antisheep antiserum (ICN Immunobiologicals, Irvine, CAI and sheep PAP ...... analysis and Beverley Lindsay for secretarial assistance. We also recognize Prof.
THE JOURNAL OF COMPARATIVE NEUROLOGY 339251-268 (1994)
Projections of GABAergic and Cholinergic Basal Forebrain and GABAergic Preoptic-AnteriorHypothalamic Neurons to the Posterior Lateral Hypothalamus of the Rat IVANA GRITTI, LYNDA MAINVILLE, AND BARBARA E. JONES Istituto di Fisiologia Umana 11, Universita degli Studi di Milano, Milan, Italy 20133 (I.G.); Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4 (L.M., B.E.J.)
ABSTRACT Within the basal forebrain, y-aminobutyric acid (GABA)-synthesizingneurons are codistributed with acetylcholine-synthesizing neurons (Gritti et al. 119931 J. Comp. Neurol. 329:438457), which constitute one of the major forebrain sources of subcortical afferents to the cerebral cortex, In the present study, descending projections of the GABAergic and cholinergic neurons were investigated to the lateral posterior hypothalamus (LHp) through which the medial forebrain bundle passes and where another major forebrain source of subcortical afferents is situated. Retrograde transport of cholera toxin b subunit (CT) from the LHp was combined with immunohistochemical staining for glutamic acid decarboxylase (GAD) and choline acetyl transferase (ChAT) using a sequential peroxidase-antiperoxidase (PAP)technique. A relatively large number of GAD+ neurons (estimated at 6,2001, which represented > 15%of the total population of GAD+ cells in the basal forebrain (estimated at 39,000), were retrogradely labeled from the LHp. These cells were distributed through the basal forebrain cell groups, where C U T + cells are also located, including the medial septum and diagonal band nuclei, the magnocellular preoptic nucleus, and the substantia innominata, with few cells in the globus pallidus. In these same nuclei, a small number of C U T + cells were retrogradely labeled (estimated at -8001, which represented only a small percentage (15%) of the C U T + cell population in the basal forebrain (estimated at -18,000). Both the GAD+ and C U T + LHp-projecting neurons represented a small subset of their respective populations in the basal forebrain, distinct from the magnocellular, presumed cortically projecting, basal neurons. In addition to the GAD+ cells in the basal forebrain, GAD+ cells in the adjacent preoptic and anterior hypothalamic regions were also retrogradely labeled in significant numbers (estimated at 5,500) and proportion ( > 20%) of the total population (estimated at 30,000) from the LHp. The retrogradely labeled GAD+ neurons were distributed in continuity with those in the basal forebrain through the lateral preoptic area, medial preoptic area, bed nucleus of the stria terminalis, and anterior and dorsal hypothalamic areas. Of the large number of cells that project to the LHp in the basal forebrain and preoptic-anterior hypothalamic regions (estimated at 66,0001, the GAD+ neurons represented a significant proportion ( > 15%) and the C U T + neurons a very small proportion ( - 2%). The relative magnitude of the GABAergic projection suggests that it may represent an important inhibitory influence of the descending efferent output from the basal forebrain and preoptic-anterior hypothalamic regions. This influence could suppress the activity of neurons in the posterior lateral hypothalamus that are involved in establishing the vegetative, somatomotor, and electrocortical components of the waking state and behaviors. o 1994 Wiley-Liss, Inc.
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Accepted July 30,1993. Address reprint requests to Dr. B.E. Jones, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4.
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1994 WILEY-LISS. INC.
I. GRITTI ET AL.
252 Key words: glutamic acid decarboxylase, choline acetyl transferase, cholera toxin, substantia innominata,septum-diagonalband
Lying among the cholinergic neurons of the basal magnocellular nucleus, gamma-aminobutyric acid (GABA)-ergic neurons of multiple shapes and sizes densely populate the basal forebrain (Brashear et al., '86; Gritti et al., '93; Kohler et al., '84a; Mugnaini and Oertel, '85). Of this GABAergic cell population, a certain contingent projects in parallel with the magnocellular cholinergic neurons to regions of the cerebral cortex (Fisher et al., '88; Freund and Antal, '88; Freund and Meskenaite, '92; Kohler et al., '84a; Zaborszky et al., '86a). Another may serve as interneurons and as a potential source of GABAergic innervation to the nearby cortically projecting cholinergic neurons (Gritti et al., '93; Ingham et al., '88; Leranth and Frotscher, '89; Zaborszky et al., '86b). A certain proportion may also, like the GABAergic neurons of the globus pallidus, give rise to descending projections into the caudal diencephalon andlor brainstem (Oertel and Mugnaini, '84; Schmued et al., '89; Smith et al., '90). In fact, such a descendingprojection could exert inhibitory control over the other major forebrain group of cortically projecting neurons that are concentrated in the posterior lateral hypothalamus (Kievit and Kuypers, '75;Saper, '85). Projections from basal forebrain GABAergic neurons to the posterior hypothalamus could be of particular functional importance in the control of cortical activation and behavioral state. Since the early clinical observations of von Economo ('31) and the early laboratory experiments of Nauta ('46) employing transections of the forebrain and of Hess ('57) employing electrical stimulation, it has been hypothesized that the basal forebrain and preoptic-anterior hypothalamic regions play an important role in the initiation and maintenance of sleep and act in opposition to the posterior hypothalamus, which had been shown by lesion and stimulation to be critically important for waking, as the
rostra1 part of the ascending reticular activating system (Lindsley et al., '50). More recently, support for this hypothesis has been supplied by experiments showing that neurotoxic lesions in the basal forebrain or preopticanterior hypothalamic areas produce a loss of slow wave sleep that can be reversed by injections of muscimol into the posterior hypothalamus (Sallanon et al., '89; Szymusiak and McGinty, '86a). It was the aim of the present study to determine whether GABAergic neurons in the basal forebrain project to the posterior lateral hypothalamus (LHp) and whether their neighboring cholinergic neurons project or fail to project to this same area. The area of the hypothalamus selected corresponds to the region through which the medial forebrain bundle passes (Geeraedts et al., 'gob), spanning the lateral hypothalamus from the caudal tuberal through the mammillary region, and corresponds to the area from which neurons have been shown to project in a topographically organized manner to all regions of the cerebral cortex (Saper, '85). Although peptides have been identified in some of the cortically projecting neurons in this area, the primary neurotransmitter of these cells is unknown (Kohler et al., '84b; Saper, '85; Saper et al., '86; Vincent et al., '83). Cells have been recorded in this area that are tonically active in association with cortical activation and the waking state (Sakai et al., '90a; Vanni-Mercier et al., '84). These cells of the posterior lateral hypothalamic area are distinct from the magnocellular neurons of the tuberomammillary nucleus, which are aligned on the ventral surface of the hypothalamus and give rise to diffuse cortical projections (Saper, '85). The tuberomammillary neurons, which contain the synthetic enzyme for GABA (glutamic acid decarboxylase) in addition to histamine (Ericson et al., '87; Panula et al., '89; Vincent et al., '83) and would also appear
Abbreviations
AAA ac Acb AHA Arc BST CeA CP CPU DBB
DHA DM EP f F fr FStr GP ic LH LHa LHP LPOA LS
MCPO MeA
anterior amygdaloid area anterior commissure accumbens nucleus anterior hypothalamic area arcuate hypothalamic nucleus bed nucleus of the stria terminalis central amygdaloid nucleus cerebral peduncle caudate putamen diagonal band of Broca nucleus dorsal hypothalamic area dorsomedial hypothalamic nucleus entopeduncular nucleus fornix nucleus Forel's fields fasciculus retroflexus fundus striati globus pallidus internal capsule lateral hypothalamus anterior lateral hypothalamus posterior lateral hypothalamus lateral preoptic area lateral septum magnocellular preoptic nucleus medial amygdaloid nucleus
ml MPA MS mt MTu oc ot OTu PF PHA Pir Po PV Ret SCh SIa SIP sm
so
STh
3v VMH
w
VPM
ZI
medial lemniscus medial preoptic area medial septum mammillothalamictract medial tuberal nucleus optic chiasm optic tract olfactory tubercle parafascicular nucleus of the thalamus posterior hypothalamic area piriform cortex posterior thalamic nuclear group paraventricular hypothalamic nucleus reticular thalamic nucleus suprachiasmatic nucleus substantia innominata pars anterior substantia innominata pars posterior stria medullaris supraoptic nucleus subthalamic nucleus third ventricle ventromedial hypothalamic nucleus ventroposterior thalamic nucleus ventral premammillary nucleus zona incerta
GABAERGIC/CHOLINERGIC PROJECTIONS to be important in waking processes (Sakai et al., '9Oa; Vanni-Mercier et al., '84), were not targeted in the present study. To examine the projections of the GABAergic and cholinergic forebrain neurons, cholera toxin b subunit (CT) was injected into the LHp for study of retrograde labeling. The brains were processed by a sequential dual-staining peroxidase-antiperoxidase (PAP) immunohistochemical procedure to reveal CT together with glutamic acid decarboxylase (GAD) or choline acetyl transferase (ChAT).The analysis revealed a significant proportion of GAD+ neurons that project to the LHp and only a tiny proportion of ChAT+ neurons that do so from the basal forebrain. According to their size, the ChAT+ and GAD+ LHp-projecting neurons represent a subset of the cholinergic and GABAergic neurons in the basal forebrain, made up of smaller cells. A large population of similar GAD + LHp-projecting neurons also extends across the lateral and medial preoptic-anterior hypothalamic regions. Some of these results have been presented in preliminary form (Gritti et al., '91).
MATERIALS AND METHODS Male Wistar (Charles River) rats, weighing approximately 250 g, were used in this study. For surgery or sacrifice, the animals were anaesthetized with pentobarbital anaesthesia (Somnotol; 60-67 mgikg). Cholera toxin b subunit (choleragenoid; LIST Biological Laboratories, Campbell, CA) was injected into the LHp of eight animals under anesthesia. A solution of 1% CT in Tris buffer was injected in a volume of 50 nl. The injections were made with a 1 pl Hamilton syringe to which an oil-filled glass micropipette (tip diameter 50-100 pm) was attached and sealed. The pipette was placed stereotaxically in the LHp according to the following coordinates (with reference to ear bar zero, Paxinos and Watson, '86): AP (anteriorposterior) +4.8, V (vertical) +1.4, and L (lateral) -1.5 (right side). The CT solution was injected over 15-20 minutes and the pipette subsequently left in place for about 5 minutes. Twenty-four hours after the CT injections, colchicine (Sigma, St. Louis, MO; 60 pg in 30 pl) was injected into the left ventricle (AP -0.8, V -4.3, L + 1.5) in order to maximize visualization of GAD. Forty-eight hours after the CT injections, the animals were sacrificed under anesthesia and the brains fixed by the intraaortic perfusion of 3%paraformaldehyde and 0.2% picric acid in phosphate buffer (0.1 M) (followed by 10% sucrose) as described previously (Gritti et al., '93). After removal, the brains were placed in a 30% sucrose solution overnight and subsequently frozen for storage at -80°C until processing. Coronal sections were cut at 25 pm thickness on a freezing microtome, and three adjacent series of sections were collected every 400 pm. Sections were processed for immunohistochemistry by the peroxidase-antiperoxidase (PAP) technique (Sternberger, '79), as previously described for ChAT and GAD (Gritti et al., '93). For double labeling of CT with ChAT or GAD, a sequential PAP procedure was used for the visualization of CT with 3,3'-diaminobenzidine (DAB) plus Nickel as chromogen in the first position and GAD or ChAT with DAB alone in the second position. A goat anticholeragenoid antiserum (LIST Biological Laboratories) was employed at a dilution of 1:40,000 (in Tris-saline solution containing 2% bovine serum albumin and 0.1% sodium azide) overnight at room temperature, followed by
253 rabbit antigoat antiserum (Jackson Immunoresearch Labs, West Grove, PA) and goat PAP (Dakopatts, Glostmp, Denmark). The sections were rinsed several times between steps (in Tris-saline containing 1% normal rabbit serum) and were treated with 0.05% DAB (Sigma) containing 0.005% hydrogen peroxide (HzOz)and 0.6% nickel ammonium sulphate in 0.1 M Tris-water (pH 7.4) for 10 minutes. In one series, CT stained sections were counterstained for Nissl with thionine. For sequential staining of ChAT, another series was immunostained with a monoclonal antibody from rat (Boehringer Mannheim, Mannheim, Germany) followed by a rabbit antirat antiserum (Jackson Immunoresearch Laboratories) and rat PAP (Sternberger Monoclonals, Baltimore, MD), as described previously (Gritti et al., '93). For sequential staining of GAD, a third series was immunostained with an anti-GAD antiserum raised in sheep [kindly supplied by Enrico Mugnaini (Oertel et al., '81, '82)] at a dilution of 1:2,000, followed by rabbit antisheep antiserum (ICN Immunobiologicals, Irvine, CAI and sheep PAP (Jackson Immunoresearch Labs) as described previously (Gritti et al., '93). For ChAT and GAD, the peroxidase was demonstrated by 0.05% DAB in the presence of 0.01% HzOzin 0.1 M Tris-water (pH 7.4) for 6 minutes. Controls were routinely run with each adjacent series of sections to ensure against nonspecific staining by the primary antisera or cross-reactivity of bridging antisera used in single and double immunostaining conditions. For the choleragenoid antiserum, the control sections were incubated in the presence of normal goat serum diluted at the same concentration as the goat anticholeragenoid serum. For the GAD antiserum, the control sections were incubated in the presence of normal sheep serum diluted at the same concentration as the sheep anti-GAD antiserum. For the ChAT antibody, the control sections were incubated in the absence of antibody in the first step since the ChAT antibody is a monoclonal and not associated with any background staining. In the sequential double immunostaining procedures, three sets of control sections were run in each series termed No-CT/ChAT, CTINo-ChAT, and NoCTINo-ChAT; and No-CTIGAD, CTINo-GAD, and No-CTI No-GAD in which the "No-" conditions represented the absence of the primary antibody as described above. These control sections were processed through the secondary antisera, PAP and DAB-Ni and/or DAB steps in the same manner as the other sections. By means of these control procedures, it was established that, with the dilutions of the primary antisera employed, no nonspecific single or double labeling of neuronal soma or processes occurred in these experiments. Sections were viewed with a Leitz Orthoplan microscope equipped with an xiy-sensitive stage and video camera that were connected to a computerized image-analysis system (Biocom, Paris, France). As described previously (Gritti et al., '931, labeled cells were plotted or drawn at high magnification (with a x 40 objective) upon appropriate graphic atlas templates at six levels (separated by 800 pm intervals) through the basal forebrain. By this means, the respective number or size of the plotted or drawn cells was computed for each nucleus at each level. ChAT+, GAD+, CT + and the double labeled cells ChAT + ICT + and GAD + I CT + were mapped in adjacent series of three representative animals (CT1, CT4, CT5), which had well placed injections of CT in the LHp. Morphometric measurements of cells in
254
I. GRITTI ET AL.
Fig. 1. Low-power photomicrograph of a section through the posterior hypothalamus processed by immunohistochemistry with diaminobenzadine (DAB) and nickel (DAB-Nil to reveal the cholera toxin (CT) injection site (in CT4). The injection is located in the posterior lateral
hypothalamus, lateral to the mammillothalamic tract (mt) and fornix (0, medial to the subthalamic nucleus (STh) and zona incerta (ZI), and ventral to the nucleus of Forel's field. Bar = 200 p,m.
the basal forebrain from one animal (CT1) were submitted to statistical analysis, employing analysis of variance (ANOVA). These measurements were further employed to estimate numbers of cells by application of the Abercrombie formula (Abercrombie, '46). Outside the basal forebrain, cells were also plotted in those regions where GAD+/CT+ neurons were located, and thus in the preoptic-anterior hypothalamic regions. In these areas, the lateral preoptic area, bed nucleus of the stria terminalis, and (anterior) lateral hypothalamus, which adjoin the basal forebrain cholinergic cell groups, were drawn as contours on the atlas templates for delimitation and tabulation of the GAD+ / CT+ cells. The medial preoptic and anterior hypothalamic areas located medial to the lateral preoptic and lateral hypothalamic areas respectively, were not, however, parcellated in this study. Labeled cells were thus plotted and tabulated per level in the general areas of the medial preoptic area (A8.5) and the anterior and dorsal hypothalamic areas (A7.7 and A6.9). The anatomical delineations and terms were assigned with reference to Bleier et al. ('79), Geeraedts et al. ('90a,b), and Paxinos and Watson ('86; see Gritti et al., '93).
mately 1,200-1,800 pm in height extending vertically around the micropipette tip (Fig. 1).The injection sites were centered in the posterior lateral hypothalamus and extended approximately 800 pm rostra1 to caudal from the caudal tuberal into the mammillary region of the lateral hypothalamus. They filled the area lateral to the fornix and mammillothalamic tract and medial to the cerebral peduncle and subthalamic nucleus. They extended dorsoventrally to include the entire area of the lateral hypothalamus (A 5.2; Paxinos and Watson, '861, located ventral to the nucleus of the fields of Fore1 (and superior cerebellar peduncle). In most cases, they included a portion of the tuberal lateral hypothalamic area (LHAt) in addition to the posterior lateral hypothalamic area (LHAp), as defined by Saper et al. ('86). In some cases, they extended dorsally and rostrally to include a ventromedial portion of the zona incerta. In some cases, they reached the ventral surface of the hypothalamus, but did not include in any case the tuberomammillary nucleus located more medially on the surface (Fig. 1). Within the basal forebrain, both CUT-positive and GAD-positive cells were retrogradely labeled (Fig. 2), in addition to CUT-negative and GAD-negative cells (Fig. 31, on the ipsilateral side following unilateral injections of CT into the LHp. In the presence of colchicine, which was used to enhance the GAD immunostaining, the retrogradely transported CT+ granules were often clumped in the soma
RESULTS The 50 nl CT injection resulted in a spherical deposit of the label, approximately 800 pm in width and approxi-
255
GABAERGIC/CHOLINERGICPROJECTIONS
Fig. 2. High-power photomicrographs of sections through the basal forebrain that were processed for sequential immunohistochemical staining of CT (with DAB-Ni in A and B) and choline acetyltransferase (ChAT; A) or glutamic acid decarboxylase (GAD; B). Cells retrogradely labeled with CT (CT+; solid arrows) are evident by the presence of black punctate granules in the cell soma and dendrites of ChAT+ (A) and GAD+ (B) cells. Among both the cell groups, the retrogradely
labeled CT+ cells (solid arrows) were generally smaller in size than the CT- cohorts (open arrows). In addition, the GAD+ /CT+ cells were generally smaller than the ChAT+/CT+ cells. The cells photographed were located in the medial portion of the magnocellular preoptic nucleus (MCPO; A) and the lateral portion of the nearby diagonal band of Broca nucleus (DBB; B). Bars = 10 km.
or dispersed into the dendrites of the cells, as has been described previously (Monti-Graziadei and Berkley, '911, but were nonetheless always evident within retrogradely cells labeled neurons. Retrogradely labeled ChAT ( C U T + /CT+) appeared morphologically similar to nonretrogradely labeled C U T + cells ( C U T+ /CT - 1, being round or polygonal in shape (Fig. 2A). The C U T + / C T + cells were, however, significantly smaller than the ChAT+ /CTcells across the basal forebrain (Fig. 4) and in most of the cholinergic cell groups (Table 1; see Gritti et al., '93). Retrogradely labeled GAD+/CT+ cells were also similar to
the GAD+/CT- cohorts in morphology, being oval to polygonal in shape and frequently multipolar (Fig. 2B). They were also significantly smaller than the GAD+/CTcells across the basal forebrain (Fig. 4) and in most of the cell groups (Table 2) (see Gritti et al., '93). As is the case for the total populations of GAD+ and C U T + neurons through the basal forebrain (Gritti et al., '931, the GAD+/CT+ cells were significantly smaller than the C U T + / C T + cells (Tables 1, 2), although the two populations overlapped considerably in size (Fig. 4).The CT+ but C U T - cells, which were distributed among the C U T + cells in the
+
256
I. GRITTI ET AL.
Fig. 3. High-power photomicrograph of a section immunostained for CT and ChAT showing a singly labeled CT+ cell (arrow) lying near ChAT+/CT- cells in the dorsal portion of the magnocellular preoptic nucleus (MCPO).Bar = 10 km
basal forebrain (Fig. 3), overlapped in size with the CT+ / ChAT+ and CT+/GAD+ cells (mean & s.d. 14.31 ? 3.7 km, range 9-25 pm in large diameter, as measured in CT1, n = 26). Through the basal forebrain, the retrogradely labeled ChAT+ cells were few in number (Table 3) and sparsely distributed through all of the ipsilateral cholinergic cell groups (Figs. 5-10). In contrast, the retrogradely labeled GAD+ cells were relatively numerous (Table 4) and were present in significant numbers through the areas where they are comingled with the cholinergic cells in the basal forebrain (Figs. 5-10) (Gritti et al., '93). As compared to the C U T + cells, a much larger number and proportion of the GAD+ cells in the basal forebrain were retrogradely labeled from the LHp (Table 4; Figs. 5-10). Similarly, of the total number of retrogradely labeled (CT+) cells (Table 51, the GAD+ cells represented a much greater proportion than the C U T + cells in the basal forebrain. In addition to GAD+ cells located in the region of the cholinergic cell groups in the basal forebrain, GAD+ cells distributed medially through the preoptic and anterior hypothalamic regions, as well as the bed nucleus of the stria terminalis, were also retrogradely labeled from the LHp. LHp-projecting and non-LHp-projecting GAD+ cells were thus plotted through these regions in order to assess their general distribution and to estimate their numbers (Figs. 8, 9, 10, Table 4). Retrogradely labeled GAD+ neurons appeared similar in morphology and size within the preoptic areas to those with which they were coextensive in the basal forebrain. For this reason, the average cell size of the basal forebrain GAD+ cells was used to estimate cell numbers in the preoptic-anterior hypothalamic areas. The GAD+ /CT+ cells were very densely distributed through the lateral preoptic region (Fig. 8 ) and represented a relatively large number of cells (Table 4). GAD+/CT+ cells also extended in significant numbers medially into the medial preoptic region and dorsally into the bed nucleus of the stria terminalis (Fig. 8, Table 4). Through these same areas,
large numbers of singly labeled GAD+ cells were also evident and formed part of a large GAD+ cell population in the preoptic regions (Figs. 5-10, insets; Table 4). GAD+/ CT+ cells decreased in number passing caudally into the medial anterior hypothalamic area but were more densely distributed within the dorsal hypothalamic area and the dorsal part of the LHa (Fig. 10). Of the total population of GAD+ cells in the preoptic-anterior hypothalamic regions, significant proportions were retrogradely labeled, particularly in the lateral areas (Table 4). The GAD+ LHpprojecting (GAD+ /CT+) cells in the preoptic-anterior hypothalamic regions also represented a similar proportion of the total number of LHp-projecting (CT+) neurons as that for the GAD+ LHp-projecting cells in the basal forebrain cholinergic cell areas (Table 5). Although retrogradely labeled (CT+) cells were present in the amygdala and in smaller numbers in the lateral septum and several cortical areas, no C U T + / C T + and no (or a few in the case of the lateral septum) GAD+/CT+ cells were evident in these areas, for which reason the CT+ cells were not plotted or tabulated therein.
DISCUSSION As a sequel to the study of the codistribution of cholinergic and GABAergic neurons in the basal forebrain (Gritti et al., '931, the present study examined the descending projections from these respective cell populations to the posterior lateral hypothalamus (LHp). The major findings of this study are that relatively large numbers and significant proportions of the GABAergic neurons project to the LHp, whereas only tiny numbers and proportions of the cholinergic neurons do so. For both the GABAergic and the cholinergic cells, the LHp-projecting cells are smaller than their cohorts that do not project to the LHp. In addition, an extensive population of GAD+ cells distributed through the preoptic-anterior hypothalamic areas also gives rise to descending projections to the LHp.
257 80I * 0 ChAT+/CT-
70x 0
2
Q
60-
I'
1
1
OGAD+/CTEl GAD+/CT+
70
.
ChAT+/CT+
60
.
1
0
5 50
50-
x 40 3
II
40-
I
I
LL
u-
= 30
- 30.
0,
0
20 10
*10 0O l d
n 0
50 100 150 200 250 300 350 400 450
0
50 100 150 200 250 300 350 400 450
Surface (pm*)
x
GAD+/CT-
ChAT+/CTa ChAT+/CT+
7060-
E l GAD+/CT+
0
s? 50E! 40 6-
u-
= 30 Q,
20
10 I) "
0
5
10 15 20 25 Large Diameter pm
30
0
2
5
10 15 20 25 Large Diameter pm
30
35
8 o L OGAD+/CT0 ChAT+/CTL ~
DGAD+/CT+
El ChAT+/CT+
15 20 25 10 Small Diameter pm
30
35
-
0
5
15 20 25 10 Small Diameter prn
Fig. 4. Series of histograms showing the frequency distribution of cell surface area (+m2;top panels), large diameter (pm; center panels), and small diameter (pm; bottom panels) of ChAT+/CT+ cells (shaded bars) and ChAT+/CT- (white bars) on the left and GAD+/CT+ (shaded bars) and GAD+/CT(white bars) on the right. Values were obtained from one animal (CT1) (Gritti et al., '93).
30
'
35
258
I. GRITTI ET AL.
TABLE 1. Morphometry of ChAT+/CT+ Neurons in the Basal Forebrain' No. of cells
Structure
MS DBB MCPO OTu SIa
;i Average AverageChAT+/CT-
TABLE 3. Number of C U T + and Number and Percentage of C M T + / C T + Neurons in the Basal Forebrain'
Small diameter2
Large diameter2
4 11 14 14 8 7
82k17 96525 108537 98228 114i51 83221
13.8 2 2.3 17.8 i 4.8 17.4 ? 4.6 16.8 2 3.3 21.3 + 8.4 14.7 i 1.3
77.1 2 19.6 116.8 5 59.3 139.1 t_ 75.1 114.7 i 56.8 181.8 2 125.4 59.4 ? 22.5
d
77116
17.2 ? 10.4
115.6 2 82.2
SIa
50 7
98k33
17.3
124.0 i_ 74.0
SIP
GP
119 7 106 3
Total
62 464
10 2
2
3 O*
t_
5.3
18.2 i 4.5*
Surface area2
142.8 k 6 7 . 6 *
ChAT t Structure MS
DBB MCPO OTu
ChAT+ICT t
Counts
Total
Counts
Total
Percentaee
154.0 5 21.7 209.0 2 23.4 186.3 i 26.1 165 7 i 15 9
2,865 3,888 3.466
3.0 2 2.0 5.7 + 2.9 8 . 7 i 2.5 12.0 i 4.4
57 107 164
2.0 2.8 4.7
13 0
943
43 5 25 3
2,226 1,978
991 7 2 45 9
18,450
? t_
2
3,082
7.0
i 5.6 3.6 2 2.5 2.0 i 1.0 41.7
t_
14.5
227
7.4
132
14.0
69 38
3.1 1.9 4.3
787
'Measurements (mean i s.d.1 are presented for cells located ipsilateral to the injection of CT in the LHp in one animal (CTlj. 2Accordingto results of two-way ANOVAs performed for each measurement, there is a significant differenceinsizebetweenChAT+/CT+ andChAT+ ICT- cells (*P < 0.0001j and a significant interaction for size between CT labeling and structure.
TABLE 2. Morphometry of GAD+/CT+ Neurons in the Basal Forebrain' No. of cells
Structure MS
31 41 29 69 61 9 7
DBB MCPO OTu SIa SIP
GP Average Average GAD+/CT-
247 522
Small diameter2,3 6.1 i 1.1 6.6 2 1.4 9.7 2 2.1 7.9 -t 2.6 7.6 i 2.0 7.6 t_ 1.8 6.2 t_ 1.2 7.3 + 2.1 9.2 2 2.6'
Large diameter2,3
Surface area2,3
12.5 + 2.8 12.9 i 3.2 13.3 2 3.1 14.3 2 4.3 14.1 2 3.6 16.3 k 2.5 12.1 i 2.4
54.7 i 14.8 60.8 i 23.9 75.9 2 32.3 85.0 i 48.0 77.0 2 35.7 82.4 2 17.8 57.6 2 19.9
13.7 2 3.6 16.4 i 4.4*
73.6 2 36.5 113.4 ? 58.4*
'Measurements (mean + s.d.j are presented for cells located ipsilateral to the injection of CT in the LHp in one animal (CTlj. 2According to the results of a two-way ANOVA performed for each measurement, there is a significant difference in size between the GAD+/CT+ cells and the GAD+/CT- cells (*P < 0.0001) and a significant interaction for size between CT labeling and structure. 3Accordingto the results of a two-way ANOVA performed for each measurement, there is a significant difference in size between GAD+/CT+ neurons and ChAT+/CT+ neurons ( P < 0.0001j and a significant interaction for size between GAD-CMT labeling and structure.
LHp-projectingcholinergic basal forebrain neurons Although of much lower density than those in the thalamus, cholinergic fibers have been visualized within the hypothalamus, including the lateral hypothalamus, and could originate in part from basal forebrain cholinergic neurons as well as from brainstem cholinergic cells (Jones and Beaudet, '87; Jones and Webster, '88; Sakai et al., '90b; Tag0 et al., '87; Jones, unpublished observations). As another potential source of such fibers, small cholinergic cells have also been identified in the hypothalamus (Tago et al., '87) but were not visualized in the present study. Although cortically projecting neurons within the LHp (and tuberal lateral hypothalamus) contain acetylcholinesterase (AChE) (Kohler and Swanson, '84), they do not contain ChAT and therefore are noncholinergic cells (Mesulam et al., '83a; Saper et al., '86). Evidence for a projection from the basal forebrain cholinergic neurons to the LHp was presented in the present study in the rat, since a small number of basal forebrain cholinergic neurons were retrogradely labeled following CT injections in the LHp. A small number of retrogradely labeled cholinergic cells was also reported in this region by others following CT injections in the posterior hypothalamus in the cat (Yoshimoto et al., '89). In the present study, the retrogradely labeled cells numbered 800 (according to estimates corrected for cell size) and represented 4% of all the cholinergic cells in the basal forebrain (estimated at 18,000 (Gritti et al., '93). Although this innervation may not be insignificant for the LHp, this descending projection
-
-
-
clearly represents a very small minority of the basal forebrain cholinergic neurons, the vast majority of which have been shown to give rise to ascending projections to the cerebral cortex (Mesulam et al., '83b; Rye et al., '84). Indeed previous studies reported that virtually no basal forebrain cholinergic neurons gave rise to descending projections to the brainstem and that, based on anatomical and physiological properties, brainstem projecting neurons represented a population of cells separate and different from the cortically projecting (mostly cholinergic) ones in the basal forebrain (Semba et al., '89). Interestingly, in the present study, the small contingent of cholinergic neurons that did project to the posterior lateral hypothalamus were smaller than the non-LHp-projecting neurons in most cholinergic cell groups and thus represent a subgroup of cholinergic cells different from those of the cortically projecting magnocellular basal neurons.
LHp-projectingGABAergic basal forebrain and preoptic-anterior hypothalamic neurons GABA has been shown to be a dominant neurotransmitter in the hypothalamus (Decaveland van den Pol, 'go), and GABAergic terminals have been found at moderate density within the posterior lateral hypothalamus, where no or very few GAD+ cell bodies are evident (Mugnaini and Oertel, '85; Vincent et al., '82; Jones, unpublished observations). GAD+ cell bodies are located ventral to the LHp area in the tuberomammillary nucleus and dorsal to the LHp area in the zona incerta and have been reported, like certain of the basal forebrain GAD+ neurons, to project to the cerebral cortex (Fisher et al.,'88; Lin et al., '90; Vincent et al., '83). The GABAergic input to the (non-GABAergic) cortically projecting neurons of the LHp could arise from multiple near and distant sources, which could include the basal forebrain GABAergic neurons. A relatively large number of GABAergic basal forebrain neurons were retrogradely labeled from the LHp in the present study. This number (estimated at 6,200) was distributed across the basal forebrain and represented > 15%of all the basal forebrain GABAergic neurons (which number approximately 39,000; Gritti et al., '93). Descending projections of forebrain GABAergic neurons to the caudal diencephalon and brainstem have previously been described for the GABAergic efferent cells of the basal ganglia (Oertel and Mugnaini, '84). It has thus been shown
GABAERGICICHOLINERGIC PROJECTIONS
259
CPU
A10.9 II I
Acb
Figure 5 Figs. 5-10. Atlas figures of six levels through the forebrain where the regions where retrogradely labeled GAD+ cells were found in the the major cholinergic and comingled GABAergic cell groups of the basal basal forebrain, preoptic region, and anterior hypothalamus, both forebrain have been delineated (Fig. 5: A10.9; Fig. 6: A1O.l; Fig. 7: GAD+/CT+ and GAD+/CT- cells are plotted in small-scale schematA9.3; Fig. 8: A8.5; Fig. 9: A7.7; Fig. 10: A6.9; Gritti et al., '93). ics (insets) for each level: triangles indicate GAD+/CT+ and GAD+/ Retrogradely labeled neurons were plotted (at high magnification with CT- cells (which were respectively plotted as solid and open triangles the aid of an image-analysis system) on the atlas templates through the though not distinguishable as such at this small scale). Numbers of cells basal forebrain and preopticihypothalamic areas and are presented in were counted within contours, which delineated structures (Tables the large-scale schematics: Xs indicate singly labeled CT+ neurons; 3-51, or within areas (MPA, AHA-DHA), which represented the remaincircles indicate ChAT+ /CT+ neurons; red triangles indicate GAD+/ ing territory containing retrogradely labeled cells at each level (A8.5, CT+ neurons. To show the distribution of all GAD+ neurons through A7.7, A6.9) (Tables 4, 5).
260
I. GRITTI ET AL.
Figure 6
that GAD+ neurons within the globus pallidus (and entopeduncular nucleus in the rat), which form the so-called dorsal pallidum (Heimer et al., '85), give rise to projections to the subthalamic nucleus, substantia nigra, and pontine tegmentum (Fisher, '89; Schmued et al., '89; Smith et al., '90). In the present study, the smallest number and proportion (5%) of GAD+ cells that were retrogradely labeled from
the posterior lateral hypothalamus were those within the globus pallidus. In contrast, a relatively large number and proportion (10-35%) of GAD+ neurons were retrogradely labeled within the (deep layers of the) olfactory tubercle and anterior substantia innominata, which have been collectively considered to represent ventral pallidum (Heimer et al., '85). Indeed the major GABAergic descendingprojection
261
GABAERGIC/ CHOLINERGIC PROJECTIONS
LS
/
Figure 7
to the posterior lateral hypothalamus [which would include that to the nuclei gemini, long known to be a target of olfactory tubercle neurons (see Heimer et al., '8511 from the cells in the so-called ventral pallidum would lend further support to the concept of a limbic, allocortico-ventral, striato-pallidal system that runs parallel to the sensorymotor, neocortico-dorsal striato-pallidal system (Groenewe-
gen and Berendse, '90; Haber et al., '85; Heimer and Wilson, '75). However, the LHp-projecting GABAergic neurons were distributed through regions of the basal forebrain that extend beyond what has been defined as the ventral pallidum, to include the septum-diagonal band complex and the larger substantia innominata or basal nucleus of Meynert. Indeed, within the posterior substantia
262
I. GRITTI ET AL.
GP
ic
/ A 8.5
Figure 8
innominata and magnocellular preoptic nucleus, which make up a major portion of the basal nucleus in the rat, 20-35% of the GAD+ cells projected to the LHp. Through all these areas, the GABAergic LHp-projecting neurons were commonly small in size and represented a relatively small subset of the population of basal forebrain GAl3Aergic neurons. They were clearly different from the larger
GAD+ basal forebrain neurons, which may make up part of the magnocellular basal nucleus and share with the cholinergic cells efferent projections to the cerebral cortex (see Gritti et al., '93). The smaller GAD+ LHp-projecting neurons furthermore were not confined to the basal forebrain areas but extended more broadly to occupy the entire territory of the medial
263
GABAERGIC/CHOLINERGICPROJECTIONS
A'
Figure 9
forebrain bundle, including the more medial, preoptic, and anterior hypothalamic areas. A relatively large number of GAl3Aergic neurons (estimated at 5,500 by correction according to basal forebrain GAD+/CT+ cell size) were retrogradely labeled within the preoptic-anterior hypothalamic regions. These cells were most numerous in the lateral preoptic area but were also plentiful in all adjoining
areas, including the medial preoptic area, bed nucleus of the stria terminalis, anterior and dorsal hypothalamic areas, and anterior lateral hypothalamus. The number of GAD+ cells in these regions (not previously reported) was also high (estimated at 30,000 by correction according to basal forebrain GAD+ cell size). Recently, by application of in situ hybridization techniques for revealing messenger RNA
264
I. GRITTI ET AL.
Figure 10
for GAD, a similarly abundant distribution of GABAergic neurons in these and other regions of the hypothalamus was reported, leading to the conclusion that GABA may represent the most important transmitter of hypothalamic neurons (Okamura et al., '90). In the present study, 10-30% of the preoptic-anterior hypothalamic GABAergic
neurons were found to project to the posterior lateral hypothalamus. Thus, given the number and proportion of these GABAergic projection cells, an important influence and role of the basal forebrain and preoptic-anterior hypothalamic GABAergic neurons could be as an inhibitory input to neurons in the posterior lateral hypothalamus.
265
GABAERGIClCHOLINERGIC PROJECTIONS TABLE 4. Number of GAD+ and Number and Percentage of GAD+/CT+ Neurons in the Basal Forebrain and Preoptic-Anterior Hypothalamic Areas' GAD+ I CT +
GAD+ Structure MS DBB MCPO OTu SIa SIP GP Subtotal BST MPA LPOA AHA-DHA LHa Subtotal Total
Counts 383.3 i 54.2 417.6 2 49.0 271.3 f 28.2 399.0 f 86.7 218.3 i 45.7 96.7 f 52.0 153.6 i 76.8 1,940.0 t 211.8 132.3 f 113.0 180.3 f 30.0 441.0 f 200.1 593.7 i 269.7 144.3 t 75.6 1,491.7 f 668.2 3,431.7 ? 482.6
Total
Counts
Total
Percentage
7.605 8,287 5,383 7.916 4,331 1,918 3,049 38,490 2,626 3,578 8,749 11,778 2,864 29,595 68,084
41.3 2 17.8 48.6 f 24.8 52.3 i 20.0 42.6 t 22.8 71.0 2 25.6 34.0 ? 2.6 7.3 f 2.9 297.3 i 73.9 22.0 i 19.3 52.0 t 46.9 92.3 i 58.3 71.0 2 41.9 27.0 -t_ 10.8 264.3 i 145.2 561.7 t 207.8
860 1,012 1,089 806 1,477 707 153 6,185 458 1,082 1,921 1,477 562 5,498 11,683
11.3 12.2 20.2 10.2 34.1 36.9 5.0 16.1 17.4 30.2 22.0 12.5 19.6 18.6 17.2
'Labeled cells were plotted on the (right) side ipsilateral to the CT injection and their numbers tabulated for each structure in 25 p m thick sections at 800 p m intervals in three animals (CT1, CT4, CT5). For each structure are presented the mean 1ks.d.J number of cells counted (Counts) and t h e total number of cells estimated (Total), which was calculated by multiplication o f t h e Counts by t h e number of sections per interval (32) and correction for mean cell size (15.5 pm for GAD+;13.7 pm for GAD+ /CT+) by employing Abercrombie's ('46) formula. The percent of retrogradely labeled neurons was calculated by dividing total GALI+ /CT+ by total GAD+ cells.
TABLE 5. Number of CT+ Neurons and Percentage of CT+ Neurons That Were C U T + or GAD+ in the Basal Forebrain and Preoptic-Anterior Hwothalamic Areas' CT + Structure
Counts
Total
MS DBB MCPO OTu SIa SIP GP Subtotal BST MPA LPOA AHA-DHA LHa Subtotal Total
294.7 t 176.5 177.0 f 30.5 273.0 I55.9 248.6 f 64.1 507.6 f 126.2 243.0 f 44.0 49.0 f 30.0 1,793.0 f 300.9 195.0 f 97.6 315.0 i 2 9 9 . 0 360.3 f 245.7 446.0 f 232.7 104.7 f 67.3 1421.0 t 900.4 3214.0 t 1142.2
6,035 3,625 5,591 5,091 10,396 4,977 1,004 36,721 3,993 6,451 7,379 9,134 2,144 29,102 65,823
Percentage ChAT +
Percentage GAD+
0.9 3.0 2.9 4.5 1.3 1.4 3.8 2.1
14.3 27.9 19.5 15.8 14.2 14.2 15.2 16.8 11.5 16.8 26.0 16.2 26.2 18.9 17.8
'Labeled cells were plotted on t h e (right) side ipsilateral to t h e CT injection and their numbers tabulated for each structure in 25 km thick sections a t 800 pm intervals in three animals (CT1, CT4, CT5). For each structure are presented the mean (2s.d.) number of cells counted (Counts) and the total number of cells estimated (Total), which was calculated by multiplication of the Counts by the number of sections per interval (32) and correction for mean cell size (14.1 bm for CT+ cells) by employing Abercrombie's ('46) formula. The percentage of CT+ neurons which were C U T + or GAD+ was calculated by dividing the total CbAT+/CT+ (Table 3) or total GAD+/CT+ (Table 4) cells by total CT+ cells.
Relative importance of GABAergic and cholinergic LHp projections from the forebrain The posterior lateral hypothalamus has long been known to lay in the path of the descending medial forebrain bundle and its incumbent neurons to receive terminals or boutons en passant from the multiple forebrain sources of this major fiber system (Morest, '69; Nieuwenhuys et al., '82; Simerly and Swanson, '88; Veening et al., '82). In the present study, large numbers of cells that were retrogradely labeled from the LHp were distributed through all these forebrain structures and plotted and counted in the basal forebrain and adjoining preoptic-anterior hypothalamic ar-
eas where prominent GAD+ neurons were also retrogradely labeled. Previous studies of the afferents to the posterior hypothalamus have similarly found large numbers of neurons retrogradely labeled through multiple forebrain structures, including the basal forebrain and preoptic-anterior hypothalamic regions (Ericson et al., '91; Yoshimoto et al., '89). In the latter studies, an equally large number of afferent neurons appeared to project to the tuberomammillary nucleus and to the posterior lateral hypothalamus. In the present study, the total numbers of cells retrogradely labeled from the LHp with CT in the basal forebrain and adjoining preoptic-anterior hypothalamic areas can be estimated (by correction for cell size according to ChAT-/CT+ cells in the basal forebrain) at approximately 66,000, with about 37,000 in the basal forebrain cell groups and about 29,000 in the preopticanterior hypothalamic regions. Although the nature and density of innervation of the LHp cannot be inferred from retrograde labeling of afferent neurons, it can be assumed that the retrogradely labeled cells provide collaterals or boutons en passant to the LHp, since CT has been shown to not be taken up by simple fibers of passage (Luppi et al., '90). In fact, in studies employing anterograde transport of Phaseolus vulgaris leukoagglutinin (PHA-L), varicose fibers have been visualized in the posterior lateral hypothalamus originating from the anterior and posterior substantia innominata and preoptic areas (Groenewegen and Berendse, '90; Grove, '88a; Simerly and Swanson, '88). This same fiber system may continue into the medial subthalamic nucleus, ventral tegmental area, substantia nigra, and pedunculopontine-laterodorsal tegmental nuclei (Grove, '88a; Jones and Cuello, '89; Simerly and Swanson, '88; Swanson et al., '84). Of this large population of afferent neurons to the LHp, > 15%are GABAergic and approximately 2% are cholinergic in the basal forebrain and > 15% GABAergic in the adjoining preoptic-anterior hypothalamic regions. Although certain peptides have been identified in forebrain LHp-projecting neurons in other studies (Yoshimot0 et al., '89), the primary neurotransmitter of the major number of these projection neurons remains unknown. The relatively large number of GABAergic neurons and the larger proportion relative to that of cholinergic neurons represented by GABAergic neurons in this projection system suggests that GABA may play an important role in the descending output from the forebrain. Although GABAergic cells also project alongside cholinergic cells to thalamic (reticularis and dorsomedial) nuclei and cortical areas, they do not appear to outnumber the cholinergic cells in this manner in any other efferent projection from the basal forebrain (Jourdain et al., '89; Kohler et al., '84a; Rye et al., '84; Steriade et al., '87; Zaborszky et al., '86a), and, conversely, they represent a small minority in the efferent projection from the basal forebrain to the neocortex (Rye et al.,'84; see also Gritti et al., '93).
Functional significance of GABAergic and cholinergic LHp projections from the forebrain Neurons within the posterior lateral hypothalamus lay as ''path neurons" within the medial forebrain bundle (Millhouse, '69) and thus receive inputs from multiple forebrain and brainstem systems in a topographically organized manner; they in turn project in a similarly organized, and in part reciprocal, manner to forebrain, brainstem, and spinal
1. GRITTI ET AL.
targets (Grove, '88a; Grove, '88b; Jones and Cuello, '89; Saper et al., '79; Tucker and Saper, '85; Veeninget al., '87). Lying within this system, the cortically projecting neurons in the LHp also appear to project in a topographically organized manner, like those of the basal forebrain, to the allo-and neocortex (Saper, '84, '85). Within such an ordered system, the descending projections from the forebrain GABAergic neurons, which are distributed from medial to lateral across the preoptic-anterior hypothalamic and basal forebrain regions, could serve as important inhibitory inputs to selective subsets of neurons in the posterior lateral hypothalamus and thus in the control of specific visceral (including cardiovascular and thermoregulatory), somatomotor, or cortical responses. The LHp-projecting GABAergic neurons could also function in an integrated manner to dampen activity globally in brainstem/spinally projecting and cortically projecting neurons and thus diminish concurrently the activating influences on sympathetic, somatomotor, and cortical systems, as might occur in association with sleep. An important coordinated inhibitory influence from basal forebrain and preoptic-anterior hypothalamic regions on the posterior lateral hypothalamus might explain why stimulation of these regions can evoke sleep (Sterman and Clemente, '62a,b) and why lesions of these regions can diminish sleep (McGinty and Sterman, '68; Szymusiak and McGinty, '86a). The production of sleep by injections of the GABAergic agonist muscimol into the posterior hypothalamus in the normal animal or in an animal previously rendered insomniac by destruction of the preoptic region supports the notion that a GABAergic, inhibitory influence on the posterior lateral hypothalamus originating in the preoptic region and basal forebrain may normally be important for the induction and maintenance of sleep (Lin et al., '89; Sallanon et al., '89). Furthermore, warming of the preoptic area, which evokes sleep onset, has recently been shown to suppress the discharge of wake-active neurons in the posterior lateral hypothalamus (Krilowicz et al., '92; McGinty and Szymusiak, '90). Single-unit recording studies have revealed that activity of posterior lateral hypothalamic neurons is high during waking behavior and cortical activation (Pare et al., '89; Sakai et al., '90a; Szymusiak et al., '89) and low during slow wave sleep, whereas the activity of a significant proportion of neurons in the basal forebrain and preoptic area is conversely relatively high during slow wave sleep and low during waking (Findlay and Hayward, '69; McGinty and Szymusiak, '90; Ogawa and Kawamura, '88; Szymusiak and McGinty, '86b). It is thus possible that in contrast to "wake-active" neurons, which are presumed to be cortically projecting cholinergic neurons, the "sleepactive" neurons recorded in the basal forebrain and preopticanterior hypothalamic regions (Detari et al., '84, '87; Detari and Vanderwolf, '87; Ogawa and Kawamura, '88; Szymusiak and McGinty, '86b, '89) might be made up in part of GABAergic neurons giving rise to descending projections to and exerting inhibitory actions on neurons in the posterior lateral hypothalamus that are involved in behavioral arousal and cortical activation.
ACKNOWLEDGMENTS We thank Enrico Mugnaini for kindly supplying the antibody against GAD. We express our appreciation to Colin Holmes for his advice and assistance in computer analysis and Beverley Lindsay for secretarial assistance. We
also recognize Prof. Mauro Mancia for his support of this research. The postdoctoral research of I.G. was supported by ARIN, ASSORN, and Jeanne Timmins Fund of the Montreal Neurological Institute. The research was funded by the Medical Research Council of Canada.
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