ORGANIZATION OF ASCENDING HYPOTHALAMIC PROJECTIONS TO THE ROSTRAL FOREBRAIN WITH SPECIAL REFERENCE TO THE INNERVATION OF CHOLINERGIC PROJECTION NEURONS

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William E. Cullinan* and László Záborszky

 

Departments of Otolaryngology, Neurosurgery, and Neurology University of Virginia Health Sciences Center, Charlottesville, VA 22908

 

 

                         

ABSTRACT

  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

 

 

 

 

INTRODUCTION

 

  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.

   

 

MATERIALS AND METHODS

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.

    

Immunocytochemistry

  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.

    

Data analysis

  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.

                     

 

RESULTS

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).  

 

PHA-L injections

  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.   

 

Projections from the lateral hypothalamus

  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).

 

Additional lateral hypothalamic cases

  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.  

 

Ventromedial hypothalamic 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.

 

Dorsomedial hypothalamic nucleus

  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.

 

Paraventricular hypothalamic nucleus

  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).

 

Projections from medial preoptic area

  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.                      

 

Electron microscopy

  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.

 

 

DISCUSSION

  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.

 

Advantages of the PHA-L method with reference to prior studies

  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.

      

Organization of ascending hypothalamic fibers in the forebrain

  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.

 

Comparison with efferent projections of cholinergic neurons

  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.

 

 

CONCLUSIONS

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. 

                      

 

 

 

ACKNOWLEDGMENTS

 

  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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Figure legends

 

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.

 

 

 

Abbreviations

 

                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