Download 521 THE CHOLINERGIC LIMBIC SYSTEM: PROJECTIONS TO

521
BY
P. R. LEWIS AND C. C. D. SHUTE
(From the Anatomy School, University of Cambridge)
INTRODUCTION
EVIDENCE has accumulated for a close functional relationship between
the brain-stem reticular formation and the hippocampal formation (hippocampus and dentate gyrus) of the fore-brain. On the one hand, it has been
shown that stimulation of the mid-brain reticular formation not only causes
desynchronization of the neocortical EEG, inhibiting slow rhythms and
replacing them with high frequency low amplitude activity, but also
produces a synchronized hippocampal EEG, with slow high amplitude
waves (e rhythm: Jung and Kommuller, 1938; Green and Arduini, 1954),
on which fast activity may be superimposed. The significance, however, of
these different patterns of electrical activity is not understood. It has also
been suggested that the hippocampus may itself modify the activity of the
reticular formation and through it modulate the activity of the cerebral
cortex, but whether in such a way as to produce facilitation or suppression
is not clear (Green, 1960). Apart from the fact that reticular influences
probably reach the hippocampus via the septum, the mechanism by which
the reticular formation and the hippocampus are able to interact is quite
unknown.
Our studies on the distribution of acetylcholinesterase (AChE) in rat
brain have shown: (1) that ascending reticular pathways from the brainstem contain AChE along their length and are probably cholinergic, and
(2) that AChE-containing neurones, proven to be cholinergic, project on to
the hippocampal formation from the septum (for references, see preceding
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BRAIN—VOL. XC
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THE CHOLINERGIC LIMBIC SYSTEM:
PROJECTIONS TO HIPPOCAMPAL FORMATION, MEDIAL
CORTEX, NUCLEI OF THE ASCENDING CHOLINERGIC
RETICULAR SYSTEM, AND THE SUBFORNICAL ORGAN
AND SUPRA-OPTIC CREST.
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P. R. LEWIS AND C. C. D. SHUTE
paper Shute and Lewis, 1967). We have also been struck by the fact that a
number of regions in the mid-brain and diencephalon which are known to
receive projections from the hippocampus are rich in AChE, and so may be
cholinergic centres. In this paper we describe in detail the routes taken by
the cholinergic hippocampal afferent fibres, the locations of AChE-containing nuclei receiving hippocampal efferents, the connexions made by
some of these nuclei with the AChE-containing ascending reticular system,
and the projections of others on to the areas of medial cortex which
constitute the so-called limbic lobe.
MATERIALS AND METHODS
OBSERVATIONS
(i) The septal radiation to the hippocampalformation
Hippocampal afferents other than those arising from the septal region,
e.g. those derived from the entorhinal cortex (temporo-ammonic fibres),
from the dentate gyms terminating on the hippocampus proper (mossy
fibres), and from the hippocampus of the opposite side (commissural
fibres) contain no cholinesterase and may be presumed to be non-cholinergjc. The fibres which arise from the medial septal nucleus and the nucleus
of the diagonal band, on the other hand, have been shown to be cholinergic
(Lewis, Shute and Silver, 1964), and supply the hippocampus proper, the
dentate gyms and the transitional region known as the subiculum. These
fibres, constituting what we have termed the septal radiation (Shute and
Lewis, 19636, c), travel to the hippocampal formation via the medial
supracallosal stria of Lancisi, the dorsal fornix, the alveus and the fimbria
(fig. A). In the fimbria the cholinergic fibres are concentrated on its outer
side. Afferent hippocampal-fibres also travel in the inner part of the
fimbria, but these are mainly commissural fibres.
Some details of the regional distribution of the septal radiation can be
made out from normal material. The supra-callosal stria is presumably
afferent to the dorsal hippocampal rudiment, which lies at the base of the
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Stereotaxic lesions were placed so as to interrupt the cholinergic afferent hippocampal pathways in anesthetized rats, which were then allowed to survive long enough
to produce accumulation of AChE on the cell body side and loss of AChE on the
opposite side of the cut fibres. The rationale of this method has already been discussed
(Shute and Lewis, 1967). AChE was rendered visible in serial frozen sections by a
modification ofthethiochoUne method, with 10-*M ethopropazine added as an inhibitor
of non-specific cholinesterase (ChE). Since a number of nuclei receiving hippocampal
projections were found to contain ChE as well as AChE, in many series alternate
sections were incubated with butyrylthiocholine instead of acetylthiocholine as
substrate. As in our previous study, a propionylthiocholine substrate was occasionally
used, combined either with 10~*M ethopropazine to show AChE or with 5 x 10—*M
62 C 47e (an AChE inhibitor) to show ChE. Suppression of AChE staining made
the connexions of ChE-containing nuclei much easier to detect.
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CHOLINERGIC LIMBIC SYSTEM
SEPTAL RADIATION
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Fio. A.—Expanded diagram (natural spacing of transverse sections increased five
times) showing the septal radiation of cholinergic fibres arising from the medial septal
nucleus (MS) and the nucleus of the diagonal band (DB), and supplying the subfomical
organ (SFO), cingulate cortex via the supracallosal stria (SS), dorsal hippocampus (DH)
via the dorsal fornis (DF) and alveus (AL), and ventral hippocampus (VH) via the
fimbria (FI). The fasciculus retroflexus, with cholinesterase-containing fibres running
from the habenular nuclei (H) to the interpeduncular nucleus (IP), is also included.
cingulate cortex immediately above the corpus callosum. Some fibres of the
stria turn dorsally in layer i of the cingulate cortex to innervate its superior
part. Otherfibre'sreach the subiculum by looping down behind the splenium
of the corpus callosum. The dorsal fornix innervates the medial part of the
dorsal hippocampal formation from its anterior end. Using the nomenclature of Lorente de N6 (1933, 1934), the more medial fibres of the dorsal
fornix supply hippocampal area CAi and the adjacent subiculum, also the
dentate gyms, especially at the apex of its curvature, and the anterior tip of
hippocampal area CA4, which lies in the hilum of the dentate gyms. The
more lateral fibres of the dorsal fornix diverge in a lateral direction to
supply hippocampal area CAa. The alveus and upper part of the fimbria
supply especially area CAa, and also areas CA2 and CA« of the dorsal
hippocampus, and the hilum of the dentate gyrus adjacent to CA4. The
bulk of the fimbria innervates the ventral hippocampal formation. "The
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N. DIAGONAL BAND
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P. R. LEWIS AND C. C. D. SHUTE
regional innervation of the dorsal hippocampus and dentate gyms are
illustrated in fig. B.
The cholinergic hippocampal afferents break up into a preterminal,
neuropil Which contains most of the AChE of the hippocampal formation,
(Shute and Lewis, 1966). The exizyme is located in definite layers (Shute
and Lewis, 1961). In the stratum oriens of the hippocampus proper, AChE
is aggregated at the base of the pyramidal cell layer, i.e. in the region of the
basal pyramidal dendrites, and extends between the bodies of the pyramidal
cells. The staining is far heavier in area CA3 than in other hippocampal
areas. Enzyme is also found in the stratum radiatum in the region of the
mossy fibres, i.e. in association with the bases of the apical pyramidal
dendrites, and in the stratum lacunosum, which is formed by horizontal
branches of the apical dendrites. In the dentate gyrus, AChE is found on
either side of the granular layer, and the staining is heavier on the side
remote from the hilum. Little enzyme can be seen between the granular
cells, which are very tightly packed. In addition to the staining due to
neuropil, AChE is found in the cell bodies of Golgi type II neurones
scattered in the stratum oriens, and more densely aggregated in the hilum
of the dentate gyms.
Regional differences in the intensity of staining for AChE in different
parts of the hippocampal formation seem to be determined by the mode of
approach of the cholinergic fibres. Thus, the heavy staining in area CA 3
deep to the pyramidal cells lies opposite the main inflow from the alveus
and fimbria, while that of the anterior ends of the subiculum, area CAX and
the apex of the dentate above the granular cells, is close to the site of entry
of fibres from the dorsal fornix (fig. B). In regions of the hippocampal
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Fio. B.—Transverse section through the dorsal hippocampal formation showing the
route taken by cholinergic afferents from the dorsal fornix (DF), alveus (AL) and
flmbria (FI) to the hilum (HI) of the dentate gyrus and to hippocampal areas CAU CA,
CA, and CA,. Cholinergic fibres in the ventral part of the fimbria supply the ventral
hippocampus (VH).
CHOLINERGIC L1MBIC SYSTEM
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formation more remote from the main inflow, the cholinergic neuropil is
more dispersed.
The direction of fibre travel in the septal radiation was confirmed by
lesions involving its various components. In an experiment with a survival
period of four days, AChE accumulated rostral to the lesion in fibres of the
medial supracallosal stria and the dorsal fornix. Loss of staining occurred
caudal to the lesion in the supracallosal stria and dorsal fornix, and in the
dorsal hippocampal formation (area CAX and dentate gyrus). In another
four-day experiment, enzyme accumulated in the lateral fibres of the dorsal
fornix supplying hippocampal area CA,.
Lesions of the alveus and fimbria were produced in animals which were
allowed to survive for 4, 5, 6, 7, 25 and 30 days. In the short term experiments accumulation of AChE occurred rostral to the lesion (PI. LXVIII,fig.
1) and in the six-day animal could be traced as far forwards as the medial
septal nucleus. Some residual excess of enzyme was still present in the
twenty-five-day animal. A comparison of the normal and operated sides
in the five-day experiment showed that not all AChE-containing fibres in
the fimbria were accumulating enzyme as a result of being divided.
Approximately one quarter of the total number of cholinergic fibres
visible by light microscopy appeared to respond in this way.
In all the animals subjected to interruption of the alveus and fimbria,
AChE was found to have disappeared from fibres caudal to the lesion, and
especially from the layers of terminal neuropil in the hippocampal formation.
The AChE-containing Golgi cells of the hippocampal stratum oriens and
those of the hilum of the dentate gyrus were unaffected (PL LXVm, fig. 2).
In cases where the fimbria alone was involved, as in the five-day experiment,
the loss of hippocampal staining was confined to the ventral hippocampal
formation below the flexure (PL LXV1TI, fig. 4, VH). In the seven and
twenty-five-day experiments much of the alveus (especially that part
adjacent to area CA3) was involved as well as fimbria, and the extent of
enzyme loss included the posterior end of the dorsal hippocampal formation, immediately above the flexure. These animals also showed loss of
staining in the posterior end of the medial amygdaloid nucleus, which
becomes continuous with the ventral hippocampus.
In the animals with fimbrial lesions which survived for twenty-five and
thirty days, adjacent sections were stained by the thiochohne technique and
by a modified Bielschowsky silver method. In each case an attempt was
made to compare on the operated side, in sections in front of the lesion,
the number of AChE-containing fibres in the fimbria with the number of
fimbrial fibres which still stained with silver. Accurate counting was only
possible along the lateral margin of the fimbria. Here the number of fibres
present in thiocholine and silver stained sections were found to be approximately equal, suggesting that, in this part of the fimbria, the afferent
hippocampal fibres are predominantly cholinergic.
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P. R. LEWIS AND C. C. D. SHUTE
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(2) Presumed cholinergic nuclei innervated by the hippocampal fornix
projection system.
The main projection pathway from the hippocampus, which arises from
the pyramidal cells, travels in the fornix and ends mainly on the medial
mammillary nuclei, does not contain AChE. The same is true of the
mammillo-thalamic and mammiUo-tegmental tracts, which arise from the
medial mammillary nuclei, and project respectively to the anterior
thalamic nuclei and to the dorsal and deep tegmental nuclei, forming
second neurones on the efferent pathway. One may conclude, therefore,
that the initial outflow from the hippocampus is non-cholinergic. The
non-cholinergic neurones, however, impinge upon a number of nuclei
whose cells are rich in cholinesterase (often containing ChE as well as
AChE), and give rise to cholinesterase-containing fibres. These nuclei,
which are presumably cholinergic, either project directly to medial cortical
areas or to the olfactory bulb, or connect with relays on the ascending
cholinergic reticular system. Other AQiE-containing neurones which are
probably innervated by hippocampal efferents supply the subfornical
organ.
(a) Nuclei supplying medial cortex.—The anterior thalamic nuclei are
supplied directly by fornix fibres as well as indirectly through the
mammillothalamic tract (Guillery, 1966; Nauta, 1956). The anteroventral nucleus is rich in both AChE and ChE, which is located in
the cells and extracellularly in terminal neuropil; in the antero-medial
nucleus, on the other hand, the enzymes are entirely extracellular in neuropil (PI. LXEX, fig. 6). The cells of the antero-dorsal nucleus are unusual in
that they contain far more ChE than AChE, and there is less extracellular
enzyme than in the other anterior thalamic nuclei. We have found a
similar preponderance of ChE in the cells of the dorsal motor nucleus of the
vagus (Navaratnam, Lewis and Shute, 1964). The anterior thalamic nuclei
project on to the cingulate cortex (Rose and Woolsey, 1948). Fibres from
the antero-medial nucleus travel farthest anteriorly to the anterior limbic
area and area infralimbica, respectively equivalent to Krieg's (1946) areas
32 and 25 of the rat cortex. Those from the antero-ventral nucleus supply
the cingular area corresponding to Area 23 (not Area 24 as stated by Krieg),
and those from the antero-dorsal nucleus travel farthest posteriorly to the
retro-splenial area which is equialent to Krieg's cortical Area 29b. None
of the projections from the anterior thalamic nuclei on to medial cortex
could be detected in normal material prepared by the thiocholine technique, but it was noticeable that in the retrosplenial area, supplied by the
antero-dorsal nucleus, prominent bands of ChE were present in cortical
layers i and iii. We were able to prove that these bands of enzyme were
produced by terminals of neurones located in the antero-dorsal nucleus,
since the normal staining was absent on the operated side (PI. LXIX, fig.
11, CC) twenty-nine days after a lesion which totally destroyed this nucleus
CHOLINERGIC LIMBIC SYSTEM
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Another nucleus closely related to hippocampal projection fibres in the
fornix is the nucleus of the anterior commissure, the cells of which likewise
contain ChE as well as AChE, although the ChE is present in lesser
amounts than in those of the interstitial nucleus of the ventral hippocampal
commissure. The cholinesterase-containing cells of this nucleus are
located, in the rat, mainly on the dorsal aspect of the anterior commissure,
immediately lateral to the descending columns of the fornix. They give
rise to fibres which project forwards on to the lateral part of the nucleus
accumbens, reaching it by looping over the anterior limb of the anterior
commissure. Their terminals contribute to the accumbens neuropil, which
is derived mainly from AChE-containing cells of the lateral preoptic area
and of the olfactory tubercle. The accumbens nucleus also receives direct
projections from the hippocampus via the precommissural fornix (Sprague
and Meyer, 1950; Carman, Cowan and Powell, 1963). The medial portion
of the accumbens nucleus, which appears to be an extension of the deep
or polymorph layer of the olfactory tubercle, contains cells which are also
rich in ChE as well as AChE (PL LXVIII, fig. 5, A). Their axons appear to
project to olfactory areas.
The main cholinesterase-containing, presumed cholinergic, links
between the hippocampal projection system and medial cortex are listed
in Table I and illustrated in fig. C.
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(PL LXTX, fig. 6. AD) without involving the other anterior thalamic nuclei.
There is, therefore, a correspondence between the type of enzyme present
in the nerve terminals and the enzyme contained in the cell body. A similar
relationship is found in the vagus, where the terminals in the cardiac
ganglia also contain ChE (Navaratnam, Lewis and Shute, 1964).
The interstitial nucleus of the ventral hippocampal commissure is also
rich in ChE as well as AChE (PL LXVIII, fig. 3, C). The connexions of this
large nucleus have not been previously described. From its position, it is
probable that afferents, derived from the hippocampus, reach it via the
fornix bundles. In material stained specifically for ChE, the axons derived
from the interstitial nucleus form a prominent discrete bundle which can
be followed forwards through the dorsal part of the septum below the corpus
callosum (PL LXVm, fig. 5). Immediately in front of the genu of the corpus
callosum, this bundle joins a group of AChE-containing precallosal cells
which form a dorsal extension of the AChE-containing cells of the olfactory
tubercle (fig. C). From this point, fibres derived from the precallosal cells
continue forwards on the medial side of the frontal lobe, and are distributed
to the area infralimbica and anterior limbic area (i.e. to cortex supplied by
the non-cholinergic anrero-medial thalamic neurones). The ChE-containing bundle derived from the interstitial nucleus of the ventral hippocampal commissure can be traced on forwards below the rhinal fissure into
the olfactory bulb.
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P. R. LEWIS AND C. C. D. SHUTE
TABLE I.—ANALYSIS OF THE CHOLINERGIC LIMBIC SYSTEM
Group 1 neurones: afferent to the hippocampal formation
nucleus of diagonal band T
f dentate gyrus
medial septal nucleus
dorsal and ventral
hippocampus
J
Group 2a neurones: afferent to medial cortex
retrosplenial area
antero-ventral thalamic nucleus
cingular area
interstitial nucleus of ventral
hippocampal commissure
anterior limbic area and
area infralimbica
precallosal cells
olfactory bulb
nucleus accumbens
olfactory tubercle
Group 2b neurones: afferent to the cerebellum
latero-dorsal tegmental nucleus
(1.d.t.n.) —>- brachium conjunctivim
dorsal tegmental nucleus
(d.t.n.)
nucleus reticularis tegmenti pontis
(n.r.t.p.) —*• brachium pontis
Group 1c neurones: afferent to the ascending cholinergic reticular system
(1) to the dorsal tegmental pathway:
d.t.n. —*• 1.d.t.n. —*• nucleus cuneiformis
(2) to the ventral tegmental pathway:
d.t.n. and deep tegmental nucleus
dorsal and median raphe nuclei
interpeduncular nucleus
habenular nuclei
d.t.n. —*• l.d.t.n.
—*• nucleus cuneiformis
d.t.n. —*• n.r.t.p.
-•-
—»• substantia nigra
ventral tegmental area
Group Id neurones: afferent to non-neural structures
cells in dorsal fornix "I
f subfornical organ
cells in septal raphe J
(_ supra-optic crest
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antero-dorsal thalamic nucleus
CHOLINERGIC LIMBIC SYSTEM
529
FIG. C.—Diagram showing cholinesterase-containing nuclei of the mid-brain and
fore-brain (indicated by stipple) connected with the hippocampus, their projections to
the medial cortex, and their connexions with the ascending cholinergic retiailar system.
Abbreviations: A, nucleus accumbens; ATH, antero-ventral and antero-dorsal thalamic
nuclei; BC, brachium conjunctivum; BP, brachium pontis; C, interstitial nucleus of
the ventral hippocampal commissure; CBL, cerebellum; CC, cingulate cortex (cingular
and retrosplenial areas); CU, nucleus cuneiformis; DB, diagonal band; DE, deep
tegmental nucleus (ventral tegmental nucleus of Gudden); DO, dorsal tegmental
nucleus; F, fornix; FC, frontal cortex (area infralimbica and anterior limbic area);
FR, fasciculus retroflexus (habenulo-interpeduncular tract); H, habenular nuclei;
HF, hippocampal formation; IP, interpeduncular nucleus; LD, laterodorsal tegmental
nucleus; LP, lateral preoptic area; M, mammillary body; MS, medial septal nucleus;
MT, mammillo-tegmental tract; MTH, mammillo-thalamic tract; OB, olfactory bulb;
OT, olfactory tubercle; PC, precallosal cells; R, dorsal and median nuclei of raphe
(nucleus central is superior); SFO, subfornical organ; SH, stria habenularis; SR, septal
radiation; TP, nucleus reticularis tegmenti pontis (of Bechterew); VT, ventral tegmental
area.
(b) Nuclei connecting with the cerebellum.—The dorsal tegmental and
the deep tegmental nucleus (nucleus of Gudden) are both innervated by
mammiJlo-tegmental fibres and so come under the influence of the hippocampal-fornix projection system. The cells of both nuclei are rich in ChE
as well as AChE (PI. LXTX, fig. 7, DO, DE). Cholinesterase-containing
fibres emanating from the dorsal tegmental nucleus can be seen in normal
material to project to the latero-dorsal tegmental nucleus and to the nucleus
reticularis tegmenti pontis (fig. C). The two last-named nuclei both send
cholinesterase-containing fibres to the cerebellum, the latero-dorsal
tegmental nucleus via the brachium conjunctivum and the nucleus reticu-
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-ToOT
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P. R. LEWIS AND C. C. D. SHUTE
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laris tegmenti pontis via the brachium pontis (Shute and Lewis, 1965).
These connexions, which are presumably cholinergic, between the hippocampal-tegmental projection and the cerebellum are illustrated in fig. C.
(c) Nuclei connecting with the ascending cholinergic reticular system.—
The latero-dorsal tegmental nucleus sends cholinesterase-containing fibres
not only to the cerebellum but also to the nucleus cuneiformis (fig. C). In
this way the hippocampal-tegmental projection is linked with the dorsal
tegmental pathway of the ascending cholinergic reticular system (Shute and
Lewis, 19636, 1967). Furthermore, a continuous chain of cholinesterasecontaining neurones can be traced from the dorsal and deep tegmental
nuclei via the dorsal and median nuclei of the raphe, which also contain
AChE and ChE, to the interpeduncular nucleus. This nucleus contains
large amounts of AChE, particularly around its periphery (PI. LXIX, fig.
8). It also contains more choline acetylase than has been recorded so far
in any other region of the mammalian brain (Lewis, Shute and
Silver, 1967). The AChE of the nucleus interpeduncularis is located
in nerve cells as well as in terminals. The nucleus contains a little
ChE, mainly at the posterior end, at the site of entry of the fibres derived
from the dorsal and deep tegmental nuclei. The AChE-containing neurones
of the interpeduncular nucleus appear to connect with those of the ventral
tegmantal area (fig. Q . Our findings here conflict with the report that the
dorsal and deep tegmental nuclei project mainly to the mammillary nuclei
via the mammillary peduncle (Cowan, Guillery and Powell, 1964). Unlike
the axons of the dorsal and deep tegmental neurones, the fibres which make
up the mammillary peduncle do not contain cholinesterase, and so may be
regarded as non-cholinergic. We conclude that the projections of the
dorsal and deep tegmental nuclei on to the interpeduncular nucleus, and
through it on to the AChE-containing neurones of the ventral tegmental
area, provide a cholinergic link between the hippocampal fornix projection
system and the ventral tegmental pathway.
The anterior end of the interpeduncular nucleus is supplied by fibres of
the fasciculus retroflexus or habenulo-interpeduncular tract containing
ChE as well as AChE. In its dorsal part these fibres form only the central
core of the fasciculus retroflexus. More ventrally, i.e. immediately above
its entry into the interpeduncular nucleus, they constitute the whole of the
bundle. The non-cholinergic fibres of the dorsal part are presumably those
which leave the fasciculus retroflexus and project to the mid-brain tegmental
nuclei (Nauta, 1958). The direction of the cholinesterase-containing fibres
in the fasciculus retroflexus was investigated experimentally. Destruction
of the lateral habenular nucleus in an animal surviving for six days (PI.
LXIX, fig. 10 H) produced diminished fibre staining in the fasciculus on the
operated side (PI. LXIX, fig. 12, FR). The persisting fibres could be seen to
emerge from the intact medial habenular nucleus. The fasciculus retroflexus
itself was damaged in animals surviving for 3, 4, 7 and 10 days. In all
CHOLINERGIC LIMBIC SYSTEM
531
The main chohnesterase-containing links between the hippocampal
projection system and the nuclei associated with the dorsal and ventral
tegmental pathways of the ascending cholinergic reticular system are listed
in Table I and illustrated in fig. C.
(d) Innervation of the subfomical organ and supraoptic crest.—The
subfornical organ or intercolumnar tubercle is a cellular structure, highly
vascularized, which lies beneath the ventral hippocampal commissure.
AChE is present in large amounts, particularly around the periphery of the
organ (PI. LXIX, fig. 9). In normal material prepared by the thiocholine
technique the subfornical organ can be seen to be innervated by AChEcontaining cells, some of which are situated in the dorsal fornix above
the hippocampal commissure, while others he close to the mid-line raphe
in the upper part of the septum. The axons of these cells reach the subfornical organ by passing through and on either side of the hippocampal
commissure (Shute and Lewis, 1963a). Other AChE-containing cells in
the ventral part of the septum supply the histologically similar organ
known as the supraoptic crest, which lies immediately rostral to the optic
chiasma. The axons supplying the supraoptic crest travel on either side of
the anterior commissure (fig. D).
The subfornical organ was destroyed in an animal which was then
allowed to survive for six days. Accumulation of AChE was produced in
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cases, enzyme accumulated dorsal to the lesion, and staining was diminished
ventral to it. In the 7-day experiment the lesion was placed medial
to the fasciculus retroflexus. Increase of staining dorsally and diminution
ventrally still occurred in response to the lesion, although the fibres were
not actually interrupted. Increased staining was much more marked in the
animal which survived for 20 days (PI. LXIX, fig. 13) than in the shorter
term experiments. It was considered that the late accumulation of enzyme
in the fasciculus retroflexus might be associated with the small calibre of its
fibres. A comparable response occurred in the fine terminal branches of
the cingulate radiation, some weeks after a lesion of the superior cortex
(Shute and Lewis, 1967):
These experiments proved that the cholinesterase-containing fibres of the
fasciculus retroflexus arise from the habenular nuclei. In normal material
it can be seen that a proportion of the cells which form these nuclei contain
AChE and ChE. Some fornix fibres are known to reach the habenular
nuclei via the habenular striae (Votaw, 1960), so it is possible that these
neurones too, like the other chohnesterase-containing nuclei mentioned
above, are in the sphere of influence of the hippocampus. If the interpeduncular nucleus projects on to cholinergic neurones of the ventral
tegmental area, the fasciculus retroflexus can be regarded as providing an
additional route by which the hippocampus can act on the ventral
tegmental pathway.
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P. R. LEWIS AND C. C. D. SHUTE
DF
FIG. D.—Mid-line sagittal section through the anterior wall (lamina tenninalis) of the
third ventricle (in V) and through the optic chiasma (CH), showing the course, in
relation to the ventral hippocampal commissure (HC) and the anterior commissure
(AC), taken by AChE-containing fibres, derived from cells in the dorsal fornix (DF)
and septal raphe (RS), which supply the subfornical organ (SFO) and the supraoptic
crest (SOC).
fibres from the septum arid dorsal fornix passing ventral to and through the
hippocampal commissure. In a long-term experiment lasting fourty-four
days, a lesion of the dorsal fornix resulted in diminished staining of the
subfornical organ at its posterior end. Loss of staining in the supraoptic
crest was produced by a septal lesion in an animal which survived for six
days. These experiments proved that the massive AChE content of the
subfornical organ and supraoptic crest is derived from incoming fibres.
The presence within the subfornical organ of unmyelinated axons and
nerve endings in a synaptic relationship with the parenchymal cells was
confirmed by electron microscopy (Shute and Lewis, 1963a).
DISCUSSION
We apply the term "cholinergic limbic system" to the groups of AChEcontaining neurones which are intimately related to the hippocampal
formation and its projection pathways in the fore-brain and mid-brain.
Our results show that the cholinergic limbic system consists of two groups
of nuclei (Table I). The nuclei of Group 1 are afferent with respect to the
hippocampal formation. Those of Group 2 are innervated by the hippocampal system and project (a) to medial cortex, (6) to the cerebellum,
(c) to nuclei of the ascending cholinergic reticular system which probably
forms the structural basis of the ascending reticular activating system of
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ISOC
CHOLINERGIC LIMBIC SYSTEM
533
Group 1 neurones (hippocampal offerents).
The Group 1 neurones whose cell bodies are located in the nucleus of the
diagonal band he on the course of the ventral tegmental pathway of the
ascending cholinergic reticular system. It would seem reasonable, therefore, to suppose that these neurones play some part in detenriining the
electrical activity of the hippocampus during arousal. In considering
what their effects may be, it must be remembered that the cholinergic
fibres are not the only afferent projection to the hippocampus travelling
via the septum and fornix. In addition there is an extensive innervation by
monoamine-containing fibres (the monoamine being mainly noradrenaline)
which arise from cells of the mid-brain lying in the mid-line dorsal to the
nucleus interpeduncularis (Dahlstrom and Fuxe, 1964; Fuxe, 1965). The
monoamine-containing terminals, which are presumably monoaminergic,
do not have the same distribution in the hippocampal formation as those
of the cholinergic system. Monoaminergic terminals are found mainly in
the stratum radiatum and stratum lacunosum of the hippocampus in
relation to apical dendrites of pyramidal cells, and in the subgranular
layer of the dentate gyrus in relation to basal dendrites of granule cells
(Fuxe, 1965). Cholinergic terminals, on the other hand, are located
predominantly in the stratum oriens of the hippocampus in relation to
basal dendrites of pyramidal cells (although they are also found in the
stratum radiatum and lacunosum), and in the supragranular layer or the
dentate gyrus in relation to apical dendrites of granule cells (Shute and
Lewis, 1961).
It is possible that the effects of the cholinergic and monoaminergic
systems on hippocampal activity are in some degree opposed to one another.
Such a view is favoured by the report that hippocampal neurones are
consistently facilitated by acetylcholine whereas those which show sensitivity to either noradrenaline or 5-hydroxytryptamine are depressed
(Salmoiraghi and Stefanis, 1966). The firing rate of hippocampal cells is
also accelerated by the cholinomimetic drug eserine (Green, Maxwell,
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neurophysiologists (Shute and Lewis, 1963c, 1967), and (d) to the problematical subfornical organ and supraoptic crest. In the rat, the nuclei of
Group 1 and Group Id contain AChE only, whereas those of Groups la, 1b
and 1c contain ChE as well as AChE. The neurones of the interpeduncular
nucleus contain AChE only. This nucleus may form a link between the
cholinergic limbic system and the ventral tegmental pathway of the ascending cholinergic reticular system, which also contains only AChE. The
dorsal tegmental pathway of the ascending cholinergic reticular system,
which contains ChE as well as AChE, receives connexions from ChEcontaining nuclei of Group 2c. We have suggested that ChE may assist in
the hydrolysis of acetylcholine at sites of high activity (Shute and Lewis,
19636).
534
P. R. LEWIS AND C. C. D. SHUTE
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Schindler and Stumpf, 1960). It is not easy to deduce, however, from the
pharmacological responses of unit cells what effect the cholinergic and
monoaminergic systems are likely to have on the electrical activity of the
hippocampus as a whole, as revealed in the EEG recoid. In favour of the
monoaminergic system being concerned in the production of 6 rhythm,
the following pieces of evidence can be cited. (1) 6 rhythm is depressed by
reserpine, which depletes the brain of monoamines (Killam and Killam,
1957). (2) Slow hippocampal rhythms can be produced by electrical
stimulation of the medial preoptic area, medial hypothalamic region, and
the central grey matter and the dorsilateral tegmentum of the mid-brain
(Torii, 1961). None of these areas, except the last, are sites of cholinergic
cells, whereas some at least may coincide with monoaminergic pathways
and monoaminergic cell groups (e.g. the mid-line Groups AlO and Al 1 of
Dahlstrom and Fuxe (1964): Group A l l occupies the central grey). (3)
Although it has been shown that some unit cells in the hippocampus are
active during 9 rhythm (Green and Machne, 1955), the firing rate of other
hippocampal cells may be depressed (Green, Maxwell, Schindler and
Stumpf, 1960), and it is possible that slow rhythms in the hippocampus
are associated with a relatively low overall level of activity (Grastyan,
Lissak, Madarasc and Donhoffer, 1959), such as might result from an
inhibitory innervation from the monoaminergic system.
In favour of the cholinergic system also playing a part in 9 rhythm is the
fact that slow waves can be induced in the hippocampus by administering
eserine, so long as the septum is intact (Green, Maxwell, Schindler and
Stumpf, 1960). The effects of eserine may be exerted at the septum, if the
hippocampal afferents at that level, cholinergic or otherwise, are cholinoceptive. It has been claimed that neurones located in the medial part of
the septum, which we have shown to be a source of cholinergic fibres
supplying the hippocampus, act as a pacemaker and produce 9 rhythm
through their own slow rhythmical discharge (Petsche, Stumpf and
Gogolak, 1962; Stumpf, Petsche and Gogolak, 1962). These neurones may
themselves be influenced by the monoaminergic system, since monoaminergic endings are reported in the septum (Fuxe, 1965).
Another possible role for the cholinergic Group 1 neurones of the medial
septum and diagonal band may be to produce fast low amplitude activity
in the hippocampus. Such activity is superimposed on 9 rhythm during
moderate degrees of reticular stimulation and after administration of
eserine, and replaces the 9 rhythm when reticular stimulation is very strong
(Stumpf, 1965). Hippocampal responses would in this way be brought into
line with those of the neocortex, where cholinergic projections are probably
responsible for the desynchronized fast activity associated with increased
unit firing which occurs during arousal. Although the hippocampal record
commonly shows 6 rhythm when the neocortical EEG is of the alert type,
fast activity only is said to occur in unanaesthetized animals confronted
CHOUNERGIC LIMBIC SYSTEM
535
Group 2 neurones of the cholinergic limbic system
A major output from the hippocampus passes either directly or through
a relay in the mammillary body to elements of the cholinergic limbic
system which connect directly with medial cortex (Group 2a neurones) or,
through links with the ascending cholinergic reticular system, with the
lateral cortex of the cerebral hemispheres (Group 2c neurones). These
connexions provide a means by which the hippocampus can influence
electrical activity in other regions of fore-brain cortex, as in arousal. The
importance of the 2a innervation is emphasized by the finding that stimulation of medial cortex, particularly the anterior limbic area, produces
manifestations of arousal in unanaesthetized animals (Kaada, Jansen and
Andersen, 1953). Green and Arduini (1954) noted that, during the transition from drowsiness to alertness, hippocampal arousal rhythms usually
precede those of the neocortex, while with behavioural changes in the
reverse direction the hippocampal 6 rhythm associated with arousal is
suppressed before the appearance of neocortical "sleep spindles." These
time sequences suggest that the hippocampus may be concerned in initiating
the reticular activity associated with the alert state. Experimental ablation
of the hippocampal formation, whether carried out surgically (Votaw, 1959)
or produced by seizure activity (MacLean, Flanigan, Flynn, Kim and
Stevens, 1955-6), tends to result in a state of sluggishness and placidity
which may be an expression of reticular hypoactivity. Green (1957)
reported that in hippocampectomized cats electrocortical arousal was
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> with a novel unconditioned stimulus, as part of a "startle response"
(Grastyan, Lissak, Madarasz and Donhofler, 1959). Fast, low amplitude
activity can in fact be produced experimentally in the hippocampus by
stimulating the medial septum (Torii, 1961). The same result is obtained
from stimulation of the medio-ventral tegmentum and lateral hypothalamus— areas traversed by cholinergic neurones of our ventral tegmental
pathway, which probably connect with the cholinergic neurones of the diagonal band and septum. Fast activity produced in this way is abolished by
atropine (Longo, 1956). It has, however, been found that the fast activity
resulting from reticular stimulation or administration of eserine can
survive septal lesions (Mayer and Stumpf, 1958; Stumpf, 1965). This must
mean either that fast hippocampal activity can be produced by cholinoceptive projections on to the hippocampus which do not traverse the
septum (e.g. those from entorhinal cortex), or that some of the septal
neurones responsible escaped damage. Destruction in the septal region
would need to be very extensive to involve all the Group 1 cells. 9 rhythm,
unlike fast activity, is always abolished by septal lesions. It is possible that
at the level of the septum the monoaminergic supply to the hippocampus,
which arises more caudally in the brain-stem, forms a more compact and,
therefore, more vulnerable bundle than the cholinergic fibres.
536
P. R. LEWIS AND C. C. D. SHUTE
SUMMARY
(1) The hippocampal formation (hippocampus and dentate gyrus) receives
a cholinergic innervation from the medial septum and diagonal band.
Hippocampal efferents travelling by the fornix project, directly or
indirectly on to cholinesterase-containing, presumably cholinergic neurones
in the hippocampal commissure, anterior thalamus, habenular and
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difficult to produce by reticular stimulation and, unlike the arousal obtained
in normal animals, lasted only for the duration of the stimulus. Hippocampal stimulation does not usually lead to generalized neocortical
desynchronization (Green, 1957), but so long as it is not so intense as to
cause a seizure discharge, has been found to produce a generalized reaction
stimulating behavioural arousal in anaesthetized monkeys (Votaw, 1959)
and attentive or searching behaviour in free-running rabbits with
implanted electrodes (Cazard and Buser, 1963).
In addition to effects on arousal, it is possible that Groups 2a and 2c
neurones may be involved in the reputed role of the hippocampus in
learning and memory, both in man (Penfield and Milner, 1958) and in
experimental animals (Thompson, Duke, Malin and Hawkins, 1961;
Thompson, Langer and Rich, 1964; Flexner, Flexner, Roberts and de la
Haba 1964; Nielson, Mclver and Boswell, 1965). It is likely that hippocampal effects on memory processes are achieved by influences acting on
some part of the brain external to the hippocampus, rather than as an
intrinsic property of the hippocampus itself (Green, 1964). Some evidence
of the importance of cholinergic mechanisms is provided by the observation
that in man memory impairment, coupled with loss of attention, drowsiness
and decrease in spontaneous speech, follows the administration of atropine
(Ostfield, Machne and Unna, 1960).
Little can be said at present of the significance of other Group 2 neurones
of the cholinergic limbic system. The Group 2b projection would account
for the hitherto unexplained evoked potentials produced in the cerebellum
by stimulating the fornix (Green and Morin, 1953). The function of the
organs supplied by the Group Id fibres is not known. Some of the dendrites
of cells of the supraoptic nucleus are said to reach the equivalent of the
subfornical organ in the frog, and on these grounds it has been concluded
that it may act as an osmoreceptor responding to changes in the osmotic
pressure of the blood and cerebrospinal fluid (Dierickx, 1963). The
subfornical organ and supraoptic crest may possibly be concerned in the
increased water intake which results in rats from hippocampal stimulation
(Fisher and Coury, 1962). Some preliminary observations which we have
made by electron microscopy have shown that many of the nerve endings
in the subfornical organ contain AChE, and that the synapses on
parenchymatous cells of the organ are of motor rather than of sensory
type.
CHOLINERGIC LIMBIC SYSTEM
537
interpeduncular nuclei, and the mid-brain tegmentum. In the rat, these
neurones contain non-specific cholinesterase as well as acetylcholinesterase.
They project to medial cortex, to nuclei of the ascending cholinergic
reticular system, and to the subfornical organ and supraoptic crest.
Together with the cholinergic neurones of the medial septal nucleus and
the nucleus of the diagonal band, they constitute the cholinergic limbic
system.
ACKNOWLEDGMENTS
We thank Mrs. Annette Bond for technical assistance, Messrs. J. F . Crane and
G. Oakes for photography, and Messrs. May and Baker Ltd. and the Wellcome
Research Laboratories for gifts of inhibitors. The work was supported by a grant
from the Medical Research Council.
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LEGENDS FOR PLATES
PLATE LXVm
Transverse sections of rat fore-brain showing locations of cholinesterases. AChE
appears in sections incubated in a medium containing acetylthiocholine (AThCh)
as substrate with 10~'M ethopropazine as a ChE inhibitor. ChE appears in sections
incubated in a medium containing either butyrylthiocholine (BuThCh) as substrate or
propionylthiocholine (PrThCh) with 5 x 10~6M 62C 47e as an AChE inhibitor.
Fio. 1.—Accumulation of enzyme in cholinergic hippocampal afferent fibres
following a fimbrial lesion (L). Five days survival. AThCh substrate, ChE inhibitor,
x 48.
FIG. 2.—Golgi type II cells in the hilum of the ipsilateral dentate gyms following
a lesion of the dorsal fornix. Six days survival. AThCh substrate, ChE inhibitor.
xlOO.
FIG. 3.—Normal brain. Localization of ChE in the interstitial nucleus of the
ventral hippocampal commissure (Q. PrThCh substrate, AChE nhibitor. x 18.
FIG. 4.—Same animal as Fig. 1. Loss of AChE staining of the ventral hippocampus
(VH). Five days after a fimbrial lesion. AThCh substrate, ChE inhibitor, x 12.
FIG. 5.—Normal brain. Localization of ChE in the islets of Calleja (I), in the nucleus
accumbens (A) and in the projection (marked by arrow) from the interstitial nucleus of
the ventral hippocampal commissure to the anterior cingulate area of the frontal lobe.
BuThCh substrate, x 18.
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, (1960)/. comp. Neurol., 114,283.
540
P. R. LEWIS AND C. C. D. SHUTE
PLATE LXIX
Transverse sections of rat fore-brain and mid-brain showing locations of AChE
and ChE. Incubation media as for Plate LXVIII.
FIG. 6.—Animal with unilateral destruction of the antero-dorsal thalamic nucleus
(AD). The antero-ventral nucleus (heavily stained for ChE) and the antero-medial
nucleus (moderately stained) were spared. Twenty-nine days survival. BuThCh substrate, x 24.
FIG. 7.—Normal brain. Dorsal (DO), deep (DE) and latero-dorsal (LD) tegmental
nuclei. LD contributing to the brachium conjunctivum (BQ. PrThCh substrate,
AChE inhibitor, x 251.
FIG. 9.—Normal brain. Subfornical organ, heavily stained for AChE. AThCh
substrate, ChE inhibitor. X 40.
FIG. 10.—Animal with unilateral destruction of the lateral habenular nucleus (H).
Six days survival. AThCh substrate, ChE inhibitor. x l 8 .
FIG. 11.—Same animal as Fig. 6. Loss of ChE staining in layer iii of the retrosplenial
field of the cingulate cortex (CQ, twenty-nine days after destruction of the ipsilateral
antero-dorsal thalamic nucleus. BuThCh substrate, x 24.
FIG. 12.—Same animal as Fig. 10. Loss of AChE staining in fibres of the fasciculus
retroflexus (on the left hand side of the picture) six days after a lesion of the lateral
habenular nucleus on the same side. AThCh substrate, ChE inhibitor, x 36.
FIG. 13.—Accumulation of AChE in fibres of the fasciculus retroflexus above
a unilateral lesion (L). 20 days survival. AThCh substrate, ChE inhibitor, x 30.
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FIG. 8.—Normal brain. Interpenduncular nucleus, heavily stained for AChE.
AThCh substrate, ChE inhibitor, x 10}.
PLATE LXVIII
^
2 '
To illustrate article by P. R. Lewis and C. C. D. Shute.
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VH
PLATE LXIX
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To illustrate article by P. R. Lewis and C. C. D. Shute.