcoeruleus. The
surrounding anatomical landmarks of this cholinoceptive
pontine generator are: locus coeruleus, dorsally, the caudal
pontine reticular nucleus, ventrally, and the dorsomedial
tegmental area and ventral nucleus subcoeruleus, medially,
and the mesencephalic trigeminal and motor trigeminal
nucleus, laterally. In all cases, small volume injections (50 nl)
of BDA in the cholinoceptive PGO generating sites resulted in
retrograde labeling of cells in many regions in the brainstem
(see Fig. 2). A slightly greater number of labeled cells was
located ipsilateral to the injection sites compared to the
contralateral side, except for midline structures. The location
and extent of BDA injection site diffusion was determined
histologically by identifying the tip of the track left by the
injection cannula and assessing the spread of the dark brown
BDA reaction product. The area of BDA diffusion at the
injection sites ranged from 50 to 100 micrometer in diameter.
The total number of BDA-labeled brainstem cells
projecting to the functionally identified P-wave generation
sites ranged between 889 and 941, with a mean of 917±24.32
(S.D.). A high number of retrogradely labeled cells were in the
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PONTINE WAVE AND AFFERENT CONNECTIONS
A.
B.
Figure 2. Distribution of labeled neurons in the brainstem. Schematic representation of selected coronal brainstem sections illustrating the distribution of labeled neurons
(each dot represents one BDA labeled neuron) after a small injection of BDA into the cholinoceptive P-wave generation site. The number in the upper left corner of
each section indicates the rostrocaudal distance from interaural line. (2A) Rostral brainstem and (2B) caudal brainstem. Abbreviations: 3, oculomotor nucleus; 7, facial
nucleus; 7n, facial nerve or its root; 8vn, vestibular root vestibulocochlear nucleus; Aq, aqueduct; BIC, nucleus brachium inferior colliculus; CnF, cuneiform nucleus;
CP, cerebral peduncle, basal; ctg, central tegmental tract; DC, dorsal cochlear nucleus; DR, dorsal raphe; Gi, gigantocellular reticular nucleus; IC, inferior colliculus;
icp, inferior cerebellar peduncle; IO, inferior olive nucleus; IP, interpeduncular nucleus; LC, locus coeruleus; LDT, laterodorsal tegmental nucleus; LL, lateral
lemniscus; LPGi, lateral paragigantocellular nucleus; LVe, lateral vestibular nucleus; M5, motor trigeminal nucleus; mcp, middle cerebellar peduncle; ml, medial
lemniscus; mlf, medial longitudinal fasciculus; MR, median raphe nuclei; MVe, medial vestibular nucleus; PB, parabrachial area; pd, predorsal bundle; PDT,
posterodorsal tegmental nucleus; Pn, pontine nuclei; PnC, pontine reticular nucleus, caudal; PnO, pontine reticular nucleus, oral; PPT, pedunculopontine tegmental
nucleus; Pr5, priciple sensory trigeminal nucleus; PrH, prepositus hypoglossal nucleus; Py, pyramidal tract; Rm, raphe magnus nucleus; ROb, raphe obscurus nucleus;
RTP, reticulotegmental nucleus pons; RVL, rostroventrolateral reticular nucleus; S5, sensory root trigeminal nerve; SC, superior colliculus; scp, superior cerebellar
peduncle; SO, superior olive nucleus; Sol, nucleus solitary tract; Sp5, spinal trigeminal tract; Sp5n, spinal trigeminal nucleus; SpVe, spinal vestibular nucleus; SubC,
subcoeruleus nucleus; SuVe, superior vestibular nucleus; VC, ventral cochlear nucleus; VTg, ventral tegmental nucleus; Xscp, decussation superior cerebellar peduncle.
pedunculopontine tegmentum (PPT, 117.83±7.47), pontine
reticular nucleus oralis (PnO, 115±11.14), parabrachial area
(PB, 98.83±9.74), vestibular nucleus (Ve, 120.83±8.38), and
gigantocellular reticular nucleus (Gi, 108.83±14.48). Within
the vestibular nucleus, the majority of those retrogradely
labeled cells were in the superior part (SuVe, 53.78%) and the
medial part (MVe, 34.45%). Only a small percentage of
retrogradely labeled cells were in the lateral (LVe, 7.57%) and
spinal part (SpVe, 4.20%) of the vestibular nucleus. A
moderate number of retrogradely labeled cells were in the
central gray (CG, 39.67±6.86), raphe group nucleus (RN,
40±6.75), laterodorsal tegmentum (LDT, 37.83±12.22), locus
coeruleus (LC, 73.33±5.72), subcoeruleus nucleus (SubC,
78.83±9.26), and parvocellular reticular nucleus alpha (PRtA,
39.83±7.08). Of those retrogradely labeled cells in the raphe
group, almost half of them were in the dorsal raphe nucleus
(DR, 51.16%), and the other half were distributed in the
median raphe nucleus (MR, 23.26%), and raphe magnus
nucleus (RM, 25.58%). There were no labeled cells in the
raphe obscurus nucleus. Some labeled cells were in the central
(3.67±2.50) and dorsal (11.50±4.09) tegmental area (ctg and
dtg) and in the cuneiform nucleus (CnF, 13±3.74). Even fewer
labeled cells were found in the pontine reticular nucleus
caudalis (PnC, 5.33±3.33) and facial nucleus (7, 6.67±3.98).
DISCUSSION
The results presented in this study provide anatomical
evidence that the cholinoceptive P-wave generation site in the
rat receives anatomical projections from other parts of the
brainstem known to be involved in the REM sleep-generation
mechanism. We chose to use local microinjection of carbachol
to identify P-wave generation sites, because in the past this
approach was successfully used to map P-wave generation
sites in the cat and rat (Datta et al., 1992, 1998). In this study,
BDA was used as the retrograde tracer because it has been
shown to be one of the most effective retrograde tracers to
study anatomical pathways (Rajkumar et al., 1993).
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DATTA ET AL.
The present study showed that the P-wave generation site
receives projections from the PPT, LDT, and CnF.
Immunohistochemical studies have shown that the majority of
cells in the PPT, LDT, and CnF of rats are cholinergic,
suggesting that the P-wave generation site may receive
cholinergic inputs (Mesulam et al., 1983; Rye et al., 1987).
Besides cholinergic inputs, the P-wave generation site also
receives anatomical projections from LC, a nucleus which
contains noradrenergic cells and RN, a nucleus which contains
serotonergic cells (Swanson, 1976; Grzanna and Molliver,
1980; Steinbusch and Nieuwehuys, 1983). Inputs from both
cholinergic and aminergic cells to the P-wave generation site
are significant, because they provide anatomical evidence for a
recent model of REM sleep generation which proposes that the
activation of brainstem cholinergic cells and inactivation of
aminergic cells activates individual REM sleep sign generators
(Datta, 1995). In the present study, we have shown that many
cells in the vestibular nucleus project to the P-wave generation
site. Earlier lesion studies have demonstrated that the
vestibular nucleus is involved in the generation of clustered
PGO waves (Morrison and Pompeiano, 1966). Vestibular
nucleus cells projecting to the P-wave generation site provide
anatomical evidence of the involvement of the vestibular
system in the generation of clustered PGO waves (Morrison
and Pompeiano, 1966). This study has demonstrated that cells
in the PnO and PB project to the P-wave generation site. It is
known that the REM sleep events like hippocampal theta
waves and autonomic fluctuations are generated by PnO and
PB cells (Vertes et al., 1993; Datta, 1995, 1997). In this study,
we have also demonstrated that the P-wave generation site
receives projections from Gi cells. In the past, other studies
have shown that the activation of Gi induces REM sleep sign
EMG atonia (Sakai et al., 1981; Chase et al., 1986; Lai and
Siegel, 1988, 1992). These data together indicate that the
individual REM sleep sign generators are interconnected.
Understanding these interconnections may help to understand
the coordinated actions that lead to the state of REM sleep.
ACKNOWLEDGMENTS
This research was supported in part by National Institutes of
Health Grant #NS34004.
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