Brain Research, 239 (1982) 265-270 Elsevier Biomedical Press
Further studies on the identification of neurons containing immunoreactive alpha-melanocyte-stimulating hormone (a-MSH) in the rat brain
J. GUY, H. VAUDRY and G. PELLETIER (J.G. and G.P.) MRC Group in Molecular Endocrinology, Le Centre Hospitalier de l' Universitd Lava/, Quebec G1 V 4G2 (Canada) and ( H. V.) Laboratoire d'Endocrinologie, Universitd de Rouen (France)
(Accepted January 21st, 1982) Key words: a-melanocyte-stimulating hormone - - immunocytochemistry - - neurotransmitter -synaptic contact
In order to identify more accurately the organelles containing immunoreactive a-melanocytestimulating hormone (a-MSH) in the rat brain, attempts were made to improve the ultrastructural preservation of the neural tissue used for immunostaining of the peptide. With the post-embedding staining technique, the quality of the preservation was not adequate since only low concentrations (0.2-0.5~o) of OsO4 could be used for post-fixation. Higher concentrations of OsO4 completely destroyed the antigenicity of a-MSH. With the pre-embedding technique in which the immunostaining is performed prior to postfixation with OsO4, a good preservation could be obtained making possible the identification of organelles and classification of endings containing immunoreactive a-MSH. In the dendrites, the staining was rather diffuse without any clear association with organelles. In the endings, the staining was mostly restricted to dense core vesicles with some degree of diffusion in the cytoplasm. None of the positive endings were seen making synaptic contact. These results support the hypothesis that a-MSH could be released in sites other than the classical synaptic junction and act as a local hormone. Since the discovery o f i m m u n o r e a c t i v e a l p h a - m e l a n o c y t e - s t i m u l a t i n g h o r m o n e ( a - M S H ) in the rat brain10,11, there have been a few reports a b o u t the d i s t r i b u t i o n o f this p e p t i d e in the central nervous system. By r a d i o i m m u n o a s s a y a n d i m m u n o s t a i n i n g techniques2,4,~,s,9,11, this peptide has been detected in several b r a i n areas o f the rat including the cortex, h y p o t h a l a m u s , p r e o p t i c areas, thalamus, m e s e n c e p h a l o n a n d pons, with the highest c o n c e n t r a t i o n in the h y p o t h a l a m u s . Very recently, with immunofluorescence a n d i m m u n o h i s t o c h e m i c a l techniques, two separate n e u r o n a l aM S H systems have been described6,7,1s, 19. T h e first one c o n s t i t u t e d o f n e u r o n s located in the a r c u a t e nucleus has projections similar to t h a t o f the e n d o r p h i n s system, whereas the second one has n e u r o n s located in the d o r s o l a t e r a l h y p o t h a l a m u s with fibers projecting into the cortex, h i p p o c a m p u s a n d striatum. W i t h i m m u n o e l e c t r o n m i c r o s c o p y p e r f o r m e d in different b r a i n areas we have shown that a - M S H could only be localized in dense core vesicles in axon endings a n d cell bodies 11. M o r e o v e r , we have recently d e m o n s t r a t e d the two a - M S H systems c o u l d n o t be distinguished at the electron m i c r o s c o p i c level 6. O n the basis o f these ultrastructural d a t a a n d the observations t h a t a - M S H when injected can exert some effects on 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
266 the behavior of the rat a, it has been suggested that this peptide could act as a neurotransmitter or neuromodulator. Due to the use of aldehydes as fixatives, it has not been so far possible to obtain an adequate preservation and then to determine if the positive endings were not making synaptic contacts with other nerve structures6,11. In order to fully characterize and tentatively classify the a-MSH containing endings, it appeared of great interest to pursue our previous ultrastructural studies with different technical approaches. Adult female rats weighing 250-300 g were used in these experiments. To perform immunostaining, the unlabeled antibody peroxidase antiperoxidase (PAP) method of Sternberger 1~ was routinely used. This technique was applied to either fixed and non-embedded tissue (pre-embedding technique) or plastic-embedded tissue (postembedding technique). For the pre-embedding technique, the animals were perfused with a mixture of' 0.1 O//oglutaraldehyde and 4 ~ paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). Coronal sections (100/~m) of the hypothalamus, thalamus, septum, periaqueductal gray matter were cut with the Vibratome (Oxford). These sections were stained according to Pickel's technique 14 and postfixed in 2 0~ OsO4 prior to dehydration and embedding in Araldite. For the postembedding technique, the animals were fixed by perfusion of 2 "j /o glutaraldehyde in 0.1 M cacodylate buffer. The hypothalamus, thalamus, septum and periaqueductal gray matter were dissected and postfixed for 1 h in different concentrations of OsO4:0.2 jo;, 0.5 ~o or l ~ , and 2 ~,,. After dehydration in ethanol, the blocks were embedded in Araldite. In these localization studies, the antiserum (no 8111-01) (ref. 17) to a-MSH was used at the dilutions of 1:500 and 1:1000 for the pre-embedding technique and 1:500 to 1:3000 for the postembedding technique. Although specificity of this antiserum has been carefully studied by radioimmunoassay 17 and immunocytochemistry 6,7 we made immunoabsortion controls with the following antigens at a final concentration of 10-6 M: synthetic a-MSH, synthetic ACTH 1-24, purified human fl-LPH (supplied by Dr. C. H. Li), and synthetic human fl-endorphin (supplied by Dr. D. Coy). Although a-MSH have been easily detected with the postembedding technique in non-osmicated tissue 6,1 t, attempts to localize this peptide in osmicated tissue were less successful. In fact, in tissue postfixed in 1 and 2 ~ OsO4, no staining could be observed, even with low dilution (1:500) of the antiserum. When lower concentrations of OsO4 (0.2 or 0.5 o~) were used, some reaction could be detected but the synaptic densities were not adequately preserved, then making difficult the classification of positive endings. With the pre-embedding technique, it was possible to detect positive axons, endings and dendrites in the different brain areas studied. Since the ultrastructure was adequate, the characteristics of the neuronal elements containing immunoreactive aMSH were studied with this technique. On the basis of previous works 14,15, we arbitrarily defined as terminals the varicosities larger than 0.5 #m in diameter and containing at least 3 large dense core vesicles as well as small clear vesicles in cross-sections. In all the areas studied, labeled intervaricose, axons and terminals could be
Fig, 1. Localization of immunoreactive a-MSH in the hypothalamic periventricular nucleus with the pre-embedding staining technique. A diffuse reaction can be observed in a dendrite (D). Note the presence of synaptic density (arrow). x 24,500.
Fig. 2. Localization of immunoreactive a-MSH in the periaqueductal gray matter. The reaction product is mostly found in dense core vesicles (arrows) and to a lesser extent throughout the axoplasm in a nerve ending. Note the presence of small clear vesicles (arrowheads). x 32,000.
Fig. 3. Localization of immunoreactive a-MSH in a nerve ending in the arcuate nucleus. Staining is almost exclusively localized in dense core vesicles (arrows). Small clear vesicles are unstained. Note the presence of a synaptic junction in an adjacent unstained ending (arrowhead). 32,000.
observed. Positive dendrites were also observed in the hypothalamus and not in the other areas examined. In the dendrites, the staining was rather diffuse without any labeling of specific organelles (Fig. 1). Mitochondria were always free of reaction. No synaptic contact was observed between labeled dendrites and unstained terminals. In positive axons and terminals (Figs. 2 and 3), the reaction product was mostly associated with dense core vesicles (60-100 nm in diameter) with some degree of diffusion throughout the cytoplasm. On the 74 labeled terminals observed in different areas, none was seen forming classical synaptic junctions. The present results indicate that a-MSH immunoreactivity cannot survive a regular osmication. This peptide is more susceptible to OsO4 than other peptides, such as enkephalin, substance P, somatostatin and L H R H which can be detected in wellpreserved tissue with the postembedding technique 12. With the pre-embedding staining technique, immunoreactive a-MSH was found in axons and terminals, confirming previous observations obtained with the post-embedding technique 7,al. The diffuse staining also observed in the positive terminals and axons has already been described for other peptides such as enkephalins 1'5, substance p15 and vasoactive intestinal peptide (VIP) 13. It is still unclear whether this diffuse staining represents an artefact diffusion due to the use of Triton or the real presence of the peptide outside the dense core vesicles.
269 The absence o f synapsis in a large n u m b e r o f r a n d o m l y sectioned endings c o n t a i n i n g i m m u n o r e a c t i v e a - M S H is different f r o m w h a t has been observed for other peptides, such as enkephalin, substance P a n d V I P which can be easily detected in endings m a k i n g synaptic contact12,13,15. It c a n n o t be totally excluded that only a small percentage o f endings containing a - M S H has synaptic specialization a n d t h a t due to the small s a m p l i n g inherent to electron m i c r o s c o p y technique, we have been u n a b l e to detect them. N o m o r e t h a n 50 % o f V I P containing endings have been r e p o r t e d to m a k e synaptic contact lz. The staining for a - M S H observed in dendrites is a new o b s e r v a t i o n which c a n n o t be o b t a i n e d with the p o s t e m b e d d i n g technique. A s previously discussed in relation with V I P localization xz, it seems that dendrites c a n n o t be stained with the poste m b e d d i n g technique. O n the basis o f the present results, it can be suggested t h a t m o s t if n o t all b r a i n aM S H is released by neurons at sites other t h a n the classical synaptic junctions, p r o b a b l y to act as a local h o r m o n e . Such a perspective has been p r o p o s e d for the noradrenergic system 1.
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