ORGANIZATION OF ASCENDING HYPOTHALAMIC PROJECTIONS TO
THE ROSTRAL FOREBRAIN WITH SPECIAL REFERENCE TO THE INNERVATION OF
CHOLINERGIC PROJECTION NEURONS
________________________
William E. Cullinan* and László Záborszky
Departments of Otolaryngology, Neurosurgery, and Neurology University of Virginia Health Sciences Center, Charlottesville, VA 22908
Axonal projections from hypothalamic nuclei to the basal
forebrain, and their relation to cholinergic projection neurons in particular,
were studied in the rat by using the anterograde tracer Phaseolus
vulgaris-leucoagglutinin (PHA-L) in combination with choline acetyltransferase
(ChAT) immunocytochemistry. Discrete
iontophoretic PHA-L injections were delivered to different portions of the
caudal lateral hypothalamus, as well as to various medial hypothalamic areas,
including the ventromedial, dorsomedial, and paraventricular nuclei, and
anterior hypothalamic and medial preoptic areas. The simultaneous detection of PHA-L labeled fibers/terminals and
ChAT-positive neurons was performed by using nickel-enhanced diaminobenzidine
(DAB) and non-enhanced DAB as chromogens.
Selected cases were investigated at the electron microscopic level.
Ascending hypothalamic projections maintained an orderly
latero-medial arrangement within the different components of the medial
forebrain bundle, as well as with respect to their terminal projection fields
(e.g. within the bed nucleus of the stria terminalis and lateral septal
nucleus). The distribution pattern of
hypothalamic inputs to cholinergic projection neurons corresponded to the
topography of ascending hypothalamic axons.
Axons originating from neurons in the far-lateral hypothalamus
reached cholinergic neurons in a zone which extended from the dorsal part of
the sublenticular substantia innominata (SI) caudolaterally, to the lateral
portion of the bed nucleus of the stria terminalis rostromedially, encompassing
a narrow band along the ventral part of the globus pallidus and medial portion
of the internal capsule. Axons
originating from cells in the medial portion of the lateral hypothalamus
reached cholinergic cells primarily in more medial and ventral parts of the SI,
and in the magnocellular preoptic nucleus and horizontal limb of the diagonal
band nucleus (HDB). Axons from medial
hypothalamic cells appeared to contact cholinergic neurons primarily in the
medial part of the HDB, and in the medial septum/vertical limb of the diagonal
band complex. EM double-labeling
experiments confirmed contacts between labeled terminals and cholinergic cells
in the HDB and SI. Individual
hypothalamic axons established synapses with both cholinergic and
non-cholinergic neuronal elements in the same regions. These findings have important implications
for our understanding of the organization of afferents to the basal forebrain
cholinergic projection system.
Key words: choline acetyltransferase,
Phaseolus vulgaris leucoagglutinin, forebrain organization, electron microscopy
The basal forebrain cholinergic projection system has been the
focus of considerable attention as a result of evidence implicating this system
in a number of behavioral functions, including learning, memory, and arousal
(Deutsch, '83; Buzsaki et al., '88; Richardson and DeLong, '88; Durkin, '89;
Steriade and McCarley, '90). In the rat
this projection system originates from a continuous collection of neurons
distributed across a number of classically defined structures, including the
medial septal nucleus (MS), nuclei of the vertical and horizontal limbs of the
diagonal band (VDB, HDB), magnocellular
preoptic nucleus (MCP), ventral pallidum, sublenticular substantia innominata
(SI), and peripallidal regions.
Detailed information has been advanced concerning the efferent
projections of these neurons (Sofroniew et al., '82; Armstrong et al., '83;
Mesulam et al., '83; Rye et al., '84; Woolf et al., '84; Carlsen et al., '85;
Wainer et al., '85; Zaborszky et al., '86a; Luiten et al., '87; Sofroniew et
al., '87; Fischer et al., '88), however, our understanding of the afferents to
forebrain cholinergic projection neurons remains relatively limited. The reasons for this are easily appreciated
in view of the anatomical complexity of the basal forebrain, in which
cholinergic neurons are intermingled among numerous non-cholinergic cells, and
are distributed in close proximity to several major ascending and descending
fiber systems (for review see Zaborszky, '89; Zaborszky et al., '90). Consequently, the determination of input
sources to these neurons requires the application of double-labeling methods
capable of examining the fine structural relationships of afferents with
identified cholinergic elements at the light and EM levels. Among the few studies employing this
approach, identified cholinergic neurons in the basal forebrain have been shown
to receive GABAergic, substance P-, and enkephalin-containing terminals of
undetermined origin, as well as afferents from the basolateral amygdala
(Zaborszky et al., '84, '86b; Bolam et al., '86, Chang et al., '87;
Martinez-Murillo et al. '88; Leranth and Frotscher, '89).
The possibility of hypothalamic input to the general forebrain
regions containing cholinergic projection neurons was initially suggested from
earlier autoradiographic studies (Conrad and Pfaff, '76a, '76b; Jones et al.,
'76; Saper et al., '76; Swanson, '76; Saper et al., '78, '79; Krieger et al.,
'79; Berk and Finkelstein, '82; Mesulam and Mufson, '84; Saper, '85), although
this issue remained somewhat unclear due to the difficulty in distinguishing
fibers from terminals with the autoradiographic technique. More recent experiments with the anterograde
tracer Phaseolus vulgaris leucoagglutinin (PHA-L) have confirmed the presence
of projections to these regions from several hypothalamic nuclei (Ter Horst and
Luiten, '86; Simerly and Swanson, '88), and a light microscopic double-labeling
study investigated the distribution of hypothalamic afferents to a subset of
cholinergic neurons (Grove, '88).
In a previous double-labeling study at the electron microscopic
level we have shown that lateral hypothalamic axons terminate on cholinergic
neurons located within the SI (Zaborszky and Cullinan, '89). In the present study we have systematically
examined the ascending projections of a number of hypothalamic areas, focusing
on their relationship to the cholinergic projection system as a whole in
combined double-labeling experiemnts involving PHA-L tracing and choline
acetyltransferase (ChAT) immunohistochemistry.
A correlated light-EM approach was used to confirm the presence of
synaptic contacts in selected cases.
PHA-L
tracing
Twenty-one male Sprague-Dawley rats, weighing 275± 10g were used
in this study. Animals were
anesthetized (Nembutal, 50 mg/kg) and mounted in a Kopf stereotaxic apparatus
adjusted to coordinates according to the atlas of Paxinos and Watson
('86). A 2.5% PHA-L (Vector) solution
in 0.1M sodium phosphate buffer pH 7.4 (PB) was back-filled into glass
micropipettes (tip diameter 15-20 um).
Discrete iontophoretic PHA-L injections were made in the caudal lateral
hypothalamus and in various medial hypothalamic nuclei according to the method
of Gerfen and Sawchenko ('84). The
glass pipette was left in place an additional 10-15 min to minimize tracer
diffusion along the pipette track.
After survival periods of 7-12 days, animals were deeply anesthetized
and perfused transcardially with 50 ml saline, followed by 350 ml of a fixative
containing 4% paraformaldehyde, 0.1-0.2% glutaraldehyde, 15% saturated picric
acid, in 0.1M PB (Somogyi and Takagi, '82).
This was succeeded by 150 ml of the same fixative without glutaraldehyde. Brains were removed immediately and
post-fixed in the second fixative for 4-12 h, before Vibratome cut into 6
series of 40 μm coronal sections.
For immunohistochemical processing, sections were rinsed several
times in ice cold PB prior to all steps.
Antibodies were routinely diluted in a PB solution to which 0.5% triton
X-100 and 0.25% carageenan (lambda type, Sigma) had been added. All steps were carried out under gentle
agitation, and except where noted, performed at room temperature.
In order to determine the location of PHA-L injections, sections
from the first series were processed with the rapid indirect immunofluorescence
technique (Coons, '58). Sections were
incubated in a goat antiserum directed against PHA-L (Vector) at a 1:750
dilution for 3 h. This was followed by
incubation in fluoroscein isothyocyante (FITC) conjugated rabbit anti-goat IgG
(Miles Biochemical) at 1:100 for 1 h.
Sections were then mounted, coverslipped, and viewed with a Zeiss Axioplan
epifluorescent microscope with appropriate filter set.
To aid in the mapping of hypothalamic projections, a second
series of sections was processed for detection of the lectin using the
avidin-biotin peroxidase (ABC) technique.
Sections were incubated in goat anti-PHA-L at a dilution of 1:2000 for
12 h at 4°C. This
was followed by a biotinylated anti-goat IgG prepared in rabbit (Vector) at
1:100 for 2 h, and the ABC complex (Vector Labs) at 1:500 for 2 h. This was succeeded by the coupled oxidation
reaction of Itoh et al. ('79) with a solution containing 50 mg
3,3'-diaminobenzidine (DAB) tetrahydrochloride, 40 mg ammonium chloride, 0.4 mg
glucose oxidase (Sigma, type VII), and 200 mg ß-D-glucose per 100 ml PB, with
0.001M nickel ammonium sulfate, for 30-40 min.
Sections were then rinsed, dehydrated in a progressive series of
alcohols, stored overnight in xylene, and coverslipped with DPX.
A third series of sections was immunostained for glutamic acid
decarboxylase (GAD) to aid in the determination of the borders of the globus
pallidus and ventral pallidum. Sections
were incubated in an anti-GAD antibody prepared in sheep (Oertel et al., '81)
at a dilution of 1:2000 for 12 h at 4°C. This was followed by biotinylated rabbit anti-goat IgG at 1:250
for 2h, the ABC complex at 1:500 for 2h, and a DAB reaction (0.06% DAB in 0.002% H2O2
in 0.05 Tris-HCl buffer, pH 7.6, 10 minutes).
Sections were then dehydrated and coverslipped as described above.
The fourth series of sections was processed with a light
microscopic double-labeling method for the sequential detection of PHA-L and
ChAT (Hsu and Soban, '82; Wouterlood et al., '87, Zaborszky and Cullinan,
'89). The first immunohistochemical
protocol (PHA-L) was similar to that of the second series of sections. The second immunohistochemical protocol
involved incubation in a rat anti-ChAT monoclonal antibody (Eckenstein and
Thoenen, '82) at 1:10 for 36 h at 4°C, followed by rabbit anti-rat
IgG (Sigma) at 1:100 for 2 h, rat monoclonal peroxidase-anti-peroxidase (PAP)
(Sternberger-Meyer) at 1:100 for 1 h, and finally a DAB reaction similar to
that used for the second series of sections, with the exception of the nickel
solution. Sections were then dehydrated
and coverslipped.
A fifth series of sections was processed for electron microscopic
double-labeling. The sequence and
antibody dilutions were the same as those of the fourth series, except that
incubation times and temperatures differed (anti-PHA-L antibody incubation of
36 h, linking antibodies for 4 h, all at 4°C), and antibodies were diluted
in PB containing only 0.04% Triton X-100.
In addition, antisera penetration was facilitated by a freeze-thaw
procedure whereby sections were floated in vials containing 10% sucrose PB
until they sunk, and successively frozen in liquid nitrogen, thawed at room
temperature, and rinsed in PB.
Following immunostaining, sections were post-fixed in 1% OsO4
for 30 min, dehydrated in a series of alcohols, stained with uranyl acetate at
the 70% ethanol stage, and flat embedded in Spurr resin (EMS) between glass
slides and coverslips that had been treated with liquid releasing agent
(EMS). Selected embedded sections
containing labeled varicosities in close apposition to cholinergic neuronal
processes were subsequently photographed, mounted on cylindrical plastic
blocks, and thin sectioned using a Reichert Ultramicrotome. Ultrathin sections were collected on single
slot Formvar-coated nickel grids and examined with a Zeiss EM 109 or Phillips
300 electron microscope.
To control for the possibility of cross-reactivity between
immunoreagents, or between secondary antibodies in the double labeling
experiments, a sixth series of sections was processed in the full
immunohistochemical sequence with one or both primary antibodies deleted, and
with anti-ChAT antibody replaced by PB and the anti-PHA-L antibody replaced
with normal goat serum or PB. Evidence
of cross-reactivity was never encountered in these experiments.
Camera lucida drawings were made at 25x from lower quadrants of
double-labeled sections (from the fourth series) at four basal forebrain
levels, corresponding to those of Figure
1 A-D. PHA-L labeled fibers and
ChAT-positive cell bodies and proximal dendritic segments were drawn. Following completion of drawings, coverslips
from mapped sections were removed, and sections were restained for Nissl in
order to further aid in delineating forebrain structures.
In order to evaluate the distribution pattern of hypothalamic
input in relation to the cholinergic forebrain projection system as a whole,
zones of putative contacts between PHA-L labeled terminals and labeled cholinergic
elements were mapped. ChAT-positive
neurons from double-labeled sections were drawn (10x) from eight standardized
forebrain levels. Grids composed of
600-800 80x80 μm pixels were subsequently placed over the drawings, and
the sections examined at 63x with the aid of an ocular reticle that precisely
corresponded to the pixel dimensions.
Grids were then scored for the presence of putative contacts using the
same criteria as taken to select cases for analysis at the EM level, i.e. a
clearly identified PHA-L labeled terminal (including associated axon) directly
abutting a labeled cholinergic cell body or dendrite in the same focal
plane. Positive zones generally had
between 1-5 of such arrangements, although occasionally greater numbers of
putative contacts could be identified.
Figures derived from the eight forebrain levels were then aligned and
merged into composite maps, such as illustrated in Figure 21 E-G.
Terminology
Figure 1
illustrates the distribution of cholinergic projection neurons in the basal
forebrain from a series of coronal sections.
For delineating regions of the rostral forebrain, we have maintained the
use of terminology outlined in the second edition of the atlas of Paxinos and
Watson ('86). Also, because many of our
injections were located along the fibers of the medial forebrain bundle (MFB),
we have referred to the parcellation of the MFB as described by Nieuwenhuys et
al. ('82).
Discrete iontophoretic PHA-L injections were made in various
portions of the caudal lateral hypothalamus, as well as a number of medial
hypothalamic regions, including the ventromedial, dorsomedial, and paraventricular
nuclei, and the anterior hypothalamic and medial preoptic areas. A typical PHA-L injection site is seen in Figure 2, in this case within the
ventromedial hypothalamic nucleus (case M065).
Figure 3 illustrates
PHA-L labeled fibers and terminals in the basal forebrain viewed under
darkfield illumination. A composite of
all injection sites included in the present experiment is presented in Figure 4.
Case L125. The PHA-L
injection in case L125 was located in the posterior lateral hypothalamus at the
level of the dorsal premammillary nucleus. The rostral projection from this
case is illustrated in Figures 5
and 6.
Ascending fibers ran in the ventrolateral portion of the MFB
(primarily in the "d" and to a lesser extent the "e"
compartment of Nieuwenhuys et al., '82), and at the level of the rostral pole
of the ventromedial nucleus, were seen to fan out toward the SI. More rostrally, a contingent of fibers
turned dorsally along the medial edge of the internal capsule and entered the
ventrolateral aspect of the bed nucleus of the stria terminalis (BSt) (Fig. 6A). A few fibers extended dorsally to the stria
terminalis. Along their course labeled
axons formed a terminal network that extended from the central nucleus of the
amygdala and dorsal part of the SI caudolaterally, to the lateral part of the
BSt rostromedially, encompassing a narrow band along the ventral part of the
globus pallidus and medial portion of the internal capsule (Fig. 6 A-B). This zone of labeling generally corresponds
to the continuum formed by the central amygdaloid nucleus/SI/lateral BSt as
described by de Olmos et al. ('85), and more recently referred to as the
"extended amygdala" (Alheid and Heimer, '88). Cholinergic projection neurons were embedded
in this terminal matrix throughout its extent, and were detected in close
apposition to labeled terminals. A
small number of fibers coursed further anteriorly, spreading out toward the
ventrolateral part of the ventral pallidum and ventral aspect of the nucleus
accumbens (Fig. 5 A-B). Only an occasional fiber could be detected as
far rostrally as the diagonal band and septum.
A sparse contralateral projection was seen which crossed in the
retrochiasmatic area and joined the supraoptic decussation, through which
fibers distributed to the SI, medial part of internal capsule, and lateral part
of the BSt. Interestingly, despite the
paucity of this contralateral projection, a number of these fibers approximated
the dendrites of cholinergic neurons in the SI in patterns suggestive of synaptic
contact.
Case L123.
The cells labeled at the injection site in this case were concentrated
at the posterior tuberal level, and were located medial, ventral, and rostral
to those in case L125. The distribution
of fibers/terminals in the rostral forebrain from this case is illustrated in Figures
7 and 8.
Ascending fibers were found to occupy much of the MFB,
particularly its medial portion ("c" compartment). Along its anterior course, fibers from this
case left the MFB dorsolaterally, ventrolaterally, laterally, dorsomedially,
and medially.
Fibers coursed dorsolaterally toward the zona incerta,
some of which penetrated the internal capsule to join the supraoptic
decussation (at approximately the level of Fig. 1F). Other
fibers reached the medial part of the internal capsule, where terminals were
detected in close approximation to cholinergic elements.
Axons directed ventrolaterally ran above the optic tract
toward the supraoptic decussation.
More rostrally, a contingent of fibers detached laterally and ran
through caudal part of the SI (at approximately the level of Fig. 1E). Labeled terminals in this region were seen
in close apposition to cholinergic cells, particularly along the ventral border
of the internal capsule.
Fibers which coursed dorsally projected to the thalamus,
where labeled fibers/terminals were seen in the paraventricular, paratenial,
and lateral habenular nuclei, as well as in the internal medullary lamina
(paracentral nucleus).
Medially, a few fibers reached the ventromedial,
dorsomedial and paraventricular hypothalamic nuclei, while others crossed in
the retrochiasmatic area to join the supraoptic decussations of the
contralateral side.
Further rostrally at about the level of Figure 8B, fibers extended
dorsally to the internal capsule, and laterally to the SI and MCP-HDB. Cholinergic neurons within these areas were
approximated by labeled terminals. More
rostrally a contingent of fibers from the MFB fanned out over much of the BSt,
with a few fibers continuing to the stria terminalis. A very heavy network of fibers and terminals was seen in the
lateral preoptic area, as illustrated in Figure 3A, which is just rostral to the level of Figure 8B. Further rostrally, labeled fibers were
detected in the medial portion of the ventral pallidum and in the nucleus
accumbens (Fig 7 A-B). Other fibers continued anteriorly, turning
dorsally through the diagonal band and MS, and terminating primarily in the
lateral septum within its intermediate division, as well as in its dorsal
division below the corpus callosum in an oval-shaped zone oriented from
ventromedial to dorsolateral. Labeled
terminals were seen in close proximity to a few cholinergic neurons located in
the ventral pallidum, and within the medial part of the MS.
Case L126. In case L126
the PHA-L injection was located
anteroposteriorly between cases L123 and L125 (Fig. 4). The labeled cells extended further ventrally than in case L125,
while medially they overlapped the caudolateral part of the area labeled in
case L123. The pattern of labeling in
the rostral forebrain showed characteristics of both L123 and L125, and can be
viewed as a mixed case. As in case
L125, fibers which coursed laterally in the MFB passed through the caudal part
of the SI, where a particularly dense network of terminals was elaborated (Fig. 9). Cholinergic neurons were
seen in close approximation to labeled terminals in this area. Also, as in case L125, this projection
continued anteriorly, providing a dense terminal field in a corridor extending
from the dorsal portion of the SI laterally, to the ventrolateral BSt medially,
and encompassing the ventral globus pallidus and medial part of the internal
capsule. However, the terminal network
in the dorsal part of the BSt was clearly shifted medially, as in case L123. Also similar to case L123, moderately dense
projections to the midline thalamus, dorsomedial, paraventricular, anterior
hypothalamic, and medial preoptic nuclei were evident. In addition, a few fibers continued
rostrally before terminating in the intermediate and dorsal division of the
lateral septal nucleus.
Case L110. This injection
was located at the midposterior level of the tuberal hypothalamus, ventral to
the fornix. This region is just medial
to the heavily myelinated portion of the MFB, and included the small-celled
medial tuberal nucleus of Bleier et al. ('79).
The PHA-L deposit also included cells in the ventrolateral part of the
ventromedial hypothalamic nucleus. The
rostral projections from this case are illustrated in Figures 10 and 11.
Fibers coursed anteriorly primarily by two routes: 1) a
lateral and dorsolateral route, toward the ventral supraoptic decussation and
zona incerta, and 2) a medial route, through the region ventral and medial to
the fornix, as well as in the ventromedial part of the MFB.
The medial projection terminated diffusely in the anterior
hypothalamic area (AH), although sparse labeling was found within its
periventricular and ventrolateral portions.
Further rostrally (Fig. 11B)
the medial preoptic area received a dense innervation. A substantial portion of the axons turned
dorsolaterally at this level and entered the posterior aspect of the BSt, where
a dense network of labeled fibers and terminals was detected. Some axons continued to the stria
terminalis, while others coursed ventrolaterally through the ventrolateral BSt
to the area of transition between the SI and dorsal MCP-HDB. Labeled terminals were found in close
proximity to cholinergic neuronal elements in this region. Further rostrally, the medial fiber group
swept dorsally to the septum, and dense labeling of terminals was noted in the
lateral septal nucleus, particularly within the more dorsal aspect of its
intermediate division. Cholinergic
cells located laterally within the MS-VDB complex were seen in close
approximation to labeled terminals.
Some more laterally coursing fibers reached the medial portion of the
ventral pallidum and nucleus accumbens (Fig. 10 A-B).
Case L124.
The tracer deposit in case L124 was centered in the ventral part of the
lateral hypothalamus at the caudal tuberal level (Fig. 4). Fibers
directed anteriorly in the MFB occupied a ventromedial position as in case
L110, and in addition, many fibers coursed medial to the MFB. From the MFB labeled fibers and terminals
reached the ventral SI, MCP, and dorsal HDB, similar to case L110, and were
seen in close approximation to cholinergic elements in these regions. Fibers ascending medial to the MFB also
reached the medial HDB, where they were seen in close approximation to
cholinergic neurons.
Further rostrally fibers
reached the septum, coursing relatively medially in the ventral part of the
septum before turning dorsolaterally and terminating principally in the
intermediate and ventral subdivisions of the lateral septum. These ascending fibers were detected in
close proximity to a few cholinergic neurons located medially in the MS-VDB
complex.
Case L104.
This injection was located in the ventral part of the lateral
hypothalamus at the posterior tuberal level (Fig. 4). The
majority of the labeled cells in case L104 were concentrated caudal to case
L110. The ascending projections from
case L104 were similar to case L110, except in the lateral preoptic area, which
received a dense innervation, similar to case L123.
Case L105.
Although the injection site in this case partially overlapped that of
case L104, it extended more ventrally, medially, and caudally (Fig. 4). This was reflected in the ascending projections from this case,
which were similar to L104 except that: 1) labeling was seen in a more ventral
position in the SI, although fewer fibers were labeled there, 2) fibers in the
lateral preoptic area were located medial to those of case L104, 3) labeling of
fibers/terminals in the BSt was shifted medially.
Case L168.
In this case a small PHA-L deposit was localized dorsally at the
posterior tuberal level. Only a very
sparse projection was followed anteriorly from the injection site: a few
individual fibers were detected in the supraoptic decussation, SI, BSt, HDB and
VDB. A few scattered fibers were also
seen in the lateral septal nucleus.
Case M066. The PHA-L
injection in case M066 was located in the dorsomedial division of the
ventromedial hypothalamic nucleus. The
distribution of labeled fibers/terminals in the rostral forebrain from this
case is presented in Figure 12
and 13.
From the injection site fibers coursed anteriorly through
the periventricular layer, the medial
hypothalamus, and the ventromedial portion of the MFB. The supraoptic, suprachiasmatic,
paraventricular nuclei were labeled at their perimeters, although only a few
fibers were found within these nuclei (Fig. 13B).
Extremely heavy labeling of fibers/terminals was seen in the AH,
particularly within its lateroanterior cell condensation (Fig. 13B). Moderately dense labeling was evident in the
medial preoptic nucleus (Fig. 13A).
Among fibers coursing anteriorly through the medial hypothalamus,
some turned dorsolaterally to terminate heavily in the the medial division of
the BSt (Fig. 13A), with a few
fibers extending to the stria terminalis itself. Other fibers continued anteriorly and dorsally, turning sharply
laterally around the decussation of the anterior commissure, coursing between
the MS and lateral septal nucleus, before terminating primarily in the
anteromedial portion of the BSt and in the lateral septum (Fig 12 A-B). In the lateral septum the majority of fibers
terminated in the ventral subdivision, and to some extent in the ventral aspect
of the intermediate subdivision.
Labeled terminals were also detected in the ventrolateral porton of the
MS, and could be seen in close proximity to cholinergic neurons.
Axons passed dorsally from the injection site through the
periventricular layer to the midline thalamic nuclei (reuniens, paraventricular
nuclei). Along this course a few fibers
with en passant varicosities were distributed in the dorsomedial
hypothalamic nucleus.
A considerable lateral projection was noted from the
injection site. At caudal levels fibers
were followed along the border of the optic tract, coursing dorsolaterally
between the optic tract and internal capsule and passing over many cholinergic
cells and their dendrites, before turning in a lateroventral arc to the
amygdala. These fibers bore few
varicosities, and thus did not appear to contact the cholinergic neurons. More rostrally a contingent of fibers passed
through and above the MFB and turned ventrolaterally, coursing through the SI (Fig. 13B). These axons were smooth with very few
varicosities, and were seen to cross over cholinergic cells and dendrites
without apparent contact, before reaching the amygdala. Another prominent lateral projection was
located ventrally just above the optic chiasm and below the heavily myelinated
part of the MFB. These fibers ran along
the dorsal and lateral aspects of the supraoptic nucleus, and extended to the
medial part of the HDB where a small terminal field was apparent (Fig. 13B). Some of these fibers extended further
laterally to the amygdala.
Sparse contralateral projections were noted, with a few fibers
found to cross in the retrochiasmatic area or through the anterior commissure
to terminate in the contralateral, AH, preoptic area, and ventral part of the
BSt. In addition, some fibers were seen
to follow the ventral supraoptic decussation to the contralateral lateral
hypothalamic area, SI, and anterior amygdaloid area.
Case M065.
The injection in this case was located in the ventromedial portion of
the ventromedial hypothalamic nucleus (Fig. 4), at a level slightly more rostral to case M066. The
nucleus was free of labeled cells both ventrolaterally and dorsomedially. Although the forebrain distribution of
fibers and terminals was generally similar to case M066, a few differences were
evident which were primarily quantitative: 1) a more prominent fiber contingent
projected laterally toward the amygdala, both through the SI and ventral
supraoptic decussation, 2) less dense labeling was noted in the posterior BSt
and in the lateroanterior division of the AH.
Case M517. The injection
site in case M517 was centered in the dorsomedial hypothalamic nucleus,
although it enchroached upon the dorsal and posterior hypothalamic areas
caudally (Fig. 4). The efferent projections of the dorsomedial
hypothalamic nucleus using the PHA-L technique have recently been described in
detail (Ter Horst and Luiten, '86), and the ascending projections described in
that study corresponded well with our data.
Sparse projections from the dorsomedial hypothalamic nucleus were found
to reach the vicinity of cholinergic neurons in the dorsal portion of the
MCP-HDB and at the ventral aspect of the brain near the organum vasculosum of
the lamina terminalis (OVLT) and ventral VDB, however, labeled terminals from
these projections were not encountered in direct apposition to cholinergic
elements in patterns suggestive of synaptic contact.
Case M506. In this case the injection site was centered in the medial and
lateral parvocellular portions of the paraventricular nucleus (delineated
according to Swanson and Kuypers, '80) (Fig. 4). A few
magnocellular neurons were also labeled in its posterior subdivision, as well
as in the central cell condensation of the AH.
In general, the rostral projection from case M506 was characterized by
sporadic fibers bearing en passant varicosities which were diffusely
distributed in many forebrain areas, without prominent terminal
arborizations.
A considerable projection emanated laterally from the
injection site, passing above the fornix before turning in a ventrolateral arc
through the SI. Fibers in the ventral
part of SI and dorsal part of the lateral hypothalamus were seen to cross
cholinergic dendrites without apparent contact, and could be followed to the
amygdaloid body. The most medial
component of this ventrolateral arc continued to the caudal medial portion of
the HDB, where a few labeled terminals were seen in close proximity to cholinergic
neurons.
Case M074 and M077.
In these cases the injection sites
labeled the dorsal and lateral parvocellular portions of the paraventricular
nucleus, and included a few magnocellular cells in its posterior subdivision,
as well as several neurons located laterally in the region above the fornix (Fig. 4). In addition, several fusiform neurons encapsulating the nucleus
were also labeled. The projections from
these cases were similar to case M506.
In addition to the cholinergic neurons located medially in the HDB, at
more caudal levels labeled terminals were found in close approximation to
cholinergic cells situated along the supraoptic decussation.
Caudal
preoptic and anterior hypothalamic areas (dorsal cases)
Case M067.
The majority of PHA-L labeled cells in case M067 were located dorsally
in the caudal part of the medial preoptic region, and in the central cell
condensation of the AH (see Saper et al., '78). A few labeled neurons were also noted within the posteroventral
part of the BSt. The distribution of
PHA-L labeled fibers and terminals in the rostral forebrain from this case is
illustrated in Figures 14 and 15. In agreement with earlier findings (Conrad and Pfaff, '76b),
anterior, lateral, and dorsal projections were noted.
Fibers directed anteriorly coursed through the
periventricular and medial preoptic nuclei, terminating heavily within these
regions (Fig. 15 A-B). Further rostrally fibers coursed through the
VDB, and turned dorsolaterally around the decussation of the anterior
commissure, supplying fibers/terminals to the lateral part of the MS, before
terminating heavily within the anteromedial part of the BSt and of the lateral
septal nucleus (Fig. 14 A-B). In the latter structure, densest labeling
was seen within the ventral subdivision, and in the lateral part of its
intermediate division. A few fibers
terminated in the medial part of the ventral pallidum. Cholinergic elements in the medial HDB,
transition zone between the HDB and VDB, as well as those along the lateral
border of the VDB and MS, were found in close proximity to labeled fibers and
terminals.
Another prominent fiber group coursed dorsolaterally from
the injection site, labeling the posterior intermediate part of the BSt, with
many fibers continuing to the stria terminalis (Fig. 15B). More
rostrally this projection primarily labeled the medial division of the BSt (Fig. 15A). Other fibers which coursed dorsolaterally
from the injection site turned in an arc to the SI, apparently as collaterals
of fibers directed toward the BSt.
These fibers bore few varicosities, and could frequently be seen to pass
over labeled cholinergic elements in the SI along their course to the amygdala
(Fig. 15B).
Fibers extending laterally from the injection site
terminated heavily in the lateral preoptic area, with some continuing to the
ventral SI (Fig. 15B).
Cholinergic neurons in the ventral portion of the SI were approximated by
fibers and terminals. More ventrally,
above the supraoptic nucleus (SON) another fiber contingent was seen to course
laterally to the medial HDB, where labeled terminals were detected in close
proximity to cholinergic neuronal elements.
Some fibers from this projection could be followed further laterally and
caudally to the amygdala.
Dorsally directed
fibers bearing en passant varicosities passed through the reuniens,
paraventricular, and paratenial thalamic nuclei (Fig. 15B).
PHA-L labeled fibers and terminals were also detected
contralaterally in sparse amounts rostrally within the lateral septum, VDB, and
BSt. At more caudal levels a few scattered
fibers were found in the medial preoptic area and AH.
Case M068.
The injection site in case M068 (Fig. 4) was similar to that of case M067, except that M068
included cells caudodorsally between the fornix and paraventricular nucleus, as
well as a few cells within the paraventricular nucleus itself. No significant differences were apparent in
the patterns of labeling in the rostral forebrain between the two cases.
Case M076.
In case M076 (Fig. 4),
the labeled cells at the injection site were centered dorsally in the central
AH, and also in its posterior subdivision.
Rostrally the injection site overlapped that of case M067. The main characteristics of the rostral
projections from M076 were also similar to M067, although a few differences
were noted. For example, the medial
HDB, VDB, and intermediate portion of the lateral septal nucleus received
relatively stronger projections than in case M067. In contrast, a relatively lighter projection to the SI was noted.
Caudal
preoptic and anterior hypothalamic areas (ventral cases)
Cases M040, M043, M044. In cases
M040, M043, and M044 labeled cells at the injection sites (Fig. 4) were concentrated in the
more ventral portion of the AH, involving to varying extents the lateroanterior
or central cell condensations of the AH and caudal part of the medial preoptic
area. In general, the forebrain projections
from these cases were remarkably similar to M067, although some differences
were noted. For example, considerably
lighter labeling was found within the posterior (lateral and intermediate)
parts of the BSt. Also, only a few
fibers passed through the ventral part of the SI, apparently as collaterals of
fibers projecting toward the BSt. In
the lateral septum the terminal labeling from these cases was less dense, and
did not extend as far dorsally as in the more dorsal cases (M067, M068).
Case M078. The PHA-L
injection in case M078 was centered in the lateral portion of the medial
preoptic nucleus and medial portion of the medial preoptic area (as defined by
Simerly and Swanson, '88), and the distribution of fibers and terminals in the
rostral forebrain from this case is illustrated in Figures 16 and 17. Fibers were directed anteriorly, laterally, dorsally, and
caudally from the injection site.
Fibers running anteriorly coursed along the lateral border
of the VDB and MS, terminating primarily within the most ventral aspect of the
intermediate subdivision of the lateral septal nucleus (Fig. 16A). A few fibers bearing en passant varicosities
were detected in the diagonal band, and labeled fibers and terminals were also
distributed in the medial portion of the ventral pallidum and rostral portion
of the BSt. Cholinergic neurons located
in the transition zone between the HDB and VDB, and along the lateral margin of
the MS, were closely approximated by terminal varicosities.
The dorsal and lateral projections from this case were similar to
case M067, although a more massive innervation of the lateral preoptic area was
evident, and labeling within the BSt was concentrated ventrally (Fig. 17A), with fewer fibers
extending to the stria terminalis.
Fibers which ran caudally were collected: 1) along the
medial border of the MFB, 2) ventrally in the medial hypothalamus, just above
the optic chiasm, and 3) in the periventricular layer. The fibers at the medial border of the MFB
fanned out laterally, with the most dorsal ones turning sharply laterally
toward the ventral part of the SI and ventral part of the globus pallidus (Fig. 17B). These fibers bore en passant
varicosities, and appeared to cross over cholinergic dendrites without
contact. Ventrally, just above the
optic chiasm, a more dense fiber group passed above the SON (Fig 17B), supplying the SON with
a few fibers/terminals before reaching the medial tip of the HDB where a
terminal network was evident.
Cholinergic neurons in the medial HDB were closely approximated by these
labeled terminals. Diffuse labeling of
fibers/terminals was apparent throughout most of the AH, and a moderately dense
innervation of the paraventricular hypothalamic (parvocellular division) and
periventricular hypothalamic nuclei was seen (Fig. 17B). At more
caudal levels, a few fibers were seen to pass around the optic tract, joining
the ventral supraoptic decussation and extending toward the amygdala.
Contralateral labeling from case M078 was sparse, and generally
found in patterns which mirrored those of the ipsilateral projections.
Topography
and terminal arborizations of hypothalamic axons in relation to cholinergic
neurons
Ascending hypothalamic axons showed three basic arborization
patterns: 1) smooth fibers bearing essentially no varicosities, 2) fibers
bearing en passant varicosities, and 3) fibers that were highly branched
with multiple varicosities or grape-like arrangements. The different fiber types were seen in
variable proportions in the forebrain, and are referred to below as they relate
to forebrain areas containing cholinergic projection neurons.
Substantia innominata. The courses
taken by hypothalamic fibers en route to the SI varied according to the
location of the cells of origin, and are described below. Fibers originating from more rostral,
medial, and ventral cell groups within the hypothalamus appeared to maintain these
relative positions within the SI.
Projections to the SI from cells located in the lateral
hypothalamus arrived via the MFB.
Axons originating from far-lateral hypothalamic neurons (case L125)
occupied a lateral position in the MFB ("d" and "e" compartments)
before coursing through the more dorsolateral part of the SI. Further rostrally, a contingent of these
fibers extended through the dorsomedial SI to the lateral portion of the BSt. Axons that reached the SI from mid-lateral
(case L123) or more medial portions (cases L104, L110) of the lateral
hypothalamus initially coursed obliquely through the MFB before running
anteriorly in its lateral portion.
These fibers were then distributed to the SI in its dorsomedial
part. Other axons from these cases
coursed medially within the MFB, before turning to the SI at more rostral
levels. These appeared to be
collaterals of axons destined for the BSt, similar to those from medial
hypothalamic cases (see below).
Both fibers bearing en passant varicosities as well as
more highly branched axons bearing multiple terminals were seen in the SI from
lateral hypothalamic cases, and were seen in close proximity to cholinergic
cells. The arborization pattern of
fibers and terminals in the dorsal SI is illustrated in Figure 18 from the far-lateral
hypothalamic case (L125). Sometimes varicosities surrounding two cholinergic
cells arose from the same axon as shown in Figure 21C from the mid-lateral hypothalamic case (L123).
Projections to the SI from the medial hypothalamus arrived
by two routes:
1)
Axons
which initially coursed dorsolaterally from the injection site arched above the
heavily myelinated portions of the MFB, and then turned to the SI, apparently
as collaterals of fibers which ran to the BSt.
The majority of these axons were of the fibers of passage type, bearing
almost no (e.g. case M066) or few (e.g.case M067) en passant
varicosities, and were directed toward the amygdala.
2)
Axons
reached the more ventral portions of the SI by coursing across the MFB through
a more straight lateral route. These
fibers tended to be more branched, contributing more terminal varicosities,
some of which could be seen to approximate cholinergic neuronal elements.
The two systems cannot be
sharply separated within the SI, although the contributions of individual cell groups showed some differences
according to their location. For example,
axons which took the more direct lateral route emanated from cells located more
ventrally within the medial hypothalamus.
It was interesting to note from both medial and lateral
hypothalamic cases that the density of the terminal field from a given
projection did not always show a simple relationship to the number of visible
fibers. For example, in case M067,
relatively few terminal varicosities were detected in the rostral SI (level of Fig. 15B), while more caudally
(not shown) the density was considerably higher despite a similar number of
axons in the two regions. It was
generally the case, however, that cells located progressively more laterally
within the hypothalamus contributed increasing numbers of varicosities to the
SI.
The topography of fiber projections to the SI from hypothalamic
cell groups was reflected in the distribution of putative contact sites on
cholinergic elements. For example,
terminal varicosities which originated from neurons in the far-lateral
hypothalamus (case L125) were detected in close proximity to cholinergic
elements in the more dorsal and lateral SI.
Varicosities approximating cholinergic neuronal elements from more
medially and ventrally located lateral hypothalamic neurons (e.g. case L110)
were found in the SI medial and ventral to those from far-lateral hypothalamic
cells (case L125). Terminals from
medial hypothalamic groups (cases M067, M078) approached cholinergic elements
that were located most medially and ventrally in the SI.
Magnocellular preoptic nucleus-horizontal limb of the diagonal
band nucleus.
Fibers originating from the mid-lateral and medial portions of the lateral
hypothalamus (cases L123, L110) projected to the more dorsal aspect of the
MCP-HDB (Fig. 8 A-B and 11 A-B). Labeled terminals were seen in proximity to
cholinergic elements from these cases, particularly within the MCP. In contrast, axons from far-lateral
hypothalamic neurons largely avoided the MCP-HDB, with only a few axons
reaching its ventrolateral part.
Among the regions of the medial hypothalamus investigated,
neurons in the more caudodorsal regions (dorsomedial and paraventricular
nuclei) were found to project to more caudolateral portions of the MCP-HDB,
while cells from more ventral or rostral areas (ventromedial, anterior
hypothalamic, preoptic) projected to more medial portions of the HDB. The projections to the MCP-HDB from the
caudodorsal groups were generally sparse.
In contrast, a common feature of the projections from the more ventral
and rostral areas was a relatively prominent projection to the medial portion
of the HDB. Fibers reached the medial
HDB by coursing either ventrolaterally (from the more dorsal cases- M067,
M044), or directly laterally (from more ventral cases-e.g. M043) from the
injection site. A small, dense terminal
field was generally found, in which labeled varicosities were seen abutting
cholinergic neuronal elements, as shown in Figure 19 from case M067.
A single axon often gave rise to several varicosities approximating the
same cholinergic profile (Fig. 19C,
21D). A contingent of fibers
from these cases continued laterally beneath the HDB, and could be followed to
the amygdala.
Medial septum-vertical limb of the diagonal band complex.
Projections to the MS-VDB complex from the lateral hypothalamus were
generally restricted. Fibers from
far-lateral hypothalamic neurons generally did not reach as far anteriorly as
this area. Axons from neurons in the
mid-lateral hypothalamus (e.g. case L123) ascended through the medial half of
the MFB ("c" compartment) before coursing dorsally through the
septohypothalamic and septofimbrial nuclei to the septum, where they terminated
primarily within the dorsal and intermediate subdivisions of the lateral septal
nucleus. Along this course fibers
passed through the MS. These axons bore
primarily en passant varicosities and appeared to represent fibers in
transit to the more dorsal portions of the lateral septal nucleus or the
cortex. A few cholinergic neurons in
the MS were approximated by labeled terminals from this case.
Axons from the medial hypothalamus ran anteriorly in the
region medial to the MFB, coursing along the lateral aspect of the VDB and
turning sharply dorsolaterally to the septum.
Within the septum these fibers coursed through the lateral portion of MS
before continuing on to terminate heavily within the ventral and intermediate
subdivisions of the lateral septal nucleus.
Along this course, fibers and terminals were seen in close proximity to
cholinergic neuronal elements in the VDB, and in particular, the most lateral
part of the MS. Although both fibers
bearing en passant varicosities as well as those that were more highly
branched were seen in these areas, the latter type was more commonly seen
laterally in the MS. In general, axons
emanating from cells in the more rostral parts of the medial hypothalamus
(anterior hypothalamic, preoptic nuclei) appeared to contribute more of such
terminal branches, and were more frequently seen apposing cholinergic neuronal
elements, than axons from more dorsal or caudal medial hypothalamic groups
(dorsomedial, ventromedial, paraventricular hypothalamic nuclei). Examples of
arborization pattern of fibers and terminals in the ventral portion of
the MS are shown in Figures 20 and 21A, following tracer injections in the medial hypothalamus
(cases M067 and M078, respectively).
Reconstruction of the forebrain cholinergic projection system with putative hypothalamic afferents
Several of the hypothalamic cases described above were selected
for high magnification analysis at the light microscopic level (Fig. 21), in which forebrain sections
were systematically examined for appositions between PHA-L labeled terminals
and ChAT-positive elements that were suggestive of synaptic contact (e.g. Fig 21 A-D, see Materials and
Methods). The composite maps of Figure 21 E-G illustrate the
relationships between hypothalamic terminals and the forebrain cholinergic
projection system following PHA-L injections in the far-lateral hypothalamus
(case L125, Fig. 21E),
mid-lateral hypothalamus (case L123, Fig. 21F), and medial hypothalamus (case M067, Fig. 21G). The distribution of these putative contact
zones in each case corresponded to the topography of the ascending hypothalamic
fibers. As seen in Figure 21E, areas of putative
contact following PHA-L delivery to the far-lateral hypothalamus were
predominately in the SI. After tracer
delivery to the mid-lateral hypothalamus (Fig. 21F), zones of putative contact were detected in the
SI, internal capsule, HDB, and to a lesser extent within the more medial
portion of the medial septal nucleus.
From the medial hypothalamus (Fig. 21G), such arrangements were seen most often in the
MS-VDB complex, medial HDB, and to a lesser, extent within the SI.
Based upon light microscopic screening from four medial
hypothalamic cases we reconstructed 12 cholinergic neurons in serial thin
sections which were located in the septum, HDB or substantia innominata. Of the 12 neurons, synaptic contact could be
confirmed between the labeled terminals and the cholinergic neurons in 6. In the remaining cases, although the labeled
varicosity could be identified, we were unable discern synaptic contact with the
respective cholinergic cell. In those
cases synapses were either identified with unlabeled postsynaptic elements, or
obscured by dense immunoprecipitates at membrane borders.
The results of two of such correlated light/EM experiments are
shown in Figures 22 and 23. Figure 22
illustrates a cholinergic neuron in the medial part of the horizontal limb of
the diagonal band that is approached by a PHA-L labeled axon originating from
the AH (case MO76). The axon is seen to
distribute several terminal varicosities
that are in close apposition to the dendrite of this neuron (arrows in Fig. 22A). Figure 22 (parts B-D) shows at progressively higher magnification
one of these appositions. Although the double-labeling technique allows a
satisfactory distinction of the two types of profiles: the PHA-L labeled bouton
is strongly electron dense as compared to the cholinergic dendrite containing
the flocculent DAB precipitate, due to the heavy accumulation of reaction
products within both pre- and postjunctional profiles, it was not possible to
clearly identify the synaptic contact. Figure 23 shows a cholinergic neuron in the ventral SI
which is approximated by a PHA-L labeled terminal following injection of the
tracer in the medial preoptic area (case M078). This terminal was seen to form a symmetric type synapse with the
soma of this cholinergic neuron (Fig.
23D).
Many PHA-L labeled varicosities were seen at the EM level to
contact neuronal processes that were ChAT-negative. One such example is shown in Figure 23. In this
case, an unlabeled dendrite was contacted by a terminal (arrowhead in Fig. 23C) belonging to the same
axon which was seen to establish contact with the cholinergic cell body.
The present findings suggest that neurons within different
portions of the caudolateral and medial hypothalamus provide afferents to the
basal forebrain cholinergic projection system.
The putative contact sites of these inputs were distributed in relation
to the cholinergic projection system in a manner that reflected the gross
topography of the ascending hypothalamic projections. Electron microscopic investigation confirmed that medial
hypothalamic axons establish synaptic contact with cholinergic projection
neurons. A previous electron
microscopic study has demonstrated that lateral hypothalamic neurons also
contact cholinergic neurons in the SI (Zaborszky and Cullinan, '89). The topography of hypothalamic input to
these neurons may be important to our understanding of the organization of the
cholinergic projection system, however, before discussing this, it is important
to consider several other aspects of our results.
The PHA-L technique offers a number of advantages over other
anterograde tracing methods which have been the subject of a recent review
article (Gerfen et al., '89), therefore, we will limit our discussion to a few
points that are relevant to the present experiments.
The ability to more precisely define neurons contributing to a
projection represents one such advantage over the autoradiographic method. In earlier autoradiographic studies the
diffusion of isotope at the injection site often prevented this, a point which
is likely to account for a number of inconsistencies with data from PHA-L
labeled material. For example, using
autoradiography, Pfaff and coworkers (Conrad and Pfaff, '76a; Krieger et al.,
'79) reported that medial preoptic injections resulted in labeling over much of
the septum, with the highest concentration of silver grains over the dorsal
septum. According to these studies, AH
injections produced the heaviest concentration of labeling over the mid-lateral
septum, and projections from the ventromedial hypothalamic nucleus were
restricted somewhat more ventrally. In
the present study, while efferents from the ventromedial nucleus were indeed
found to project to relatively ventral parts of the lateral septum, medial
preoptic fibers were found to terminate ventral to AH axons in the lateral
septum. These findings are consistent
with those of Simerly and Swanson ('88) using the PHA-L method. The discrepancy may be due to the
involvement of the lateral preoptic area or part of the AH in the isotope
injections in the medial preoptic case.
Another example of a discrepancy with the present findings which may
have resulted from the difficulty in discerning the zone of tracer uptake using
autoradiography concerns the septal projections of the AH. According to Saper et al. ('78), the septal
projection from the AH originates mostly from the ventral cell
condensation. According to our data, AH
axons terminate in the different septal subdivisions in a complicated fashion,
but in general, both dorsal and ventral AH cells contribute to this
innervation.
Another clear advantage of the PHA-L technique over
autoradiographic tracing is the clear differentiation of terminal arborizations
and fibers of passage. For example,
Saper et al. ('76) concluded that the silver grains over the AH following
isotope injections in the ventromedial hypothalamic nucleus represented passing
fibers. However, it is evident from our
material, particularly in case M066, that in addition to fibers of passage,
axons terminate heavily in the AH.
Finally, identification of PHA-L labeled terminals can be
combined with transmitter determination of postsynaptic neuronal elements, both
at the light (Wouterlood et al., '87; Luiten et al. '88) and electron
microscopic levels (Zaborszky and Cullinan, '89; Zaborszky and Heimer, '89)
using double-labeling methods. Indeed, a major finding of the present study is
that medial hypothalamic axons terminate on cholinergic projection neurons.
Although the descending projections of the hypothalamus have been
previously characterized (Pfaff and Conrad, '78), no study has systematically
explored the organization of ascending hypothalamic projections, with the
exception of some preliminary observations of Veening et al. ('82). The present data, together with that from
fiberarchitectural studies, may have important implications for the
organization of afferents to the forebrain cholinergic projection system.
Ascending fibers from hypothalamic cases traveled within the MFB
and region medial to it, where a strict lateral to medial order was
maintained. Axons from far-lateral
hypothalamic neurons (case L125) coursed laterally in the MFB (in the
"d" and "e" compartments of Niewenhuys et al., '82)
terminating primarily within the SI and BSt, with only a few scattered fibers
reaching as far anteriorly as the septum.
Axons emanating from more medially located lateral hypothalamic neurons
(e.g. cases L105, L110) traveled more medially in the MFB. Finally, ascending axons from medial
hypothalamic nuclei traveled primarily in the medial portion of the MFB and
region medial to it. Although overlap
existed, a continuous lateral to medial shift was evident with respect to the
locations of ascending hypothalamic axons in the MFB. This principle of organization is consistent with the notion of
Nieuwenhuys et al. ('82) that compartmentalization within the MFB is generally
maintained throughout its extent.
The latero-medial topography of hypothalamic axons ascending
within the MFB was reflected in the distribution of fibers and terminals in the
bed nucleus of the stria terminalis, where more laterally located cells
(eg. cases L125, L123) projected to more lateral areas, while more medially
placed neurons (e.g. cases M066, M067) terminated in progressively more medial
regions, although considerable overlap was apparent.
The latero-medial order of ascending hypothalamic fibers was
maintained as far rostrally as the ventral part of the septum. However, an inverse relationship was
apparent more dorsally in the septum, where axons that reached the septum from
more lateral hypothalamic neurons were found medial to those from more medial
hypothalamic cells. Lateral
hypothalamic fibers were seen to terminate in the intermediate and dorsal
portions of the lateral septal nucleus, while medial hypothalamic axons turned
in a dorsolateral arc before terminating primarily in the ventral and
intermediate divisions of the lateral septum.
In general, topographical relationships according to the
rostro-caudal and dorso-ventral locations of hypothalamic injections were
difficult to distinguish, although some subtle differences were noted. For example, in the lateral septum, lateral
hypothalamic axons which originated more dorsally (cases L123, L126) were
detected in the dorsal part of the lateral septal nucleus, while more ventrally
located cells (L104, L124) projected to more ventral and intermediate
portions. Another example is the BSt,
where axons emanating from more dorsal portions of the medial hypothalamus
(e.g. case M067) terminated more dorsally than those originating from more
ventral regions (e.g. case M078).
Organization
of hypothalamic input to the forebrain cholinergic projection system
An important finding from the present study is that the
distribution pattern of hypothalamic inputs to the cholinergic projection
system corresponded to the topography of the ascending fibers. These relationships become more clear when
the cholinergic cells and their afferents are viewed with respect to parcellation of the MFB as defined by Nieuwenhuys
et al ('82). Figure 24 illustrates the
terminal arborizations from three cases projected onto a single coronal section
in relation to the positions of cholinergic neurons and the compartments of the
MFB. The terminal field from the
far-lateral hypothalamic case (L125) was located in the SI, an area which
corresponds to the "d" and "e" compartments of the
MFB. In this case, the labeled cells at
the injection site were located mainly in the area corresponding to the
"e" compartment, and the ascending axons were restricted to the
"d" and "e" compartments.
The majority of the projection from the mid-lateral hypothalamic case
(L123) terminated in the lateral preoptic area, which corresponds to the
"c" compartment.
Interestingly, it was the "c" compartment which contained most
of the labeled cells at the injection site, as well as the ascending
fibers. However, ascending fibers from
case L123 were also seen within other components of the MFB, which were likely
to contribute to the terminal labeling in the SI, ventral globus pallidus,
internal capsule and MCP-HDB seen at more rostral forebrain levels from this
case. Medial hypothalamic fibers that
coursed in the ventromedial portion of the MFB ("b" compartment) or
region medial to it, were found to distribute terminal networks in a
corresponding region, the medial portion of the HDB.
The results of the present experiment appear not only to confirm
the suggestions of Nieuwenhuys et al. ('82) and Veening et al. ('82) that there
is a high degree of constancy in the distribution of the various components of
the MFB, but also add several new findings which are likely to be relevant to
the organization of the basal forebrain in general. Firstly, the particular compartment occupied by a given
hypothalamic axonal projection was not strictly related to the location of the
cells of origin of the projection, but more to the position of these fibers
within the MFB after their initial course.
Secondly, of fiber contingents that coursed caudo-rostrally in the MFB,
those that were confined to a particular compartment caudally tended to have
terminal zones in the rostral forebrain corresponding to the same
compartment. Thirdly, projections in
which fibers ascended through multiple compartments of the MFB tended to
innervate the forebrain more diffusely.
It seems that these principles of organization may apply not only
to axons of hypothalamic origin, but also to those from the brainstem which
course through the MFB for considerable distances. For example, the study by Veening et al. ('82) demonstrated that
parabrachial efferents have a clear preference for the dorsolateral portion of
the MFB ("e" compartment) and
retain this position throughout the MFB.
Grove ('88) showed using the PHA-L technique that efferents from the
lateral parabrachial area terminate in the corresponding forebrain region (the
SI), similar to our case L125. In
contrast, locus coeruleus efferents take a relatively diffuse course through
the MFB, and are distributed in extensive regions of the basal forebrain
(Zaborszky et al., in preparation). A
precise localization of different ascending brainstem systems in relation to
the compartments of the MFB (see Satoh and Fibiger, '86) may therefore be of
value in guiding studies directed at defining inputs to chemically specific
neurons in the basal forebrain, including those of the cholinergic projection
system.
Technical considerations. Previous evidence has indicated that cholinergic neurons receive
relatively few inputs along their cell bodies and proximal dendrites, although
the density of contacts was found to increase on more distal dendritic segments
(Ingham et al., '85). Our capacity for
identifying PHA-L labeled terminals in contact with these neurons was enhanced
by the fact that our ChAT immunostaining labeled dendrites for several hundred
microns. Conversely, in order to
preserve reasonable ultrastructure, only low amounts of detergent could be used
in EM preparations, resulting in a dramatic decline of detectable PHA-L
labeling. It therefore became clear
that to assess the organization of hypothalamic inputs in relation to the
cholinergic projection system, an alternative approach was required in which
potential sites of contact were mapped under high magnification light
microscopy (e.g. Fig. 21). In this case, relatively high detergent
levels were used during processing to facilitate immunolabeling, and identified
terminals in direct apposition to cholinergic neuronal elements were recorded
using the same criteria as that used to select cases from embedded sections in
correlated light/EM experiments (see Materials and Methods). This approach is subject to several
limitations, however. For example, not
all of the contacts so identified are likely to represent true synapses, since
at the EM level labeled terminals directly abutting cholinergic processes were
sometimes found to terminate on unlabeled elements. Using the present criteria in EM studies, however, we were able
to confirm synaptic contact in half of
the cases. In addition, despite
increased levels of detergent, penetration of the PHA-L antibody was limited to
a few microns of the section, unlike the ChAT antibody which appeared to label
cholinergic elements throughout most of the section. In view of this constraint, together with the fact that the more
distal portions of the cholinergic dendrites were not labeled, many putative
contacts are likely to have gone undetected.
In any case, the present approach can give insight only into the pattern
of innervation from a given hypothalamic locus, rather than generate
quantitative data for comparison. It
should also be noted that some cholinergic dendrites that received putative
contacts could not be traced to the cells from which they originated. However, since cholinergic dendrites
appeared to be labeled for up to 200-300 um, such contacts would have occurred
within this distance of their parent cell bodies, and therefore this factor is
unlikely to have significantly distorted the innervation pattern. In light of the limitations of the present
study, it is clear that more detailed electron microscopic analyses are
required to assess the significance of hypothalamic input to the forebrain
cholinergic system at the single neuron level, and await the application of
sophisticated combined techniques capable of revealing more complete portions
of cholinergic dendrites together with their afferents.
The topography of corticopetal cholinergic projections has
been extensively investigated (Lamour et al., '82; Mesulam et al., '83; Saper
et al., '84; Rye et al., '84; Woolf et al., '84, '86; Luiten et al., '87), and
a comparison of this data with the present findings suggest a few general
conclusions. For example, anterior and
medial cortical areas, which are innervated by cholinergic cells located in the
more rostral and medial SI, may be preferentially innervated by hypothalamic
neurons located more medially within the lateral hypothalamus. Posterior and lateral cortical areas, which
appear to innervated by cholinergic neurons situated more caudally and
laterally within the SI, may receive afferents preferentially from cells
located more laterally within the lateral hypothalamus.
Hippocampopetal cholinergic neurons are located in the
MS-VDB complex (Amaral and Kurz, '85; Gaykema et al., '90) and are likely to
receive input from medial hypothalamic groups, particularly from the anterior
hypothalamic and medial preoptic areas.
Mid-lateral hypothalamic cells may also influence the hippocampus
through connections with cholinergic neurons in the dorsomedial portion of the
septum.
A more complicated arrangement appears to involve the medial
portion of the HDB. A close comparison
of available retrograde tracing data indicates that cholinergic neurons within
this area may project to the cingulate cortex, occipital cortex, or olfactory
bulb (Rye et al., '84; Woolf and Butcher, '84; Amaral and Kurz, '85; Zaborszky
et al., '86a). A common feature of
medial hypothalamic cell groups is a projection to this region, and it remains
to be determined whether these afferents discriminate among cholinergic neurons
in the HDB on the basis of the efferent targets of the cholinergic cells. This question requires investigation through
combined techniques capable of revealing cholinergic neurons, their projection
targets, as well as their afferent connections in the same experiment.
Functional organization of the forebrain cholinergic projection system.
The basal forebrain cholinergic projection system has been
suggested to be involved in an number of behavioral processes, including
attention, learning, and memory (for reviews see Deutsch et al. '83; Hagan and
Morris, '88), although the precise neuronal circuits involved are presently
unclear. Electrophysiological
experiments have also implicated cholinergic mechanisms in cortical sensory
processing (Sillito and Kemp, '83; Sato et al., '87; Donoghue and Carol, '87;
Sillito and Murphy, '87; Metherate et al., '88a,b; Ma et al., '89). Although many of these studies have
suggested a permissive role for acetylcholine, generally facilitating the
responses of cortical neurons to other inputs, recent data has suggested that
ACh may influence information processing in a more specific manner. For example, frequency-specific alterations
in receptive field properties of neurons in the auditory cortex have been
reported following application of cholinergic agents in the unanesthetized cat
(Ashe et al., '89; McKenna et al., '89).
In addition, morphological evidence indicating a) a topographical
organization of efferent projections of forebrain cholinergic projection
neurons (McKinney et al., '83; Mesulam et al., '83; Rye et al., '84; Amaral and
Kurz, '85), b) regionally specific patterns of cortical cholinergic innervation
(Eckenstein et al., '88; Lysakowski et al., '89), as well as c) differential
distributions of afferents to subsets of cholinergic projection neurons as shown
in the present study, and suggested in several other recent papers (Semba and
Fibiger, '89; Zaborszky, '89; Zaborszky et al., '90), are consistent with the
notion that this system may be organized to allow for relatively selective
information flow to functionally distinct cortical regions. On the other hand, more global cortical
functions (e.g. arousal) have been suggested to be mediated, at least in part,
by the forebrain cholinergic projection system (Buzsaki et al., '88; Steriade
and McCarley, '90). Such proposed
effects might be subserved by the cholinergic projection system through more
diffuse afferents, such as those recently described from brainstem
catecholaminergic systems (Zaborszky et al., submitted). In any case, specific vs global functions of
this system may not be mutually exclusive alternatives, in that either
mechanism might predominate depending on the current prevailing state of afferent control.
Significance of hypothalamic input to forebrain cholinergic projection neurons.
Behavioral
and electrophysiological experiments may provide clues to the nature of the
information relayed to forebrain cholinergic projection neurons from the
hypothalamus, particularly from the lateral hypothalamus. Studies in primates have revealed that nucleus
basalis, presumably cholinergic, neurons respond to the sight of food only if
the animal is hungry (Burton et al., '76; Mora et al., '76; Rolls, '79). Changes in the discharge rates of these
neurons have also been found to be related to sensory stimuli that are novel,
appetitive, aversive, or rewarding, suggesting that the responses reflect these
dimensions rather than encoding specific sensory stimuli (DeLong, '71; Rolls,
'87; Richardson and DeLong, '88). In
view of a role for the lateral hypothalamus in the regulation of consummatory
behavior (Oomura and Yashimatsu, '84; Dunnett et al., '85; Fukuda et al., '86)
and as an integrative center for visceral information (Norgren, '70; Oomura,
'80; Jeanningros, '84; Cechetto, '87),
it is possible that lateral hypothalamic projections may be a route by which
this viscerosensory input reaches the cholinergic projection neurons. It is not known whether these hypothalamic
projections to forebrain cholinergic neurons are primary projections or
collaterals of fibers destined for the cortex, and thus part of a 'diffuse
corticopetal system' as described by Saper (1985).
The situation for the medial hypothalamus is less clear, as this
region is thought to participate in the control of a number of neuroendocrine,
autonomic, and behavioral mechanisms (Zaborszky, '82; Swanson, '87). Insight into nature of the information
sampled and relayed to cholinergic neurons from hypothalamic cell groups is
likely to come from knowledge of the neurotransmitters involved in these
projections, as well as an understanding of the functional impact of these
afferents obtained through further detailed anatomical, pharmacological, and
physiological studies.
The authors wish to express their sincere appreciation to Mr. F.
L. Snavely and Ms. V. Alones for expert technical assistance with the electron
microscopy, Ms. C. Allen for assistance with camera lucida drawings, and Mr. L.
Clarke of the University Printing Services for photographic expertise in
production of figures. This work was
supported by USPHS grant NS 23945 and 17743, and a training grant in behavioral
neurosciences MH 18411 (W.E.C.).
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Záborszky, L., V.N. Luine, W.E. Cullinan, and L. Heimer (1989) Direct catecholaminergic-cholinergic interactions in the basal forebrain: Morphological and biochemical studies. Neuroscience (submitted).
Fig. 1. A-F: Series of drawings made from coronal sections
through a rat brain (rostral to caudal) that have been immunostained for ChAT,
illustrating the distribution of cholinergic neurons (dots). Striatal cholinergic neurons (including
those in the ventral striatum) have been omitted for simplicity. Adjacent sections were Nissl counterstained
and/or immunostained for GAD to aid in the delineation of nuclear and regional
borders. Approximate distance from bregma in mm. A-F: +0.5, +O.1, -0.4, -1.0,
-1.9, -3.1.
Fig. 2. Typical PHA-L injection, in this case located in the
ventromedial hypothalamic nucleus (case M065), as shown using the avidin-biotin
peroxidase method. Scale bar: 0.5 mm.
Fig. 3. Darkfield photomicrographs of the forebrain distribution of
PHA-L labeled fibers and terminals following tracer delivery in two lateral
hypothalamic cases (A: case L123, B: case L110). Note the differential distribution of PHA-L
labeled fibers approximately at the same forebrain level. Scale bar: 100
μm.
Fig. 4. Composite diagram of hypothalamic injection sites.
Fig. 5 and 6. Camera
lucida drawings of coronal forebrain
sections, arranged from rostral (5A) to
caudal (6B) to illustrate the distribution of PHA-L labeled fibers in relation
to cholinergic neurons following tracer deposit in the far-lateral hypothalamus
(case L125). The hatched area in the inset shows the maximum extent of the
region containing neurons labeled at
the injection site. In these and the following figures (Figs. 7-17) only the most proximal dendritic segments of cholinergic
neurons were drawn, and most of the dorsal and ventral striatal cholinergic
neurons have been omitted for simplicity. Sections were Nissl counterstained,
and adjacent sections immunostained for GAD for delineation of pallidal
borders.
Fig. 7 and 8. Camera lucida drawing of forebrain coronal sections, arranged from
rostral (7A) to caudal (8B), to show the distribution of PHA-L labeled fibers
originating from the mid-lateral hypothalamus (case L123; inset) in relation to
cholinergic projection neurons.
Fig. 9. Distribution of PHA-L labeled fibers originating from the
lateral hypothalamus (case L126) in relation to cholinergic projection neurons
at the level of the caudal globus pallidus.
Fig. 10 and 11. Camera
lucida drawing of forebrain coronal sections, arranged from rostral (10A) to
caudal (11B), to illustrate the distribution of PHA-L labeled fibers
originating from injection site L110 (inset) in relation to cholinergic
projection neurons.
Fig. 12 and 13. Distribution
of PHA-L labeled fibers originating in the dorsomedial portion of the
ventromedial nucleus (case M066, inset) in relation to cholinergic neurons
plotted onto line drawings of four representative coronal sections (12A,
rostral; 13B, caudal).
Fig. 14 and 15. Distribution
of PHA-L labeled fibers originating in the dorsal part of the anterior
hypothalamic area (case M067, inset) in
relation to cholinergic projection neurons in four forebrain levels (14A, rostral; 15B, caudal).
Fig. 16 and 17. Distribution of PHA-L labeled fibers originating in the medial preoptic
area (case M078, inset) in relation to cholinergic projection neurons plotted
from four coronal sections, arranged from rostral (16A) to caudal (17B).
Fig. 18. D: Camera lucida drawing (63x) made
from a region shown in box in (A), illustrating the relationship of PHA-L
labeled fibers/terminals and ChAT labeled neurons in the sublenticular
substantia innominata following a tracer injection in the lateral hypothalamus
(case L125). B,C: Micrographs of neurons in boxed areas in (D)
shown at higher magnification. Arrows
denote PHA-L labeled terminal varicosities in close apposition to cholinergic
neurons. Scale bar: 10 μm
Fig. 19. D: Camera lucida drawing (63X) made
from region shown in box in (A) illustrating the relationship of PHA-L
labeled fibers/terminals and ChAT labeled neurons in the medial portion of the
HDB following tracer delivery to the anterior hypothalamic area (case
M067). B,C: Micrographs of
neurons in boxed areas in (D) shown at higher magnification. Arrows denote PHA-L labeled terminal
varicosities adjacent to cholinergic neuronal elements. Scale bar: 10 μm.
Fig. 20. D: Camera lucida drawing (63X) made
from region shown in box in (A) depicting the distribution of PHA-L
labeled fibers/terminals and ChAT labeled neurons in the medial septal
nucleus-vertical limb of the diagonal band following tracer delivery to the
anterior hypothalamic area (case M067).
B,C: High magnification micrographs of boxed regions in (D).
Arrows denote PHA-L labeled
terminal varicosities in
close approximation to cholinergic neuronal elements. Scale bar: 10
μm.
Fig. 21. A: Color micrograph illustrating ChAT
labeled cell body in the medial septum
approximated by PHA-L labeled terminal varicosities (arrows) following a PHA-L
injection in the medial hypothalamus (case M078). B: Several PHA-L
labeled terminal varicosities (arrows) from a PHA-L labeled axon are seen in
close proximity to a distal cholinergic dendrite in the substantia innominata
following a PHA-L injection in the lateral hypothalamus (case L125). C: PHA-L labeled terminal
varicosities are seen abutting proximal dendrites (arrows) of two ChAT labeled
neurons in the substantia innominata following a tracer injection in the
lateral hypothalamus (case L123). The terminal varicosities shown are from the
same axon (arrowhead). D: PHA-L labeled terminal varicosities in
proximity to a proximal dendrite of ChAT labeled cell in the dorsal HDB from
case L123. The grid simulates the
proportions of the ocular reticle used to screen sections from high
magnification light microscopic analysis.
Scale bar (A-D): 16 μm (one division of grid in D). E-G:
Composite maps illustrating putative zones of contact between afferent fibers
and cholinergic neuronal elements following PHA-L injections into the (E)
far-lateral hypothalamus (case L125), (F) mid-lateral hypothalamus (case L123),
and (G) medial hypothalamus (case M067).
These maps were composed from 8 camera lucida drawings which were
aligned and superimposed to generate the final figure. Cholinergic neurons are represented by black
dots. Zones of putative contacts
between cholinergic elements and PHA-L labeled terminals are depicted as red
squares (corresponding to 80x80 μm areas in the section).
Fig. 22. A: Reconstructed ChAT labeled neuron located
in the medial portion of the HDB (indicated by asterisk in upper right inset)
whose long dendrites are approximated by a number of PHA-L labeled varicosities
(arrows) originating in the anterior hypothalamic area (case M076). Curved arrows point where the left dendritic
branch of this cholinergic neuron is
lost from the plane of focus, but the same axon distribute several additional
varicosities (arrowheads). B: Low power electron micrograph showing
the perikaryon of the identified neuron (arrowhead). Vessel in boxed area indicated by asterisk
is same as in A and C. C: Higher
power electron micrograph from boxed area in B. D: PHA-L labeled terminal bouton (star) abutting on the
immunolabeled dendrite (arrowhead) from region boxed in C. Arrow points to
synaptic vesicles in the nickel intensified axonterminal. Scale bars: A=10
μm, B=10 μm, C= 2.5 μm, D=1 μm.
Fig. 23. B: High power light micrograph shows
a ChAT-positive neuron (from region
denoted by asterisk in A) which
is approached by a PHA-L labeled fiber bearing several terminal varicosities,
one of which is seen in apposition to the soma (arrow). The PHA-L injection
site was located in the medial preoptic area (case M 078). C: Low power
electron micrograph showing the ChAT-positive cell body. Boxed area contains
the PHA-L varicosity indicated by arrow
in (B). Arrowhead denotes the other PHA-L labeled terminal shown with the same symbol in (B), which contacts an unlabeled
dendrite. D: High power electron
micrograph from boxed
region in (C). Symmetric contact
between PHA-L labeled terminal and ChAT-positive cell is denoted by arrowheads.
Scale bar: 1 μm.
Fig. 24. A: Location of cells labeled at the
injection site from case L125 in relation to the compartments of the MFB (bold
lower case letters) as defined by Nieuwenhuys et al. ('82). B: Location
of neurons labeled at the injection site from case L123 in relation to the MFB.
C: Areas of terminal arborization from cases L125, L123, and M067
plotted at a single forebrain level in relation to the compartments of the MFB
(a-e) and the positions of cholinergic neurons (dots). Compartments a, b and c, which are
easily visible under dark field illumination, are delineated by
continuous lines, interrupted lines mark the arbitrary borders of compartments
d and e. Hatching represents areas of dense fiber/terminal labeling, whereas
stippling denotes area of less dense labeling in case L123.
ac anterior commissure
Acb accumbens nucleus
AD anterodorsal thalamic nucleus
AH anterior
hypothalamic area
Arc arcuate nucleus
AV anteroventral
thalamic nucleus
AVP anteroventral
preoptic nucleus
BL basolateral
amygdaloid nucleus
BSt bed nucleus of the
stria terminalis
CA central
amygdaloid nucleus
CP caudate putamen
DM dorsomedial
hypothalamic nucleus
f fornix
GP globus pallidus
HDB horizontal limb
diagonal band nucleus
HI hippocampus
ic internal
capsule
LA lateroanterior
hypothalamic nucleus
LH lateral
hypothalamus
lo lateral olfactory
tract
LOT nucleus lateral
olfactory tract
LPO lateral preoptic
area
LSd lateral septal
nucleus, dorsal
LSi lateral septal
nucleus, intermediate
LSv lateral septal nucleus, ventral
LV lateral ventricle
MCP magnocellular
preoptic nucleus
MD mediodorsal
thalamic nucleus
ME median eminence
MFB medial forebrain
bundle
MP medial preoptic
nucleus
MPa medial preoptic area
MS medial septal
nucleus
mt mammillothalamic
tract
ot optic tract
ox optic chiasm
PM premammillary
nucleus
PT paratenial
thalamic nucleus
PV paraventricular
hypothalamic nucleus
Re reuniens thalamic
nucleus
Rt reticular
thalamic nucleus
SCh suprachiasmatic
nucleus
SHy septohypothalamic
nucleus
SI sublenticular
substantia innominata
sm stria medullaris
SO supraoptic nucleus
sox supraoptic
decussation
st stria terminalis
Sth subthalamic
nucleus
SubI subincertal nucleus
Tu olfactory
tubercle
VDB vertical limb
diagonal band nucleus
VM ventromedial
hypothalamic nucleus
VP ventral pallidum
ZI zona incerta
3V third ventricle