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ACTH-immunoreactive buotons form synaptic contacts in the hypothalamic arcuate nucleus of rat: evidence for local opiocortin connections
Brain Research, 263 (1983) 142-146
142
Elsevier Biomedical Press
ACTH-immunoreactive boutons form synaptic contacts in the hypothalamic arcuate nucleus of rat: evidence for local opiocortin connections J. Z. KISS and T. H. WILLIAMS
Department of Anatomy, Collegeof Medicine, Universityof lowa, Iowa City, 1,4 52242 (U.S.A.) (Accepted November 1lth, 1982)
Key words: ACTH immunoreactivity - arcuate nucleus - opiocortin - hypothalamic ACTH
ACTH-like immunoreactive structures were localized in rat hypothalamic arcuate nucleus using the unlabeled antibody, peroxidase-antiperoxidase method. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites and with ACTH-like immunoreactive perikarya. The results obtained are consistent with the hypothesis that ACTH in the brain acts as a synaptic regulator or transmitter, contributing not only to long projection pathways but also to a local circuit in the arcuate nucleus where ACTH cell bodies are localized.
It is now well recognized that adrenocorticotropin (ACTH), along with other opiocortin peptides, i.e., fi-endorphins (fl-END), a-melanotropin (a-MSH) and fl-lipotropin (fl-LPH), which were observed initially in the pituitary, are also present in neuronal elements in the brain (see reference in review 13). Ultrastructural immunocytochemical studies have provided evidence to support the claim that opiocortins are mainly confined to dense cored vesicles present in nerve fibers and endings ~3. There is also pharmacological evidence indicating that these peptides interact with specific receptor sites in the brain and can be released by electrical stimulation (for review see refs. 13, 22). Therefore, opiocortin peptides can be regarded as putative neurotransmitters and neuromodulators. They are derived from a common precursor (31 K pro-opiocortin) and have been shown to coexist in hypothalamic arcuate neurons 13. These opiocortin arcuate-ventral premammillary neurons apparently innervate a variety of hypothalamic, thalamic and brain stem structures [see reference in review 15]. Using light microscopic immunocytochemistry, a dense arborization of ACTH fibers and varicosites has been demonstrated in the arcuate nucleus 3J3 in addition to immunoreactive cell 0006-8993/83/0000 0000/$03.000 1983 Elsevier Biomedical Press
bodies. Because of limited resolution, light microscopic methods are incapable of distinguishing between such elements as classical synaptic contacts and simple 'boutons en passant'. Since ACTH-like immunoreactive neurons are predominantly or exclusively located in or adjacent to the arcuate and ventral premammillary nucleP the additional demonstration that ACTH-positive nerve endings are engaged in synaptic contacts here would provide structural evidence to support the hypothesis that an intrinsic opiocortin circuit is present. Furthermore, although there is electrophysiological evidence for a direct postsynaptic effect12 by opioid peptides, the demonstration that opiocortin peptide-containing nerve terminals engage in classical synaptic contacts in any brain area has been successfuP insofar as we are aware. To answer this question, ACTH-immunoreactive neurons and their connections were studied in the arcuate nucleus by electron microscopic immunocytochemistry. Male Sprague-Dawley rats, 200 _ 10 g b. wt., were anesthetized and their brains fixed by transaortic perfusion of 4% paraformaldehyde, 0.05% glutaraldehyde and 0.2% picric acid in 0.167 M phosphate buffer (pH 7.3) 17. The brains were cut into blocks which were placed in ice-
143 cold fixative for 3 h and then washed in several changes of phosphate buffered saline (PBS). To enhance penetration by reagents the blocks were frozen in liquid nitrogen and subsequently thawed in PBS 17. Coronal sections of 20-50 /~m thickness were cut with a Vibratome (Oxford) and processed by a modified Sternberger 18 immunoperoxidase technique. Sections were incubated in ACTH antiserum (dilution 1:1000) at 4 °C for 24 h; washed in PBS, incubated in goat anti-rabbit IgG (Cappel) 1:40 for lh, washed again in PBS, and placed in rabbit peroxidase antiperoxidase complex (Cappel) 1:100 at room temperature for an hour. The chromagen used was 3,3-diaminobenzidine tetrahydrochloride (Hach). Triton-X was not used in any of the solutions. Sections used for light microscopy were mounted on chromealum-coated slides, dehydrated and cover-slipped. Adjacent sections to be used for electron microscopy were osmicated, dehydrated and embedded in Epon 812 (Ladd Research Industries) between two sheets of transparent Aclar (Allied Chemical). Thin sections were examined (without counterstaining with uranyl acetate or lead citrate) in a Philips 201 electron microscope. Forty-eight hours prior to sacrifice, two of the rats used in these experiments were injected in the lateral ventricle with colchicine (Sigma) 50 /xg dissolved in 25 #10.9% NaC1. ACTH antiserum used in this study was generated in rabbit against ACTHt_32 (Richter, Budapest) conjugated to bovine serum albumin and purified by affinity chromatography4,5. This antibody has been shown to recognize the N-terminal portion of the ACTH molecule and to cross-react with a-MSH. The specificity test used in our immunocytochemical control experiment revealed no cross-reactivity with fl-endorphin, fl-lipotropin (fl-LPH), m e t h i o n i n e and leucine-enkephalin, corticotropin-like intermediate lobe peptide (CLIP) and bovine serum albumin. Absorption of the ACTH serum by either ACTHt_39 or ACTHI_24 eliminated all immunocytochemical staining. After omitting the primary antiserum or replacing with normal rat serum, peroxidase reaction product was localized in dense bodies in some glial cells but no
reaction product was seen in neuronal elements. Using light microscopy to study serial sections immunostained with ACTH antiserum, immunoreactive cell bodies were observed only in the arcuate nucleus and the adjacent ventral premammillary nucleus. Although we found that colchicine treatment enhanced cell body staining, no immunoreactive perikarya were observed elsewhere following its administration. In addition to ACTH-like cell bodies, a dense pattern of ACTH-positive nerve fibers and varicosities was present throughout the arcuate-ventral premammillary area (Fig. 1A). At the ultrastructural level, immunopositive cell bodies, dendrites and bouton profiles were all identified. In ACTH-positive neurons, peroxidase reaction product was localized in large dense cored vesicles as well as on the surfaces of agranular vesicles and all intracellular organelles (Fig. 1C), despite the low concentration of antiserum used. Although many labeled bouton profiles were found, those which possessed 'classical' synaptic membrane specializations were relatively uncommon. However, there was clear evidence that ACTH-immunoreactive boutons made synaptic contacts with various other elements within the arcuate nucleus (Figs. 1C-F). As observed in the present study, labeled elements only made connections at symmetrical (Gray type II) synapses (Fig. 1C-F). Most commonly, labeled terminal profiles synapsed with unstained dendrites (Fig. 1D-F) but a number of examples of immunoreactive boutons making synaptic contact with ACTH-Iike immunostained perikarya were seen also (Fig. 1C). In the present study we have demonstrated that ACTH immunoreactive boutons in the arcuate nucleus participate in synapses. This finding may apply generally to opiocortin neurons, since it has been claimed that ACTH and other biosynthetically related opioid peptides (flEND/fl-LPH/fl-MSH) are parts of the same neuronal system t3. However, this may be an imperfect generalization because a separate aMSH immunoreactive neuron population has been localized outside the arcuate nucleus ~3 (e.g., in the dorsolateral posterior hypothalamus). The ACTH antiserum used in the present
144
Fig. 1. Light (A) and electron (B-F) micrographs of ACTH-immunoreactive neuronal elements in the a rcuate nucleus of rat. A: light microscopic field showing several immunopositive neurons (white stars) and varicosities (arrows) in a 50 ttm thick vibratome section. Nomarski optics. Bar scale = 5/tm. B: an immunoreactive bouton packed with labeled synaptic vesicles. Arrow points to a large, immunoreactive vesicle. Bar scale = 0.5/~m. C: an ACTH-immunopositive bouton (white star) makes a symmetrical synaptic contact (arrow) with a labeled perikarya. Arrowheads point to large, immunoreactive, granulated vesicles. Bar scale = 0.5/xm. D-F: immunoreactive boutons form symmetrical synaptic contacts (arrows) with unlabeled dendrites (D). In E, triple arrows point to a subsynaptic specialization. Bar scale = 0.5/~m.
145 study has not visualized this latter a-MSH positive neuron system, indicating that the presynaptic elements identified in the arcuate nucleus most probably originate from neighboring cell bodies and therefore represent local, intrinsic connections. Previous morphological studies have led to the observation of a variety of intrinsic connections in the arcuate nucleus 2.8-2°. The question as to whether some of the opiocortin neurons are specifically local circuit neurons which lack any long projecting axons, or whether they are projection neurons which also possess recurrent collaterals, has not been resolved. From a study of Golgi preparations, Van den Pol and Cassidy2° reported axons with both origins and terminations within the rat arcuate nucleus. They also described a different class of neuron with bifurcating nerve fibers, with one branch leaving the nucleus and the other terminating within it. Which of these patterns is followed by opiocortin neurons still has to be determined. Both non-labeled and labeled postsynaptic elements were found to receive inputs from identifiable opiocortin terminals. The chemical properties of the postsynaptic non-labeled neurons remains to be clarified, though there are numerous candidates since perikarya containing dopamine, serotonin acetylcholine, neurotensin, GABA, somatostatin, substance P and enkephalin have been localized in the arcuate nucleus (for recent review, see ref. 15). It should be noted, however, that even some non-labeled postsynaptic elements could belong to opiocortin neurons and remained unstained because of the limitations and variables inherent in the immunocytochemical methods. The observed labeled axosomatic contact may represent the morphological basis of an 'ultrashort' feedback loop from opiocortin neurons back to the same or to other neurons of the same transmitter type.
The idea that there may be an 'ultrashort' feedback loop within the hypothalamus was originally conceived in the context of vasopressin, oxytocin and releasing as well as release-inhibiting hormones I°. It was postulated that these substances could control the activity of neurons producing them either through changes of their titers in the general circulation or through direct neuronal feedback, e.g. recurrent axon collaterals. Ultrashort feedback loops of oxytocin, vasopressin I° and somatostatin 9 have already been demonstrated in pharmacological experiments. Furthermore, a number of Golgi ~9-2~ and deafferentation 2,6-s studies have provided evidence that the arcuate as well as other hypothalamic nuclei have a recurrent axon collateral system. Recurrent axon collaterals along with interneurons are the morphological substrates of local neuronal circuits, which represent a standard form of organization which is reduplicated extensively in the brain. Indeed, according to Rak i c L6, 'it is probably safe to state that there is not a single structure in the mammalian central nervous system that lacks LCN's' (local circuit neurons). It is now widely maintained ~6 that these local neuronal circuits integrate morphologically delineated aggregates of neurons (e.g. nucleus) to operate as a functional unit, i.e. to integrate the function of many neurons of the same type and to influence the activity of different types of neurons within the same nucleus. Our results suggest that opiocortin neurons in the arcuate nucleus may contribute not only to output channels to the median eminence, hypothalamic and extrahypothalamic areas ~5but also participate in a local neuronal circuit. Subsequent physiological and pharmacological experiments may help to illuminate the functional correlates of this morphological finding.
1 Guy, J., Vaudry, H. and Pelletier, G., Futher studies on the identification of neurons containing immunoreactive alpha-melanocyte-stimulating hormone (c~-MSH) in the rat brain, Brain Research, 239(1982) 265-270. 2 Halfisz, B2, Koves, K., Rethelyi, M., Bodoky, M. and Koritsanszky, S., Recent data on neuronal connections between nervous structure involved in the control of the ad-
enohypophysis. In W. E. Stumpf and L. D. Grant (Eds.), New York, Karger, 1975 pp. 9-14. 3 Joseph, S. A., Immunoreactive adrenocorticotropin in rat brain: a neuroanatomical study using antiserum generated against synthetic ACTH (1 39), Amer. J. Anat., 158 (1980) 533 548. 4 K~rteszi, M., Makara, G. B. and Stark, E., The rise of
146
5
6
7
8 9 10
12
plasma ACTH induced by ether is mediated through neural pathways entering the medial basal hypothalamus, A cta endocrinol., 93 (1980) 129-133. K~irteszi, M., Stark, E., Rappay, Gy., L~iszl6, F. A. and Makara, G. B., Corticoliberin activity of rat neurohypophysis is distinct from vasopressin, Amer. J. Physiol., 240 (1981) E685 E693. Kiss, J. Z., Palkovits, M., Zaborszky L., Tribollet, E., Szab6 D. and Makara, G. B., Quantitative histological studies on the hypothalamic paraventricular nucleus in rats. II. Number of local and certain afferent nerve terminals, Brain Research, in press. L6r~inth, Cs., Z~iborszky, L., Marton, J. and Palkovits, M., Quantitative studies on the supraoptic nucleus in the rat. I. Synaptic organization, Exp. Brain Res., 22 (1975) 509 523. Matsumoto, A. and Arai, Y., Morphological evidence for intranuclear circuits in the hypothalamic arcuate nucleus, Exp. Neurol., 59 (1978) 404-412: McCann, S. M., Control of anterior pituitary hormone release by brain peptides. Neuroendocrinologv, 31 (1980) 355 363. Motta, M., Fraschini, F. and Martini, L., 'Short' feedback mechanism in the control of anterior pituitary function. In W. F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinologv, 1969, pp. 211-255. North, R. A., Opiates, opioid peptides and single neurones, Life Sci., 24 (1979) 1527-1546.
13 O'Donohue, T. L. and Dorsa, D. M., The opiomelanotropinergic neuronal and endocrine systems, Petides, 3 (1982) 353-395. 14 Palkovits, M., Neurotransmitter distribution in the brain, Front. Hormone Res., 10 (1982) 15-32. 15 Palkovits, M., Topography of chemically identified neurons in the central nervous system: Progress in 19771979, Med. Biol., 58 (1980) 188-227. 16 Rakic, P., Local circuit neurons, Neurosci. Res. Prog. BulL, 13 (1975) 291-446. 17 Somogyi, P. and Hiroshi, T., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytocb,emistry, Neuroscienc~, 7 (1982) in press. 18 Sternberger, L. A., lmmunocytochemistry, Prentice-Hall, Englewood Cliffs, N. J., 1974. 19 Van den Pol, A. N., The magnocellular and parvocellular paraventricular nucleus of rat: intrinsic organization, J. comp. Neurol., 206 (1982) 317 345. 20 Van den Pol, A. N. and Cassidy, J. R., The hypothalamic arcuate nucleus of rat - A quantitative Golgi analysis, J. comp. Neurol., 204 (1982) 65 98. 21 Van den Pol, A. N., The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy, J. comp. Neurol., 191 (1980) 661-702. 22 Watson, S. J., Akil, H. and Walker, J. M., Anatomical and biochemical studies of the opioid peptides and related substances in the brain, Peptides, 1, Suppl. 1 (1980) 11-20.
142
Elsevier Biomedical Press
ACTH-immunoreactive boutons form synaptic contacts in the hypothalamic arcuate nucleus of rat: evidence for local opiocortin connections J. Z. KISS and T. H. WILLIAMS
Department of Anatomy, Collegeof Medicine, Universityof lowa, Iowa City, 1,4 52242 (U.S.A.) (Accepted November 1lth, 1982)
Key words: ACTH immunoreactivity - arcuate nucleus - opiocortin - hypothalamic ACTH
ACTH-like immunoreactive structures were localized in rat hypothalamic arcuate nucleus using the unlabeled antibody, peroxidase-antiperoxidase method. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites and with ACTH-like immunoreactive perikarya. The results obtained are consistent with the hypothesis that ACTH in the brain acts as a synaptic regulator or transmitter, contributing not only to long projection pathways but also to a local circuit in the arcuate nucleus where ACTH cell bodies are localized.
It is now well recognized that adrenocorticotropin (ACTH), along with other opiocortin peptides, i.e., fi-endorphins (fl-END), a-melanotropin (a-MSH) and fl-lipotropin (fl-LPH), which were observed initially in the pituitary, are also present in neuronal elements in the brain (see reference in review 13). Ultrastructural immunocytochemical studies have provided evidence to support the claim that opiocortins are mainly confined to dense cored vesicles present in nerve fibers and endings ~3. There is also pharmacological evidence indicating that these peptides interact with specific receptor sites in the brain and can be released by electrical stimulation (for review see refs. 13, 22). Therefore, opiocortin peptides can be regarded as putative neurotransmitters and neuromodulators. They are derived from a common precursor (31 K pro-opiocortin) and have been shown to coexist in hypothalamic arcuate neurons 13. These opiocortin arcuate-ventral premammillary neurons apparently innervate a variety of hypothalamic, thalamic and brain stem structures [see reference in review 15]. Using light microscopic immunocytochemistry, a dense arborization of ACTH fibers and varicosites has been demonstrated in the arcuate nucleus 3J3 in addition to immunoreactive cell 0006-8993/83/0000 0000/$03.000 1983 Elsevier Biomedical Press
bodies. Because of limited resolution, light microscopic methods are incapable of distinguishing between such elements as classical synaptic contacts and simple 'boutons en passant'. Since ACTH-like immunoreactive neurons are predominantly or exclusively located in or adjacent to the arcuate and ventral premammillary nucleP the additional demonstration that ACTH-positive nerve endings are engaged in synaptic contacts here would provide structural evidence to support the hypothesis that an intrinsic opiocortin circuit is present. Furthermore, although there is electrophysiological evidence for a direct postsynaptic effect12 by opioid peptides, the demonstration that opiocortin peptide-containing nerve terminals engage in classical synaptic contacts in any brain area has been successfuP insofar as we are aware. To answer this question, ACTH-immunoreactive neurons and their connections were studied in the arcuate nucleus by electron microscopic immunocytochemistry. Male Sprague-Dawley rats, 200 _ 10 g b. wt., were anesthetized and their brains fixed by transaortic perfusion of 4% paraformaldehyde, 0.05% glutaraldehyde and 0.2% picric acid in 0.167 M phosphate buffer (pH 7.3) 17. The brains were cut into blocks which were placed in ice-
143 cold fixative for 3 h and then washed in several changes of phosphate buffered saline (PBS). To enhance penetration by reagents the blocks were frozen in liquid nitrogen and subsequently thawed in PBS 17. Coronal sections of 20-50 /~m thickness were cut with a Vibratome (Oxford) and processed by a modified Sternberger 18 immunoperoxidase technique. Sections were incubated in ACTH antiserum (dilution 1:1000) at 4 °C for 24 h; washed in PBS, incubated in goat anti-rabbit IgG (Cappel) 1:40 for lh, washed again in PBS, and placed in rabbit peroxidase antiperoxidase complex (Cappel) 1:100 at room temperature for an hour. The chromagen used was 3,3-diaminobenzidine tetrahydrochloride (Hach). Triton-X was not used in any of the solutions. Sections used for light microscopy were mounted on chromealum-coated slides, dehydrated and cover-slipped. Adjacent sections to be used for electron microscopy were osmicated, dehydrated and embedded in Epon 812 (Ladd Research Industries) between two sheets of transparent Aclar (Allied Chemical). Thin sections were examined (without counterstaining with uranyl acetate or lead citrate) in a Philips 201 electron microscope. Forty-eight hours prior to sacrifice, two of the rats used in these experiments were injected in the lateral ventricle with colchicine (Sigma) 50 /xg dissolved in 25 #10.9% NaC1. ACTH antiserum used in this study was generated in rabbit against ACTHt_32 (Richter, Budapest) conjugated to bovine serum albumin and purified by affinity chromatography4,5. This antibody has been shown to recognize the N-terminal portion of the ACTH molecule and to cross-react with a-MSH. The specificity test used in our immunocytochemical control experiment revealed no cross-reactivity with fl-endorphin, fl-lipotropin (fl-LPH), m e t h i o n i n e and leucine-enkephalin, corticotropin-like intermediate lobe peptide (CLIP) and bovine serum albumin. Absorption of the ACTH serum by either ACTHt_39 or ACTHI_24 eliminated all immunocytochemical staining. After omitting the primary antiserum or replacing with normal rat serum, peroxidase reaction product was localized in dense bodies in some glial cells but no
reaction product was seen in neuronal elements. Using light microscopy to study serial sections immunostained with ACTH antiserum, immunoreactive cell bodies were observed only in the arcuate nucleus and the adjacent ventral premammillary nucleus. Although we found that colchicine treatment enhanced cell body staining, no immunoreactive perikarya were observed elsewhere following its administration. In addition to ACTH-like cell bodies, a dense pattern of ACTH-positive nerve fibers and varicosities was present throughout the arcuate-ventral premammillary area (Fig. 1A). At the ultrastructural level, immunopositive cell bodies, dendrites and bouton profiles were all identified. In ACTH-positive neurons, peroxidase reaction product was localized in large dense cored vesicles as well as on the surfaces of agranular vesicles and all intracellular organelles (Fig. 1C), despite the low concentration of antiserum used. Although many labeled bouton profiles were found, those which possessed 'classical' synaptic membrane specializations were relatively uncommon. However, there was clear evidence that ACTH-immunoreactive boutons made synaptic contacts with various other elements within the arcuate nucleus (Figs. 1C-F). As observed in the present study, labeled elements only made connections at symmetrical (Gray type II) synapses (Fig. 1C-F). Most commonly, labeled terminal profiles synapsed with unstained dendrites (Fig. 1D-F) but a number of examples of immunoreactive boutons making synaptic contact with ACTH-Iike immunostained perikarya were seen also (Fig. 1C). In the present study we have demonstrated that ACTH immunoreactive boutons in the arcuate nucleus participate in synapses. This finding may apply generally to opiocortin neurons, since it has been claimed that ACTH and other biosynthetically related opioid peptides (flEND/fl-LPH/fl-MSH) are parts of the same neuronal system t3. However, this may be an imperfect generalization because a separate aMSH immunoreactive neuron population has been localized outside the arcuate nucleus ~3 (e.g., in the dorsolateral posterior hypothalamus). The ACTH antiserum used in the present
144
Fig. 1. Light (A) and electron (B-F) micrographs of ACTH-immunoreactive neuronal elements in the a rcuate nucleus of rat. A: light microscopic field showing several immunopositive neurons (white stars) and varicosities (arrows) in a 50 ttm thick vibratome section. Nomarski optics. Bar scale = 5/tm. B: an immunoreactive bouton packed with labeled synaptic vesicles. Arrow points to a large, immunoreactive vesicle. Bar scale = 0.5/~m. C: an ACTH-immunopositive bouton (white star) makes a symmetrical synaptic contact (arrow) with a labeled perikarya. Arrowheads point to large, immunoreactive, granulated vesicles. Bar scale = 0.5/xm. D-F: immunoreactive boutons form symmetrical synaptic contacts (arrows) with unlabeled dendrites (D). In E, triple arrows point to a subsynaptic specialization. Bar scale = 0.5/~m.
145 study has not visualized this latter a-MSH positive neuron system, indicating that the presynaptic elements identified in the arcuate nucleus most probably originate from neighboring cell bodies and therefore represent local, intrinsic connections. Previous morphological studies have led to the observation of a variety of intrinsic connections in the arcuate nucleus 2.8-2°. The question as to whether some of the opiocortin neurons are specifically local circuit neurons which lack any long projecting axons, or whether they are projection neurons which also possess recurrent collaterals, has not been resolved. From a study of Golgi preparations, Van den Pol and Cassidy2° reported axons with both origins and terminations within the rat arcuate nucleus. They also described a different class of neuron with bifurcating nerve fibers, with one branch leaving the nucleus and the other terminating within it. Which of these patterns is followed by opiocortin neurons still has to be determined. Both non-labeled and labeled postsynaptic elements were found to receive inputs from identifiable opiocortin terminals. The chemical properties of the postsynaptic non-labeled neurons remains to be clarified, though there are numerous candidates since perikarya containing dopamine, serotonin acetylcholine, neurotensin, GABA, somatostatin, substance P and enkephalin have been localized in the arcuate nucleus (for recent review, see ref. 15). It should be noted, however, that even some non-labeled postsynaptic elements could belong to opiocortin neurons and remained unstained because of the limitations and variables inherent in the immunocytochemical methods. The observed labeled axosomatic contact may represent the morphological basis of an 'ultrashort' feedback loop from opiocortin neurons back to the same or to other neurons of the same transmitter type.
The idea that there may be an 'ultrashort' feedback loop within the hypothalamus was originally conceived in the context of vasopressin, oxytocin and releasing as well as release-inhibiting hormones I°. It was postulated that these substances could control the activity of neurons producing them either through changes of their titers in the general circulation or through direct neuronal feedback, e.g. recurrent axon collaterals. Ultrashort feedback loops of oxytocin, vasopressin I° and somatostatin 9 have already been demonstrated in pharmacological experiments. Furthermore, a number of Golgi ~9-2~ and deafferentation 2,6-s studies have provided evidence that the arcuate as well as other hypothalamic nuclei have a recurrent axon collateral system. Recurrent axon collaterals along with interneurons are the morphological substrates of local neuronal circuits, which represent a standard form of organization which is reduplicated extensively in the brain. Indeed, according to Rak i c L6, 'it is probably safe to state that there is not a single structure in the mammalian central nervous system that lacks LCN's' (local circuit neurons). It is now widely maintained ~6 that these local neuronal circuits integrate morphologically delineated aggregates of neurons (e.g. nucleus) to operate as a functional unit, i.e. to integrate the function of many neurons of the same type and to influence the activity of different types of neurons within the same nucleus. Our results suggest that opiocortin neurons in the arcuate nucleus may contribute not only to output channels to the median eminence, hypothalamic and extrahypothalamic areas ~5but also participate in a local neuronal circuit. Subsequent physiological and pharmacological experiments may help to illuminate the functional correlates of this morphological finding.
1 Guy, J., Vaudry, H. and Pelletier, G., Futher studies on the identification of neurons containing immunoreactive alpha-melanocyte-stimulating hormone (c~-MSH) in the rat brain, Brain Research, 239(1982) 265-270. 2 Halfisz, B2, Koves, K., Rethelyi, M., Bodoky, M. and Koritsanszky, S., Recent data on neuronal connections between nervous structure involved in the control of the ad-
enohypophysis. In W. E. Stumpf and L. D. Grant (Eds.), New York, Karger, 1975 pp. 9-14. 3 Joseph, S. A., Immunoreactive adrenocorticotropin in rat brain: a neuroanatomical study using antiserum generated against synthetic ACTH (1 39), Amer. J. Anat., 158 (1980) 533 548. 4 K~rteszi, M., Makara, G. B. and Stark, E., The rise of
146
5
6
7
8 9 10
12
plasma ACTH induced by ether is mediated through neural pathways entering the medial basal hypothalamus, A cta endocrinol., 93 (1980) 129-133. K~irteszi, M., Stark, E., Rappay, Gy., L~iszl6, F. A. and Makara, G. B., Corticoliberin activity of rat neurohypophysis is distinct from vasopressin, Amer. J. Physiol., 240 (1981) E685 E693. Kiss, J. Z., Palkovits, M., Zaborszky L., Tribollet, E., Szab6 D. and Makara, G. B., Quantitative histological studies on the hypothalamic paraventricular nucleus in rats. II. Number of local and certain afferent nerve terminals, Brain Research, in press. L6r~inth, Cs., Z~iborszky, L., Marton, J. and Palkovits, M., Quantitative studies on the supraoptic nucleus in the rat. I. Synaptic organization, Exp. Brain Res., 22 (1975) 509 523. Matsumoto, A. and Arai, Y., Morphological evidence for intranuclear circuits in the hypothalamic arcuate nucleus, Exp. Neurol., 59 (1978) 404-412: McCann, S. M., Control of anterior pituitary hormone release by brain peptides. Neuroendocrinologv, 31 (1980) 355 363. Motta, M., Fraschini, F. and Martini, L., 'Short' feedback mechanism in the control of anterior pituitary function. In W. F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinologv, 1969, pp. 211-255. North, R. A., Opiates, opioid peptides and single neurones, Life Sci., 24 (1979) 1527-1546.
13 O'Donohue, T. L. and Dorsa, D. M., The opiomelanotropinergic neuronal and endocrine systems, Petides, 3 (1982) 353-395. 14 Palkovits, M., Neurotransmitter distribution in the brain, Front. Hormone Res., 10 (1982) 15-32. 15 Palkovits, M., Topography of chemically identified neurons in the central nervous system: Progress in 19771979, Med. Biol., 58 (1980) 188-227. 16 Rakic, P., Local circuit neurons, Neurosci. Res. Prog. BulL, 13 (1975) 291-446. 17 Somogyi, P. and Hiroshi, T., A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytocb,emistry, Neuroscienc~, 7 (1982) in press. 18 Sternberger, L. A., lmmunocytochemistry, Prentice-Hall, Englewood Cliffs, N. J., 1974. 19 Van den Pol, A. N., The magnocellular and parvocellular paraventricular nucleus of rat: intrinsic organization, J. comp. Neurol., 206 (1982) 317 345. 20 Van den Pol, A. N. and Cassidy, J. R., The hypothalamic arcuate nucleus of rat - A quantitative Golgi analysis, J. comp. Neurol., 204 (1982) 65 98. 21 Van den Pol, A. N., The hypothalamic suprachiasmatic nucleus of rat: intrinsic anatomy, J. comp. Neurol., 191 (1980) 661-702. 22 Watson, S. J., Akil, H. and Walker, J. M., Anatomical and biochemical studies of the opioid peptides and related substances in the brain, Peptides, 1, Suppl. 1 (1980) 11-20.