Cell Tissue Res (1992) 269:107-117

Cell&Tissue Research 9 Springer-Verlag 1992

Morphological analysis of the neurons in the area of the hypothalamic magnocellular dorsal nucleus of the guinea pig O. Doutrelant, L. Martin-Bouyer*, and P. Poulain INSERM U J56, Place de Verdun, F-59045 LilleCedex, France Received November 8, 1991 / AcceptedMarch 14, 1992

Summary. In the guinea-pig hypothalamus, a group of

Key words: Hypothalamus - Magnocellular dorsal nu-

enkephalinergic cells forms a well-circumscribed nuclear area called the magnocellular dorsal nucleus (MDN). This nucleus gives rise to a prominent projection to the lateral septum: the hypothalamo-septal enkephalinergic pathway. In the present study, MDN neurons visualized by Golgi impregnation were subjected to morphological analysis in order to define the potential segregation of cellular types within the MDN. This study was complemented by additional observations of MDN neurons intracellularly injected by Lucifer yellow (LY) or horseradish peroxidase (HRP) during the in vitro incubation of hypothalamic slices. The following results were obtained from the analysis of 200 neurons: 163 Golgi-impregnated cells plus 37 injected cells (LY= 14; HRP =23). Thirteen HRP-injected cells were precisely located in the MDN and 10 were located in the perifornical area surrounding the MDN. Four different cellular types were identified. Type-I neurons (41%) displayed a globular perikaryon, a variable number of primary dendrites that were poorly ramified, no preferential orientation, and an axon emerging from the perikaryon. Type-II neurons (30.5%) had a triangular perikaryon, three well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from the perikaryon. Type-III neurons (22%) exhibited a spindle-shaped perikaryon, two opposed well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from a primary dendrite. Type-IV neurons (6.5%), showed a globular perikaryon, a variable number of primary dendrites, poorly ramified dendrites, an orientation parallel to the third ventricle, and an axon whose orientation could not be identified. Neurons labeled after intracellular injection belonged to the first three cellular types.

cleus - Golgi impregnation Horseradish peroxidase - Lucifer-yellow labeling Enkephalin - Guinea pig

* Present address: Laboratoire de Biologie, Facult6 Libre des Sciences, 13, Rue de Toul, F-59046 LilleCedex, France Correspondence to: O. Doutrelant

In the guinea-pig hypothalamus, nerve cells immunoreactive for enkephalin are located in various hypothalamic areas, such as the periventricular nucleus, the dorsomedial and ventromedial nuclei, the arcuate nucleus and the mammillary bodies (Stengaard-Pedersen and Larsson 1981 ; Tramu et al. 1981). In the anterior hypothalamus, a particularly dense cluster of enkephalin-containing neurons (Beauvillain et al. 1980; Tramu et al. 1981) constitutes a well-circumscribed structure called the magnocellular dorsal nucleus (MDN). The MDN (Mfihlen 1966; Poulain 1974) or dorsolateral magnocellular nucleus (Bleier 1983) is located dorsomedially to the ascending columns of the fornix and is clearly distinguishable from the adjacent paraventricular nucleus (PVN). Ultrastructural studies show that immunoreactivity for metenkephalin is found in granules and on some ribosomes of MDN cells (Beauvillain et al. 1982). Moreover, recent autoradiographic studies have demonstrated the presence of proenkephalin-A mRNA in these neurons (Mitchell et al. 1992), thus confirming their enkephalinergic nature. Neuroanatomical (Poulain 1983; Staiger and Nfirnberger 1989) and electrophysiological (Poulain 1986) studies have demonstrated the bilateral projection of the MDN to the lateral septal nucleus (LS) in the guinea pig. In the same species, other studies (Poulain et al. 1984) have established the existence of a robust hypothalamo-septal enkephalinergic pathway originating from the MDN. In the rat, a similar juxtafornical population of enkephalinergic neurons has also been found (H6kfelt et al. 1977; Sar et al. 1978; Wamsley et al. 1980; Finley et al. 1981 ; Sakanaka et al. 1982; Krukoff and Calaresu 1984; Merchenthaler et al. 1986), although these neurons are more scattered than in the guinea pig. Combined neuroanatomical and immunocytochemical methods (On-

108 teniente et al. 1989; S a k a n a k a a n d M a g a r i 1989) h a v e enabled a hypothalamo-septal enkephalinergic pathway to be d e s c r i b e d in the rat. I n the s a m e species, M e r c h e n t h a l e r (1991) h a s r e c e n t l y s h o w n the c o e x p r e s s i o n o f enk e p h a l i n w i t h t h y r o l i b e r i n ( T R H ) in these n e u r o n s . T h e p o s s i b l e c o l o c a l i z a t i o n o f T R H w i t h e n k e p h a l i n in the g u i n e a - p i g M D N has n o t b e e n d e m o n s t r a t e d . T h e c o m p a c t o r g a n i z a t i o n o f the g u i n e a - p i g M D N has f a c i l i t a t e d the s t u d y o f the w h o l e nucleus, w h i c h is useful for lesional ( P o u l a i n et al. 1984) a n d electrop h y s i o l o g i c a l studies in vivo ( P o u l a i n 1986) a n d in v i t r o ( C a r e t t e et al. 1990). W e envisage t a k i n g a d v a n t a g e o f this p e c u l i a r i t y to p e r f o r m in vivo a n d in v i t r o p h a r m a c o l o g i c a l studies to i d e n t i f y the n e u r o a c t i v e s u b s t a n c e s w h i c h m o d u l a t e the a c t i v i t y o f the n e u r o n s e n g a g e d in the h y p o t h a l a m o - s e p t a l p a t h w a y . In o r d e r to p r o v i d e a m o r p h o l o g i c a l basis for this s t u d y , w e h a v e c a r r i e d o u t a precise analysis o f the m o r p h o l o g y o f M D N neur o n s v i s u a l i z e d either b y G o l g i i m p r e g n a t i o n o r b y i n t r a cellular injections o f L u c i f e r y e l l o w ( L Y ) o r h o r s e r a d i s h p e r o x i d a s e ( H R P ) , p e r f o r m e d d u r i n g the in v i t r o i n c u b a t i o n o f h y p o t h a l a m i c slices.

and attached to the stand of a Vibroslice. Three or four 400-gmthick frontal slices were cut and transferred to an interface-type recording chamber. Slices were perfused with oxygenated ACSF at 34~ C and exposed to a humidified atmosphere of 95% 02 and 5% CO2; they were incubated for about 3 h before starting the experiments. The MDN area was localized under the dissecting microscope using landmarks of the third ventricle and the fornix columns.

Intracellular L Y injections. A 5% solution of LY (LY-CH, Fluka, Buchs, Switzerland) in 0.25 M lithium chloride was intracellularly injected through beveled (Amthor t984) glass micropipettes (tip resistances 90-120 Mf~) into neurons of the MDN area. When the impalement was stable, the dye was ejected by iontophoresis with 700-msec hyperpolarizing current pulses of 1-1.5 nA at 1 Hz for 1-20 rain until deterioration of the electrical properties of the neuron. After injection, slices were left for approximately 1 h in the chamber before overnight fixation with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4; they were then transferred into dimethylsulfoxide (DMSO) for 15-20 rnin. DMSO prevents the neuronal shrinkage that occurs during dehydration (Grace and Llinas 1985). The slices were dehydrated, cleared and mounted onto slides with DMSO. The coverslips were sealed with paraffin. The injected cells were observed under a Leitz epifluorescence microscope, drawn and photographed using Ektachrome 800/1600 or Ilford Hp5 films.

Intracellular HRP injections. A 6% solution of HRP (grade I, Materials and methods Golgi impregnation Silver chromate impregnations were obtained from 7 brains of female guinea pigs (380-400 g body weight). In all cases, we used the Ramon-Moliner modified technique (1957) of the Golgi method reported by MacMullen and Almli (1981). Animals were anesthetized with Nembutal (33 mg/kg, i.p.), and perfused transcardially with 50 ml 1% sodium nitrate solution (to flush out the blood and induce vasodilation), followed by the Golgi fixative containing freshly mixed 6% potassium dichromate (200 ml), 5% potassium chlorate (100 ml), 20% chloral hydrate (150 ml), and 38% formaldehyde (50 ml). After perfusion, brains were dissected out, and the hypothalami were cut into 10 mm 3 blocks that were immersed in the Golgi fixative for 24 h. The blocks were then transferred to a solution prepared as described above, but in which formaldehyde was replaced by distilled water; they were immersed in this solution for 2 x 24 h. The replacement of formaldehyde by water is known to improve the impregnation and to reduce background staining (Frontera 1964). Subsequently, blocks were soaked in a 3% potassium dichromate solution for 6 x 12 h, followed by a 14day impregnation in 1% silver nitrate solution. Blocks were cut into frontal microtome sections of 100-150 gm thickness. Sections were dehydrated in alcohols, cleared in xylene, and individually mounted between two coverstips with Eukitt. Mounting between two coverslips made it possible to observe both sides of the sections. Well-impregnated neurons of the MDN area were drawn using a Zeiss microscope equipped with a camera lucida. Perikarya were drawn with the use of a 100 x oil-immersion objective, dendritic arborizations with the aid of a 40 x objective. Impregnated cells were photographed using Agfapan 25 films.

In vitro intracellular injections Slice preparation. Slices were cut from brains of female guinea pigs (200-300 g body weight). After decapitation, brains were quickly removed and placed in cold (4-6 ~ C) artificial cerebrospinal fluid (ACSF; Yamamoto 1973) containing: 124 mM NaC1, 26 mM NaHCO3, 5 mM KC1, 1.24 mM KHzPO4, 2.4 mM CaC12, 1.3 mM MgSO4 (7 H20), 10 mM glucose. The hypothalamus was dissected

Boehringer Mannheim France, Meylan, France) in 0.5 M TRISHC1 buffer pH 7.6 was intracellularly injected through beveled glass micropipettes (tip resistances 50-100 Ms into neurons of the MDN area. The enzyme was iontophoretically ejected with 600 msec depolarizing current pulses of 1.5-2 nA at I Hz for 1015 rain until deterioration of the electrical properties of the neuron. After injection, slices were left in the perfusion chamber for 1-2 h. They were then immersed for 2 h at 4~ C in PB containing 1.25% glutaraldehyde and 1% paraformaldehyde, and afterwards subjected to overnight cryoprotection at 4~ C in PB containing 20% sucrose. Slices were frozen on a microtome and cut serially into 60-1xm-thick frontal sections. Sections were then processed for HRP visualization using 3,3'-diaminobenzidine tetrahydrochloride (DAB), as follows; after being cut, the sections were rinsed in PB, incubated for 10 rain in 5% cobalt chloride in PB, and rinsed again 3 x 5 rain in PB. Thereafter, sections were first soaked in 0.05% DAB in PB for 20 rain before being transferred to PB containing 0.05% DAB and 0.003% hydrogen peroxide for an additional 20 min. The reaction was stopped by 3 rinses of 5 min each in PB, and the sections were mounted on gelatin-coated slides and air-dried at room temperature. Nissl-staining was performed with 0.1% thionin in order to reveal the exact limits of the MDN within the MDN area. Sections were dehydrated, cleared and coverslipped with Eukitt. The somato-dendritic and axonal morphology of labeled neurons were reconstructed from camera lucida drawings of serial sections observed with a 40 x objective. Labeled neurons were photographed using Agfapan 25 films.

Results Analysis of Golgi-impregnated neurons General comments. T h e m o d i f i e d G o l g i m e t h o d ( M a c M u l l e n a n d A l m l i 1981) used in this s t u d y a l l o w e d us to stain n u m e r o u s n e u r o n s t h r o u g h o u t the h y p o t h a l a mus, especially w i t h i n the M D N a r e a (Fig. 1A). This m e t h o d gave g o o d r e s o l u t i o n o f n e u r o n a l shapes a n d o f such m o r p h o l o g i c a l details as the s o m a t i c a p p e n d ages. A t o t a l o f 163 n e u r o n s o f the M D N a r e a w a s d r a w n a n d studied. A l l these n e u r o n s p r e s e n t e d a well-

109

Fig. 1. A Survey of the area of the magnocellular dorsal nucleus (MDN) in a frontal section through the hypothalamus after Golgi impregnation. B Micrograph showing a thionin-stained 40-gmthick frontal section cut from a hypothalamic slice maintained in vitro. Fx Fornix; P V N paraventricular nucleus. The third ventricle is to the left. Bars: 300 gm

110

Fig. 2. Camera lucida drawings of three type-I neurons visualized after Golgi impregnation (GOLGI), Lucifer yellow (L Y) or horseradish peroxidase (HRP) intracellular injections and located within the MDN area as indicated by symbols on the schematic drawing of a frontal section through the hypothalamus. Only the HRPlabeled neuron was located within the MDN proper. Arrowheads point to the axons. Note the axonal bifurcation observed on the LY-filled cell (arrows). Fx Fornix; PVN paraventricular nucleus; 3V third ventricle. Bar: 50 gm

impregnated cell body and dendrites, and in most cases, the emerging axon. Four cell-types were distinguished in the M D N area with regard to (1) the shape of the perikaryon, (2) the number of primary dendrites, (3) the degree of arborization of the dendrites, and (4) the general orientation of the neuron to the third ventricle.

Description of neuronal types. Type-I neurons ( n = 69; 42.3% of the total number of studied cells) were the most prevalent (Figs. 2, 3A). The shapes of the cell bodies varied from globular to ovoid; the average size of these type-I neurons was 23.5_+ 3.7 Bm along the longer axis, and 14.6-+ 1.6 I~m along the shorter axis (means-+ SD). Two or three poorly ramified primary dendrites emerged from the perikaryon. They were impregnated over a short distance and extended in all directions; thus, the neurons of this type did not display a specific orientation with regard to the third ventricle. Twenty-eight cells exhibited an identifiable axon. Of these 28 cells, 11 had their axon emerging from the perikaryon, 10 had their axon originating from a primary dendrite at a distance less than 10 ~tm from the perikaryon, and 7 had their axon emerging from a dendritic trunk at a distance greater than 10 ~tm from the perikaryon. These axons were preferentially directed to the dorsomedial hypothalamic area, to the periventriculo-fornical area, or to the perifornical area. Type-II neurons ( n = 4 4 ) constituted 27% o f the 163 impregnated M D N neurons (Figs. 3 B, 4). The shape of the cell bodies was globular, but the presence of 3 primary dendrites gave them a triangular aspect; the average

size of these type-II neurons was 25.9_+4.2 lain along the longer axis, and 16.3__+1.5 lain along the shorter axis ( m e a n s + SD). O f the 3 primary dendrites, the one impregnated over the greatest length was well developed with a poorly spread arborization, the other two being shorter and widely ramified. The preferential orientation of the neurons was perpendicular to the third ventricle. Twenty-seven cells exhibited an identifiable axon. O f these 27 cells, 10 had their axon emerging from the perikaryon, 7 had their axon emerging from a primary dendrite at a distance less than 10 gm, and 10 had their axon emerging from a dendritic trunk at a distance greater than 10 gm from the perikaryon. The axons had two main directions: toward the dorsal hypothalamus or the perifornical area. Type-III neurons ( n = 3 7 ) constituted 22.7% of the 163 studied cells (Figs. 3 C, 5). Type-III neurons had spindle-shaped perikarya; the average size of these cells was 28.0_+4.0 gm along the longer axis, and 14.6+ 1.1 gm along the shorter axis (means_+ SD). Perikarya occasionally displayed spines with a globular head. A strong primary dendrite emerged at each pole of the perikaryon. Dendrites had a well-developed and wellspread arborization. The long axis of the perikarya and the general orientation of the neuron were perpendicular to the third ventricle. Thirteen cells exhibited an identifiable axon. O f these 13 cells, 4 had their axon emerging from the perikaryon, 4 had their axon originating from a primary dendrite at a distance less than 10 gm, and 5 had their axon emerging on a dendritic trunk at a distance greater than 10 gm from perikaryon. Axons were preferentially oriented following two diametrically opposed directions: toward the perifornical area or the zona incerta. Type-IV neurons ( n = 13) constituted 8% of the 163 studied neurons. The shape of the cell bodies was ovoid; the average size of the longer axis was 26.0-+1.5 gm and the shorter axis was 15.5_+0.7 gm (means _+SD). Two or three primary dendrites emerged from the perikaryon and were impregnated over a considerable length. The dendrites were poorly ramified. The preferential orientation of the neurons was parallel to the third ventricle. About one half of these neurons exhibited an identifiable axon emerging from a dendritic trunk at a distance greater than 10 gm from perikaryon. The number of type-IV neurons was too small to determine whether their axons were oriented in a particular direction. Some of these neurons apparently projected toward the thalamus or the periventriculo-fornical area. Table 1 summarizes the principal features of the four cellular types. There was no obvious topographic distribution of any of these four neuronal types throughout the M D N area.

Intracellular injections of L Y Intracellular LY injection experiments were performed to complement data obtained from the Golgi-impregnated neurons. Twenty-one neurons were injected. A m o n g these 21 cells, 14 well-labeled cells were assigned to one

111

Fig. 3. High-magnification

micrographs of type-I (A, D), type-II (B, E), and type-III (C, F) neurons. A-C Golgi-impregnated neurons located within the MDN area; D-F HRP-filled neurons located within the MDN proper. The perikarya of type-I, type-II, and type-III cells are multipolar, triangular and spindle-shaped (with two opposed dendrites), respectively. Bar: 40 gm

of the four groups described above (Table 1). These 14 LY-labeled neurons included a group of three closely neighboring neurons that were filled by the dye after injection of only one cell (Fig. 6A). Five cells had features c o m m o n to type-I neurons (Figs. 2, 6B): globular perikarya (23 x 22 gm average size) from which a variable (2-7) number of primary dendrites emerged, poorly ramified dendrites, and no preferential orientation. Labeled axons were directed toward the medio-dorsal hypothalamus or the lateral hy-

pothalamic area. One cell had its axon bifurcating at a distance of 120 gm from the cell body (Fig. 2). Six cells had features c o m m o n to type-II neurons (Fig. 4): triangular perikarya (26 x 20 gm average size) possessing 3 primary dendrites that ramified densely, and preferential orientation of the cells perpendicular to the third ventricle. Labeled axons were directed toward the zona incerta or the perifornical area. Three cells had morphological features c o m m o n to type-III neurons (Fig. 5): spindle-shaped perikarya (35 x 18 gm average size) from

112

j : "

f

"

3V

"k

LY (=1

Fig. 4. Camera lucida drawings of three type-II neurons. Note the presence of spines on the dendrites (small arrowheads) and perikaryon (arrow) of the HRP-labeled cell. Key and abbreviations as in Fig. 2. Bar." 50 gm

Fig. 5. Camera lucida drawings of three type-III neurons. Key and abbreviations as in Fig. 2. Bar: 50 gm

which 2 o p p o s e d a n d well-ramified p r i m a r y dendrites emerged, a n d p e r i k a r y a a n d dendrites o r i e n t e d p e r p e n d i c u l a r to the third ventricle. Labeled a x o n s were directed t o w a r d the z o n a incerta. L Y - l a b e l e d cells with features c o m m o n to t y p e - I V n e u r o n s were n o t observed.

Intracellular injections of H R P Thirty-five n e u r o n s in the M D N area were labeled after i n t r a c e l l u l a r injections o f H R P . The t h i o n i n c o u n t e r s t a i n i n g o f the sections c o n t a i n i n g the injected cells en-

Table 1. Summary of the main morphological features of the cell types defined by the analysis of Golgi-impregnated neurons of the MDN area Type I

Type II

Type III

Type IV

Perikaryon (mean + SD)

Globular aspect 23.5_+3.7 gm x 14.6_+1.6 ~tm

Triangular aspect 25.9_+4.2 gm x 16.3+1.5 gm

Spindleshaped aspect 28.0_+4.0 gm • 14.6• 1.1 ~tm

Globular aspect 26.0_+ 1.5 gm x 15.5_+0.7 gm

Dendritic arborization

Variable abundance Various number of primary dendrites No preferential orientation

Abundant

Abundant

Variable abundance

Three primary dendrites

Two primary dendrites

Perpendicular to the third ventricle

Perpendicular to the third ventricle

On the perikaryon

On the perikaryon

On a dendritic trunk

Cell numbers Golgi (%) LY HRP

69 (42.3%) 5 8

44 (27%) 6 11

37 (22.7%) 3 4

13 (8%)

Total

82

61

44

13

Total percentage

41%

30,5 %

22 %

Point of emergence of the axon

Parallel to the third ventricle

0 0

6.5%

113 sults solely concern the cells located within the M D N or within the perifornical area. These neurons were compared with cellular types defined by the analysis of Golgi-impregnated cells.

Fig. 6A, B. LY-injected cells. A A typical image of dye-coupling: three cells were labeled after injection of a single cell. B LY-filled neuron belonging to type I. Bar." 60 btm

abled the M D N boundaries to be identified; this was an important advantage over the Golgi method (Fig. 1 B). Indeed, a m o n g the 35 labeled cells in the M D N area, 13 were located in the M D N proper, 10 were in the perifornical area surrounding the M D N , and 12 were outside the perifornical area. The following re-

Cells located within the M D N . Thirteen cells were labeled after 9 injections within the M D N : two groups of 2 cells and one group of 3 cells were obtained in addition to six individually labeled neurons. Thus, 13 neurons were analyzed (Table 1). Six cells had morphological features c o m m o n to the type-I neurons (Figs. 2, 3 D): globular perikarya (18 x 14 ~tm average size), 2 or 3 poorly ramified primary dendrites, and no preferential orientation. Axons were not observed. Six cells had features characteristic of type-II neurons (Figs. 3 E, 4) : triangular perikarya (26 x 16 gm average size), and one of which had somatic spines (Fig. 7A), 3 primary dendrites, a well-developed dendritic field, and a preferential orientation perpendicular to the third ventricle. A m o n g these 6 neurons, 5 exhibited occasional dendritic spines with a thin shaft and with no terminal head (Fig. 7B). One axon was identifiable and seen to emerge f r o m the perikaryon. One cell resembled the type-III neurons (Figs. 3 F, 5). It had a spindle-shaped perikaryon (40 x 20 i_tm average size), and 2 opposed primary dendrites with abundant arborization. The perikarya and dendrites were oriented perpendicular to the third ventricle.

Fig. 7A-D. High-magnification micrographs showing examples of specialization of neurons located within the MDN area. A Type-II HRP-injected cell exhibiting somatic spines (arrow). B Dendritic profile of HRP-injected neuron located within the MDN proper. Arrows indicate spines with thin shafts and spherical heads. Arrowhead points to a spine with no visible terminal enlargement. C, D Segment of axons coursing in the dorsolateral hypothalamus and emanating from two HRP-injected cells located within the perifornical area. These two axons (arrows) give rise to collaterals (arrowheads). The collateral in C arborizes and displays swellings. Bars: 20 gm

114 Dendrites exhibited occasional spines with long shafts terminated by spherical heads (Fig. 7B). Axons were not observed. Cells located in the perfornical area. Ten cells were labeled after 9 injections in the perifornical area surrounding the MDN: one group of 2 cells was obtained in addition to 8 individuaIly labeled neurons. Thus, 10 cells were analyzed (Table 1). Two cells had features common to type-I neurons: globular perikarya (17 x 12 gm average size) with 3 poorly ramified primary dendrites, showing no preferential orientation. Only one cell exhibited an identifiable axon that was oriented toward the perifornical area. This axon had a high level of collateralization, displaying bouton-like swellings suggestive of en passant contacts, and terminal arborizations around neighboring cells of the perifornical area. Axon collaterals projected toward the dorsolateral hypothalamus where they terminated. Five cells had morphological features common to type-II neurons: triangular perikarya (25 x 17 lam average size, and one of which had somatic spines), 3 primary dendrites, a well-developed dendritic field, and a preferential orientation perpendicular to the third ventricle. All of them displayed dendritic spines: short shafts terminated by spherical head, thin long shafts with a spherical or oval head, or short shafts without a head. Three cells had an identifiable axon, emerging either from the cell body or from a dendritic trunk. Two of these axons were oriented toward the hypothalamic-dorsolateral area. One of them exhibited boutons en passant (Fig. 7 C) over its course within the dorsolaterat hypothalamus. The third identifiable axon exhibited numerous axonic collaterals (Fig. 7D) oriented toward the adjacent PVN or hypothalamic dorsolateral area. Three cells had features common to the type-III neurons: spindle-shaped perikarya (28 x 14 gm average size) with 2 opposed primary dendrites that were densely ramiffed, and a preferential orientation perpendicular to the third ventricle. Dendritic spines of various shapes were observed on 2 cells. One axon emerging from a dendritic trunk was identified but stained over a short length only; it was oriented toward the dorsolateral hypothalamic area. Horseradish-peroxidase-labeled cells with features common to type-IV neurons were not found.

house (]981). The modified technique of MacMullen and Almli (1981), which leads to a particularly satisfactory impregnation of hypothalamic cells, was chosen for our study. Because this technique is not selective, as demonstrated by Shimono and Tsuji (1987), for the ventromedial nucleus and lateral hypothalamic area, we consider that every potentially different cellular type was impregnated in the region studied. It is unlikely that the M D N area contains a cellular type that escapes impregnation, in view of our results from LY and H R P intracellular injection experiments. Indeed, every dye-injected or enzyme-injected cell presented morphological similarities with the cell types identified by impregnation. In the present study, LY or H R P injections allowed the discrimination of neuronal specializations, such as dendritic spines or axonic bifurcations, to a greater degree than the Golgi impregnation. It has previously been noted that the H R P intracellular injection method gives a more complete view of cellular morphology than the Golgi impregnation method that possibly fails to reveal entire dendritic systems (Somogyi and Smith 1979). The H R P method has also been found to be superior to the fluorescent dye injection method in revealing cellular details (Brown and Fyffe 1984). Despite carefully chosen parameters for H R P injection and histochemical visualization procedures (Adams 1977; Bishop and King 1982), the staining of more than one cell was occasionally obtained in our material. Non-injected but HRP-filled cells, in contrast to injected cells, generally displayed faint labeling often limited to the perikaryon and to the proximal dendrites. These cells were probably artifactually labeled by the endocytosis of extracellular H R P present in the vicinity of the injected cell, as suggested by Brown and Fyffe (1984). LY intracellular injection also occasionally resulted in the staining of more than one neuron. Dye-coupling (Stewart 1978) frequently occurs in slice preparations. This phenomenon may be of functional significance since, for example, it is more frequently observed in the PVN magnocellular neurons of lactating animals (Yang and Hatton 1987). On the other hand, Gutnick et al. (1985) have suggested that partial dendrotomy caused by slicing tissue induces the formation of new intercellular junctions. Thus, the multicellular labeling that we have obtained could have resulted from either natural functional contacts between cells or contacts between neuronal fragments severed by slicing.

Discussion

With the exception of data from immunocytochemical and retrograde transport studies (Stengaard-Pedersen and Larsson 1981 ; Tramu et al. 1981 ; Beauvillain et al. 1982; Poulain 1983; Poulain etal. 1984), nothing is known about the morphological characteristics and orientation of guinea-pig M D N neurons. The results of the present study indicate that this cytoarchitectonically defined nucleus contains different cell types. Technical considerations

Many modifications of the original impregnation technique of Golgi have been proposed, as reviewed by Mill-

Morphological considerations

The study of the 163 Golgi-impregnated neurons located in the M D N area allowed us to distinguish four cellular types differing from each other with respect to four main features: the shape of the perikaryon, the number of primary dendrites, the degree of arborization of the dendrites, and the general orientation of the cell. The perikaryon sizes measured in the present study justify this nucleus being termed the magnocellular nucleus. Although a type-by-type comparison of the perikaryon sizes has revealed a significant difference between some cellular types (Student's t-test), we did not consider in

115 the present study the average size of the impregnated neurons as a parameter for defining the four cellular types. On the other hand, it would be futile to compare sizes of perikarya measured from Golgi-impregnated material and the sizes of intracellularly filled perikarya, because of the differences in the histological procedures. The division of the cellular types according to the four main features was corroborated by the data obtained from the 37 cells injected with LY or HRP; these could unambiguously be assigned to three out of the four cellular types. The fact that no injected cell exhibited the combined morphological features of type-IV cells is probably the result of sampling bias; type-IV cells were underrepresented and constituted only 8 % of the population of Golgi-impregnated cells. It should be noted that all studies were carried out on frontal sections because the M D N area cannot easily be located in sagittal or horizontal sections. Consequently, other cell types or other morphological features may be found when using a sagittal or a horizontal section plane. Comparison between M D N cell types and hypothalamic neurons in various species

In the present study, a total population of 200 neurons has been studied including 163 Golgi-impregnated cells plus 37 injected cells (Table 1). The first cellular type represented 41% (n = 82) of the total population of studied cells. These type-I neurons had multipolar globular perikarya, a variable number of primary dendrites, poorly ramified dendrites, and no preferential orientation with regard to the third ventricle. During the present study, some of these neurons were electrophysiologically characterized before being injected (results not shown). They displayed low-threshold calcium spikes (LTS) that were inactivated at resting potential and deinactivated by membrane hyperpolarization. Hypothalamic neurons with electrophysiological features identical to those of the type-I cells of the present study have been found in the ventromedial nucleus (Minami et al. 1986) and in the periphery of the PVN in the guinea pig (Poulain and Carette 1987) and the rat (Dudek et al. 1989). Morphological analysis of LTS cells found in the periphery of the PVN after post-recording dye injection reveals that they have a small globular perikaryon from which few rectilinear and poorly ramified dendrites emerge (Poulain and Carette 1987), features characteristic of the present type-I neurons. On the other hand, type-I neurons have morphological features similar to those of the "class-II cells" described by MacMullen and Almli (1981) in the perifornical area of the rat. Moreover, they also share morphological features with the "isodendritic cells" that have been described by Ramon-Moliner and Nauta (1966) in Golgi preparations, and that are particularly abundant in the hypothalamus (Leontovich and Zhukova 1963; Lefranc 1966; Barry 1972, 1975). Thus, type-I cells displaying LTS probably represent a subpopulation of neurons that are not specific to the MDN; they may belong to a network of reticular cells that are spread throughout the hypothalamus and that are in-

volved in local regulation (Leontovich and Zhukova 1963). In agreement with this suggestion, we have observed type-I neurons in the perifornical area; these neurons have axons with swellings and bouton-like terminals providing the establishment of local contacts. The second cellular type constitutes 30.5% (n=61) of the total number of studied cells. These type-II neurons have triangular perikarya, 3 primary dendrites developing an abundant arborization, and a general orientation perpendicular to the third ventricle. Because they have similar perikaryal and dendritic morphologies, type-II neurons may be compared with neurons arrayed perpendicularly to the medial forebrain bundle in the rat lateral hypothalamus and named "path neurons" by Millhouse (1969). However, the dendritic orientation of type-II neurons is slightly different from that observed for "path neurons", although some type-II cells have dendrites following a dorsomedial-ventrolateral axis as observed for "path neurons" located close to the fornix (Millhouse 1969). Neurons of the second cellular type also have features in common with the "class-I cells" described by MacMullen and Almli (1981) in the lateral preoptic and lateral hypothalamic areas of the rat brain and with the "lateral hypothalamic cells" described by Leontovieh and Zhukova (1963) in the brain of cats and dogs. The third cellular group constitutes 22% (n=44) of all studied cells. These type-III neurons have spindleshaped perikarya from which 2 opposed and well-ramified primary dendrites emerge. Their long axis and dendritic field lie perpendicular to the third ventricle. This cellular type has frequently been observed in the hypothalamus of various mammals (Barry 1972). The type-III neurons resemble the "class-III cells" of the lateral preoptic and lateral hypothalamic areas of MacMullen and Almli (1981), the lateral hypothalamic neurons described by Leontovich and Zhukova (1963), and the magnocellular PVN neurons of cats, guinea pigs and rats (Lefranc 1966; Hatton et al. 1985). H R P retrograde labeling of M D N neurons from the LS results in images of labeled perikarya arranged as fish scales oriented in the direction of the third ventricle (Poulain 1983). This observation parallels the present data showing that spindle-shaped perikarya of type-III cells are oriented perpendicular to the third ventricle. This suggests that type-III cells constitute the prevailing neuronal population giving rise to the enkephalinergic hypothalamo-septal tract. Type-IV neurons were the least abundant, constituting only 6.5% of all studied cells and were observed exclusively in Golgi-impregnated tissue. They had ovoid perikarya, a variable number of primary dendrites, dendrites that were poorly ramified but that were impregnated over a considerable length, and an orientation parallel to the third ventricle. Type-IV neurons exhibited almost the same morphological features as type-I neurons. They only differed in their parallel orientation to the third ventricle. Neurons of this type were not found after intracellular injections. A comparison of our results with those previously obtained by others in Golgi-impregnated hypothalamic tissue of the rat clearly shows that the three major cell

116 types present in the M D N area (including the M D N and nearby surrounding structures) display morphological features similar to those of the various cell types described in other parts o f the hypothalamus. Regarding the M D N proper, our observations concerning H R P filled cells located within the cytoarchitectonic boundaries of the nucleus indicate first that these cells resemble those located in the nearby perifornical area, and second that they belong to at least three out o f the four cell types. Our results thus demonstrate the unexpected heterogeneity of the M D N , in which neurons appear to be morphologically indistinguishable f r o m neurons located outside it. Consequently, the homogeneous appearance of the guinea-pig M D N on a Nissl-stained preparation is mainly the result of its c o m p a c t organization and its being surrounded by a cell-poor zone.

Axon trajectory In the present study, we have observed axonal pathways toward different hypothalamic structures: the lateral hypothalamic area, dorsolateral hypothalamic area, mediodorsal hypothalamic area, homolateral PVN, periventriculo-fornical area, perifornical area, zona incerta and thalamus. This indicates that neurons located in the M D N area innervate various hypothalamic areas. Met-enkephalinergic M D N neurons probably participate in the innervation of various hypothalamic and thalamic structures, in addition to their well-known projection to the LS. Preliminary results of anterograde transport studies, not reported here, have shown a projection of M D N neurons to the reticular and medial nuclei of the thalamus. Some axons exhibit various degrees of branching, as observed in LY-injected and HRP-injected cells. Indeed, neuroanatomical (Poulain et al. 1984) and electrophysiological (Poulain 1986) studies have demonstrated the existence of a crossed projection from the M D N to the contralateral LS. According to in vivo electrophysioIogical experiments, axonal branching in this system occurs near the cell body (Poulain 1986). In good agreement with this, we have often observed the branching of axons in the vicinity o f the parent cell body, and therefore such collateralizing axons m a y establish direct projections to the LS.

Presence of somatic and dendritic appendages Somatic spines with short shafts surmounted by developed spherical heads were sometimes observed on typeI I I impregnated cells or on type-II HRP-injected cells. Spines on dendrites were also observed on approximatively one half of the HRP-injected cells, including cells located in the M D N proper. As previously described in the rat by Millhouse (1979) and MacMullen and Almli (1981), dendritic spines of various shapes and distributions are a c o m m o n feature of perifornical and lateral hypothalamic neurons. Functionally, somatic and dendritic appendages are anchoring elements for axon terminals containing vat-

ious neuroactive substances. Studies concerning the nature of afferent inputs onto enkephalinergic M D N neurons are growing in number. At the ultrastructural level, symmetrical catecholaminergic (Mitchell etal. 1988) and ?-aminobutyric-acid(GABA)-ergic (Beauvillain et al. 1988) synapses on M D N enkephalinergic neurons have been demonstrated. Enkephalin-(Beauviltain et al. 1982), somatostatin-(H6kfelt etal. 1974; T r a m u et al. 1981) and cholecystokinin-(Ciofi and T r a m u 1990) immunoreactive fibers are present within the M D N , and these peptides also are possible candidates for regulating the activity of the M D N cells. Further pharmacological studies are needed to k n o w the roles played by the various inputs onto the specific cell types of the M D N .

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