Distribution of delta sleep-inducing peptide in the
newborn and infant human hypothalamus: an
immunohistochemical study

MOHAMED NAJIMI^1,2, MOHAMED BENNIS², EMMANUEL MOYSES³, FATIHA CHIGR^1,2

1: Unité de Génie Biologique, F.S.T de Beni Mellal, Beni Mellal, Morocco. 2: Unité Neurosciences du
Comportement, Département de Biologie, Faculté des Sciences Semlalia, 40 000 Marrakech, Morocco.
3: Laboratoire de Neurophysiologie sensorielle, la DOUA, 69100 Villeurbanne, France

ABSTRACT

The distribution of delta sleep-inducing peptide immunoreactive cell bodies, fibers, and terminal-like structures was investigated in the normal human hypothalamus during the first postnatal year, using immunohistofluorescence and peroxidase anti-peroxidase techniques. Immunolabeled perikarya were relatively few and were mostly scattered through the anterior (preoptic) and mediobasal regions (infundibular nucleus) of the hypothalamus. DSIP-immunoreactive fibers and terminal-like fibers were observed throughout the entire rostro-caudal extent of the hypothalamus. They exhibit high densities in the preoptic region, the organum vasculosum of lamina terminalis, infundibular nucleus and median eminence. Moderate to low densities of DSIP-immunoreactive fibers were observed in the other hypothalamic structures, located in the anterior and mediobasal regions of hypothalamus, such as periventricular, paraventricular, suprachiasmatic, ventromedial, dorsomedial and parafornical nuclei. In the present study, the analysis of the immunohistochemical pattern of DSIP-immunoreactive neuronal elements in the human infant hypothalamus during the first postnatal year provided evidence of the presence of several differences. We have found qualitative age-related changes in the density of DSIP immunoreactivity in several hypothalamic structures such as the anterior region and the median eminence.

Key terms : DSIP, immunohistochemistry, development, post-mortem, human brain

INTRODUCTION

The presence of a factor involved in the control of sleep has been already reported by several studies (Pappenheimer et al, 1975). These investigations lead to the isolation of two similar peptide factors, one from whole brains of rabbits (sleep-promoting factor S) (Krueger et al, 1978) and the other from human urine (sleep-promoting muramyl peptide) (García-Arraras and Pappenheimer, 1983). The characterization of these peptides has been based on their capacity to induce sleep (Pappenheimer 1979; Krueger et al, 1982; Garcia-Arraras and Pappenheimer, 1983). Other biochemical studies have isolated the nonapeptide delta sleep inducing peptide (DSIP) from the cerebral venous blood of the rabbit, and characterized it for its hypnogenic activity (Schoenenberger et al, 1977).

Pharmacological and physiological studies have shown that in addition to their implication in the control of sleep-inducing activity, brain DSIP neuronal elements, particularly hypothalamic DSIP neurons, are considered to be involved in several physiological functions such as neuroendocrine regulation. Indeed, it has been suggested in previous studies that DSIP is involved in the control of release of anterior pituitary hormones. DSIP in particular has been shown to influence the secretion of many hormones and factors in rodents and humans, such as the adrenocorticotropic hormone (ACTH), luteinizing hormone (LH) and growth hormone (GH) (Sahu and Klara, 1987; Okajima and Hertting, 1986; Iyer and Mc Cann,1987a; Bjartell et al, 1989; Chiodera et al, 1994).

Evidence that DSIP is present in the central nervous system was provided in earlier studies by biochemical investigations. They revealed that in laboratory animals and human brains, the hypothalamic region exhibits the highest concentrations of this neuropeptide (Graf and Kastin, 1986). These findings were confirmed by previous observations concerning the distribution of DSIP-immunoreactive neuronal elements in different mammalian species, which demonstrated their predominance in the hypothalamus (Costantinidis et al, 1983; Charnay et al, 1989a,c; Vallet et al, 1990; Yon et al, 1992).

In the case of humans, much less is known about their presence and distribution. Our current knowledge of the distribution of DSIP immunoreactive neuronal elements is based only on studies done primarily with adults, i.e., brain and hypophysis (Vallet et al, 1988, 1990). These rare studies demonstrated the presence of DSIP immunoreactive neurons and fibers in several brain regions including the hypothalamus (Vallet et al, 1988, 1990). However, these studies have focused only on their colocalization with luteinizing hormone releasing hormone (LHRH), rather than their precise and detailed distribution. To our knowledge, there is no report concerning the specific anatomical organization of DSIP-immunoreactive neuronal elements in the human infant hypothalamus. Thus, the aim of the present study is to establish a detailed distribution of structures exhibiting DSIP immunoreactivity in the human hypothalamus during the first postnatal year.

METHODS

The hypothalami examined for these morphological studies were obtained from seven infant human brains. A prior approval for experiments using human tissue samples was obtained from local ethics committee at the institution (F.S.T.B.M.). Therefore, the present research involving human subjects was performed in accordance with the ethical standards embodied in the 1964 Declaration of Helsinki. The infants were aged 28, 30, 40, 60, 120, 150 and 270 days and were devoid of neurological or neuroendocrinological disorders before demise (Table I). Furthermore, no neuropathological lesions were observed upon macroscopic and histological examination of the hypothalamus. The fixation was performed by the perfusion of 2 liters of ice-cold 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. The brains were post-fixed in the same fixative solution at 4°C for 1-2 weeks and subsequently in 20% sucrose (in 0.1 M PBS, pH 7.4) for another week. A tissue block containing the hypothalamic region was sliced according to a plane transverse to the intercommissural line (from the anterior commissure to the posterior commissure). The dissected samples were placed in an embedding medium (Tissue Teck) and frozen in liquid nitrogen. They were cut on a freezing microtome (20 µm-thick coronal sections) (Frigocut, Reichert Jung, Germany). The sections destined for immunohistochemistry were collected at 100 µm intervals. The consecutive sections were counterstained with Cresyl violet for identifying the hypothalamic structures.

Immunohistochemistry

Two techniques were used to identify DSIP immunoreactivity, the indirect immunofluorescence (Coons, 1958) and the peroxidase anti-peroxidase (PAP) (Sternberger et al, 1970) methods. Before processing for immunofluorescence, lipofuscin autofluorescence was eliminated with potassium permanganate (0.25% in PBS, 20 min) (Vallet et al, 1988; Guntern et al, 1989). In the two techniques, sections mounted on gelatin-coated slides were incubated with DSIP-antiserum at working dilution 1:1000 overnight at 20°C. They were then washed with PBS and incubated with IgG goat anti-rat fluoresceine isothiocyanate (Miles, Ltd. Co.) at dilution 1: 100 for 1 h, rinsed in PBS and mounted in a phosphate buffer/glycerin mixture (1/3) when the immunofluorescence technique was applied. When the PAP technique was used, the sections were incubated with anti-rat IgG (1:50; 1h 30 min) and rat PAP (1:800; 1h 30; ICN Biomedical, Costa Mesa, USA). Peroxidase activity was revealed with the diaminobenzidine chromogen (0.5 mg:ml + H₂O₂ 0.01% for 10-15 min). After staining, sections were washed, dehydrated, and embedded in Eukitt (Merck). It is interesting to note that the original protocol (Sternberger et al, 1970) was modified as follows: treatment of sections with 0.1 M PBS containing 1.0% hydrogen peroxide (pH 7.4) for 20 min at room temperature prior to the immunoreaction, adding 0.3% Triton X-100, 1.0% normal sheep serum and 0.1% sodium azide to the primary antibody and 0.5% nickel ammonium sulfate to the final solution of revelation.

This results in a diminished background and an increased intensity staining. The anti-DSIP serum used in this immunohistochemical study was produced in rats by conjugating the peptide (Bachem, Bubendorf, Switzerland) with thyroglobulin and glutaraldehyde (Charnay et al, 1989b). Its characteristics have been described in detail previously (Charnay et al, 1988).

Specificity of the immunostaining was examined by pre-adsorption of primary antibody with the following peptides: somatostatin, substance P, neurotensin, thyrotropin releasing hormone, vasoactive intestinal polypeptide and Luteinizing hormone releasing hormone at concentrations of 100 µg/ml. None of these neuropeptides modified the immunostaining obtained with the DSIP antiserum. However, the immunoreaction was abolished when conjugated and non-conjugated DSIP 10 µg/ml were added to incubation medium. In addition, no immunoreactive neuronal DSIP structures were observed in the absence of the primary antiserum.

Hypothalamic structures were identified according to an atlas of the human hypothalamus based on Cresyl violet-stained serial sections realized for this aim in our laboratory. Black and white microphotographs were taken with a Zeiss Fluorescence microscope using Ilford FP4 films.

RESULTS

DSIP-containing neuronal elements were not homogeneously distributed throughout the rostrocaudal extent of the hypothalamus, rather distribution was restricted to defined nuclei and areas. The results will be presented as maps in Figure1 and as photomicrographs showing typical immunoreactive structures for the peptide in Figures 2-6.

Figure1: Atlas of anatomical distribution of DSIP immunoreactive perikarya (star) and fibers (dots) in human newborn/infant hypothalamus. Coronal sections throughout A-E: the anterior hypothalamus, F-G: the mediobasal hypothalamus, H-I: the posterior hypothalamus.

Figure 2: Photomicrographs showing the distribution of DSIP immunoreactive neuronal elements in the infant human hypothalamus of two months old (PAP method). A : DSIP-LI perikarya in the in the septo-preoptico junction. Bar 25 µm B : high magnification of DSIP-LI perikarya in the medial preoptic area. Note that the nucleus remains unstained. Bar 25 µm.

Cell bodies

DSIP immunoreactive cell bodies were relatively sparse in the hypothalamic region. They were, however, mainly present at the anterior and mediobasal hypothalamic levels (Fig. 1).

Anterior hypothalamus

At the junction between the septum and the preoptic region, as well at the level of diagonal band of Broca, few DSIP-LI cells were found (Fig. 1A). The immunoreactive cell bodies were generally fusiform and medium in size (Fig. 2A). At the preoptic area, senso stricto, immunopositive cell bodies were also sparse, but relatively more numerous than in the septum. They were found primarily at the rostral levels of the medial preoptic area (Figs. 1B; 2B). This pattern primarily characterizes the infants aged between 1 to 4 months. The density decreased progressively between two and four months. At 5 and 9 months, no DSIP-LI cell bodies could be seen. Ventrally to the medial preoptic area, sparse immunoreactive neurons were detected in the organum vasculosum of lamina terminalis. They were generally masked by the very dense network of DSIP-immunoreactive fibers and terminals present in the structure. Occasional cells displaying DSIP immunoreactivity were found within the periventricular nucleus (Fig. 3A). Single immunoreactive cells were also found on the border of the anterior hypothalamic area. As for the preoptic area, these DSIP-LI cell bodies were not detected at 5 and 9 months of age. At the caudal level of the anterior hypothalamic region, no immunostained cell bodies were observed in the anterior and dorsal hypothalamic areas, as well as in the suprachiasmatic, supraoptic and paraventricular nuclei (Fig. 1E).

Figure 3: Fluorescence micrographs showing DSIP-like immunoreactive cell body in the anterior hypothalamic level of two month-old infant. A: high magnification of DSIP-LI cell body in the periventricular nucleus. Bar 20 µm B: DSIP-LI perikaryon in the infundibular nucleus. Bar 25 µm

Mediobasal hypothalamus

The infundibular nucleus represents the only structure of this hypoyhalamic level, containing DSIP-immunoreactive cell bodies. The perikarya were sparse, medium sized and primarily present at the rostral part of the infundibular nucleus (Figs. 1F, 1G; 3B).

Posterior hypothalamus

This hypothalamic level is generally characterized by an absence of DSIP immunopositive cells in all its anatomical components, i.e., mammillary complex and posterior hypothalamic area (Figs. 1H-I).

Fibers and terminals

Anterior hypothalamus

The medial preoptic area contained dense patches of DSIP-LI fibers; rostrally they are less dense compared to the caudal portion where they are organized in a dense network (Figs. 1C, 1D; 4A). Ventrally, high densities of DSIP-LI fibers and terminal-like structures were observed in the organum vasculosum of lamina terminalis (Figs. 1C, 1D; 4B). The supraoptic nucleus contained rare immunoreactive fibers (Figs.1D, E). In the suprachiasmatic nucleus, the DSIP-LI fibers formed a moderately dense cluster distributed homogeneously in the entire nucleus (Figs. 1D, E). The paraventricular nucleus also displayed a relatively dense network of immunoreactive fibers present in both the magnocellular and the parvocellular parts (Figs. 1D, E, F). Equivalent densities were also present in the periventricular nucleus. In the anterior hypothalamic area, low DSIP-LI density was observed (Figs. 1D, E). These fibers were present principally in the dorsal portion of the area. The group extended dorsally to the dorsal hypothalamic area and the limits of the fornix, which was devoid of immunostaining (Fig. 1E). The density of the immunostained fibers tended to decrease in the rostrocaudal direction, and only scattered fibers were found. Laterally, the staining seemed to be continuous with a patch of stained fibers occurring in the lateral hypothalamic area (Fig. 1E).

Figure 4 : Fluorescence microphotographs showing DSIP immunoreactivity for fibers in the anterior hypothalamus of 9-month-old infant. Bar 50 µm A densely network DSIP-LI fibers and terminal like structures in the medial preoptic area. B: rich rete of varicose DSIP immunoreactive fibers in the organum vascumosum of lamina terminalis

Mediobasal hypothalamus

Relatively high densities were present in the ventromedial nucleus (Fig. 1F). A few DSIP immunoreactive fibers were distributed in the dorsomedial nucleus (Fig. 1F,G), which is in contrast with the relatively high density of immunostained fibers present in the infundibular nucleus (Figs. 1F,G). These fibers were distributed homogeneously throughout the nucleus (Fig. 5A). They seemed to extend into the tuber cinereum area and the tuberal nuclei laterally and ventromedial nucleus dorsally (Figs. 1G, H). In this latter structure, a moderately dense group of DSIP-LI fibers was distributed throughout its rostrocaudal extent (Figs. 1F,G; 5B). The tuberal nuclei (medial and lateral parts) were characterized by the presence of a low density group of immunoreactive fibers (Figs. 1G,H). The median eminence displayed a very dense group of DSIP-LI fibers (Figs. 1F; 6A). The very dense network was found in the external layer where the fibers are concentrated mostly near the blood vessels. This localization was particularly observed in infants aged between 4 and 9 months. At one month, only relatively moderate density was observed in the median eminence (Fig. 5C).

Figure 5: A: high density of DSIP-LI fibers in the infundibular nucleus of 9-month-old infant. B: moderate DSIP-LI fibers density in the ventromedial nucleus of 4-month-old infant. C: relative moderate density of DSIP immunoreactive fibers and terminal-like structures in the median eminence of 1-month-old infant. Bar 55 µm (for A, B and C)

Figure 6: Light microscopic microphotograph of the very high density of immunoreactive fibers in the median eminence of 9-month-old infant. Bar 50 µm

Posterior hypothalamus

Few immunoreactive fibers were seen in the premammillary nucleus (Fig. 1H). An equivalent pattern was present in the principal mammillary part, i.e. the medial mammillary nucleus (Fig. 1I). The other components of the mammillary complex, i.e. lateral mammillary, paramammillary and supramammillary nuclei, contained rare immunostained fibers (Fig. 1I). Dorsally, the posterior hypothalamic area shows low density of DSIP-LI fibers (Figs. 1H,I).

DISCUSSION

To our knowledge, we are reporting on the first distribution of delta sleep inducing peptide immunoreactive neuronal elements in the human hypothalamic region, during the first postnatal period. The relatively good preservation of the tissue, due in great part to the fixation mode and to the relatively good postmortem delay, allowed us to provide a detailed analysis of the peptide immunoreactivity in each hypothalamic structure.

Some aspects of tissue sampling should be noted. Indeed, in the present study, post-mortem tissues were used to investigate the immunohistochemical distribution of DSIP immunoreactivity in the newborn/infant human hypothalamus. This kind of investigation is in general limited by the post-mortem delay prior to fixation (Rossor, 1986; Swaab and Uylings, 1988), which could result in a depletion of immunoreactivity. In the present study, the post-mortem delays ranged from 5 to 12 hours and did not improve the immunoreactivity in the hypothalamic region. The comparison of the individual patterns does not indicate any difference, which favors a stability of the peptide in the range of the post-mortem delays used. These findings were in agreement with previous immunohistochemical investigations of DSIP in the post-mortem human brain (Vallet et al, 1988, 1990). We reported on the same general stability for other neuropeptides and neurotransmitter-synthesizing enzymes in infant human brain. This concerns neurotensin (Michel et al, 1986), somatostatin (Najimi et al, 1989a; Chigr et al, 1989a), LHRH (Najimi et al, 1990), substance P (Chigr et al, 1991), tyrosine hydroxylase (TH) and phenylethanolamine-N-methyl transferase (PNMT) (Chigr et al, 1989b).

DSIP immunoreactive neurones were widely distributed from the septo-preoptic junction, anteriorly, to the mammillary complex in the posterior part. The immunoreactivity is present in cells, fibers and terminals. Fibers and terminals were present throughout the rostrocaudal extent of the hypothalamus, whereas perikarya were regionally localized, principally in the anterior (preoptic area) and the mediobasal (infundibular nucleus) hypothalamus. High or very high density of fibers and terminals were seen in the preoptic area, and the infundibular nucleus as well as in the organum vasculosum of lamina terminalis. A very dense network of DSIP-LI fibers was present in the median eminence. In general, the distribution of DSIP containing neuronal elements respects the anatomical boarders of nuclei and areas of the hypothalamus.

In general, the present distribution of DSIP-LI cell bodies and fibers in the newborn/infant hypothalamus shows similarities to the immunohistochemical distribution of LHRH immunoreactivity we previously reported in the same cases (Najimi et al, 1990). Indeed, we have shown that during the first postnatal year in humans, LHRH neurons were also relatively few and mainly localized in the preoptic area and infundibular nucleus. Furthermore, the regional distribution of DSIP-LI fibers and terminal like structures is in general terms, similar to that observed for LHRH. A substantial overlap between the two peptidergic systems could be suggested in the hypothalamus during this period in human. It has been shown that DSIP and LHRH coexist in the same neuronal elements in many species including adult humans (Charnay et al, 1989b,c; Vallet et al, 1990; 1991; Pu et al, 1991; Yon et al, 1992). Whether DSIP, like LHRH, participates in the control of hypothalamo-pituitary-gonadal axis during postnatal development remains to be established. Of interest, in sudden infant death syndrome (SIDS) which affects infants aged between one month and one year, LHRH immunoreactive fibers have been shown to be less abundant in the peri- and paraventricular nuclei than in controls (Najimi et al, 1989b). As the present data show comparable density of DSIP immunoreactive fibers in these structures, it would be very interesting to analyze this immunohistochemical pattern in SIDS cases.

In the present study, we have described the immunohistochemical distribution and the development of DSIP immunoreactive neurons in the newborn/infant hypothalamic region (1-9 months). We found that the majority of hypothalamic regions exhibited a similar distribution during this postnatal period. Indeed, in these structures analyzed, there were no obvious significant changes in relation to their pattern and densities. However, in this period, some areas present considerable variations in the distribution and the density of DSIP-LI neuronal elements. In the median eminence, relatively moderate density of DSIP-LI fibers were observed at one month, later their density increases sharply, and at 4 months a very dense network of DSIP-LI characterized the structure. In older infants, a similar pattern and fiber-density distribution were observed. Therefore, there appears to be a postnatal development of DSIP-LI fibers within this region.

The present study also demonstrates differences in the density of DSIP-LI cell bodies in the anterior region of the hypothalamus between 1 and 9 months. A low density of immunoreactive perikarya was present at the first postnatal month. Their number decreased remarkably at the second month after birth and then were absent after 4 months of age. In the human adult, only DSIP-LI fibers have been demonstrated in the preoptic area, but no DSIP-LI cell bodies (Vallet et al, 1990). This suggests that DSIP-LI cell bodies in this structure appeared at fetal life as it has been shown in the guinea pig (Pu and Dubois, 1992). Such cells become progressively less noticeable at later times in development.

Indeed, as development proceeds, the number of cells showing DSIP immunoreactivity decreases, and fewer cells in the preoptic area were immunoreactive. The analysis of the findings in the infant (this study), as well as those obtained in the adult (Vallet et al, 1990), suggests that the development of some hypothalamic structures is continued in the perinatal period (Swaab, 1995; 1999). This phenomenon may imply either the natural degeneration of some neurons during CNS development (Forger and Breedlove, 1987) or the processing of the peptide synthesis. It has been noted that neonatal animals have a tendency to show decreased immunoreactivity of neuronal perikarya (Pu and Dubois, 1992), and it may be for similar reasons that we were not able to detect DSIP-LI neurons so readily in human infants. We have previously noted similarly marked age-related changes in peptidergic systems in the human brain (Najimi et al, 1989a,b, 1990, 1991a, b; Sarrieau et al, 1994). Further studies of DSIP-LI neurons in human brain may well show additional changes in staining intensity and perhaps also of distribution of terminals with advancing age. One can hypothesize that in humans as in experimental animals this tendency in decreasing density may be an important step in the maturation (Ulfig et al, 2000) of the hypothalamic structures. The present findings showing the presence of developmental patterns of DSIP immunoreactivity in the human infant hypothalamus contrast with radioimmunoassay investigations of human DSIP in human infants during the first postnatal year. In these studies, no age-related differences of the plasma levels of DSIP during the first postnatal year of life have been found (Scholle et al, 1992). This suggests the presence of differential roles of DSIP in relation to its localization. Since altered plasma levels of DSIP have been measured only in hypophyseal portal blood, the unchanged peripheral plasma levels of DSIP could however be due to the fact that fibers have already reached the capillary loops of the median eminence and they start to release their peptide content. Alternatively to the presence of a dense network of DSIP-LI fibers in the median eminence, these immunoreactive axons could interact with releasing factor containing axons present in this structure in the control of pituitary function. Indeed, we have previously demonstrated in human the presence of thyrotropin releasing hormone (TRH) (Borson-Chazot et al, 1986), somatostatin (Najimi et al, 1989a) and LHRH (Najimi et al, 1990) in the median eminence. These findings call up the possibility of the coexistence of DSIP with these releasing factors. In support of this, an involvement of DSIP in the control of GH (Iyer and Mc Cann, 1987a) and LH release (Iyer and Mc Cann, 1987b; Sahu and Klara, 1983) from pituitary and somatostatin from the median eminence (Iyer and Mc Cann, 1987c) has been shown in rats. Nevertheless, these studies carried out in rodents cannot be directly extrapolated to the infant, since they concerned the adult stage only.

Functional significance of postnatal changes and activity of DSIP neurons in the hypothalamus has to be considered under several aspects related to the differentiation of the central nervous system in humans (Ulfig et al, 2000), to the activity of DSIP in the CNS during ontogeny, and the molecular and cellular targets of pharmacological or addictive molecules interacting with DSIP transmission. The function of DSIP during development is unknown, and whether the modes of action of DSIP during development progress in the same way as in the adult is an open question. Although it is unknown if these DSIP neuronal elements are actually producing and utilizing DSIP as a neuromodulator by this time, our histological observations are compatible with their proposed role in the regulation of many postnatal physiological functions.

Our findings about the distribution of DSIP in infants agrees with the results of previous immunohistochemical studies in the adult human hypothalamus (Vallet et al, 1990). Indeed, both in infants and adults, neuronal elements are predominantly distributed in the preoptic region, organum vasculosum of lamina terminalis, periventrivular and ventromedial nuclei as well as median eminence. However, some differences have been found. This particularly concerns the neuronal group demonstrated in the infant medial preoptic area that was not reported in adults. Furthermore, many structures such as suprachiasmatic, supraoptic, paraventricular and parafornical nuclei, anterior, tuber cinereum and posterior hypothalamic areas as well as mammillary complex, displaying DSIP immunoreactive fibers and terminal-like structures, have not been reported to show any immunoreactivity in adults. These differences could be due to the fact that these investigations performed in adults have focused only on colocalization of DSIP and LHRH, rather than on the detailed anatomical distribution. As another explanation, the disappearance of some DSIP-LI structures in adults could reflect the possible involvement of DSIP in neuronal maturation during development.

In summary, the present study establishes the precise distribution of DSIP-LI neuronal elements in the newborn/infant hypothalamus and shows the presence of immunoreactive elements that were not reported in adults. On the other hand, this study constitutes an additional step toward the comprehension of the neurochemical development of human hypothalamus. The investigation of specific fetal stages and prenatal periods will help us to understand the special roles of DSIP in development. Finally, this distribution in a normal human infant's hypothalamus may constitute an anatomical basis for comparison with the neuronal DSIP elements in pathological cases such as sudden infant death syndrome which involves the hypothalamic region (Najimi et al, 1989b).

ACKNOWLEDGEMENTS

We are grateful to Pr. N. Kopp (Lab. Anat. Pathol. Lyon), R. Gilly (Lyon Sud Hospital) and Dr. R. Bouvier (E. Herriot Hospital, Lyon, France) for their efficient help in obtaining the tissue samples. The authors would also like to thank Dr. Y. Charnay (Institutions Psychiatriques, Geneva, Switzerland) for the gift of the anti serum.

Corresponding author: M. Najimi: Unité de Génie Biologique. Département de Biologie & Biotechnologie, F.S.T de Beni Mellal, B.P : 523, 23000 . Beni Mellal, Morocco. Tel: (212)-23-48-51-12. Fax: (212)-23-48-52-01. Email: mnajimi1@fstbm.ac.ma

Received: October 17, 2000. In revised form: January 18, 2001. Accepted: February 20, 2001

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