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Terminal degeneration in supraoptic nucleus following subfornical organ lesions: ultrastructural observations in the rat
Brain Research, 275 (1983) 365-368
365
Elsevier
Terminal degeneration in supraoptic nucleus following subfornical organ lesions: ultrastructural observations in the rat L. P. RENAUD, J. ROGERS and S. SGRO Neurosciences Unit, Montreal General Hospital and McGill University, 1650 Cedar Avenue, Montreal, Que. H3G 1A4 (Canada)
(Accepted May 17th, 1983) Key words: subfornical organ - - supraoptic nucleus connections - - terminal degeneration - - neurosecretory neurons
A projection from the subfornical organ (SFO) to the supraoptic nucleus, recently identified in light microscopicstudies, was examined at the ultrastruetural level following lesions in SFO. After 18-36 h, axon terminal degeneration was identified in axosomatic contacts with supraoptic neurosecretory neurons, and in axodendritic contacts within and around the supraoptic nucleus. These observations confirm a monosynaptic pathway from SFO to supraoptic neurosecretory neurons that may participate in the release of vasopressin followingactivation of angiotensin II receptors in SFO. The supraoptic nucleus performs a vital role in the maintenance of body fluid balance, achieved through controlled secretion of antidiuretic hormone ( A D H ) from posterior pituitary axon terminals of its vasopressinergic neurons. Since neurohypophyseal hormone release is governed by the state of excitability and impulse frequency in neurosecretory neurons 3,4,10, it is important to identify and characterize the actions of both general factors, such as plasma osmotic pressure, and neural influences on supraoptic neuronal excitability. There is also a need for further study of structural and functional characteristics of specific neural connections to supraoptic neurons that relay information from other regions known to participate in body fluid balance. Recently, attention has been focused on a circumventricular structure, the subfornical organ (SFO), shown to undergo structural and biochemical alterations during dehydration and identified as a receptor site for intravenous or intracerebroventricular angiotensin II in the induction of drinking behaviourg,13A4,16, a centrally mediated pressor response 7, and release of pituitary vasopressinS. Relevant to the last of these items are light microscopy studies that demonstrate a projection from SFO to the supraoptic nucleus6,8,15, and electrophysiological observations that SFO stimulation influences the excitability of supraoptic neurosecretory neurons12. In order to confirm that these latter events may be mediated through a monosynaptic 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
pathway, we utilized degeneration techniques and electron microscopy to identify ultrastructural features of SFO axon terminals in the supraoptic nucleus. In male Sprague-Dawley rats under pentobarbital anesthesia, small electrolytic lesions were placed in the SFO using anodal currents of 0.5 m A delivered for 5-7 s through a glass-insulated platinum electrode with a tip exposure of 50/~m. After survival periods ranging from 18 to 96 h animals were deeply anesthetized with pentobarbital and perfused transcardially with 1% glutaraldehyde-l% paraformaldehyde in 0.1 M phosphate buffer. SFO lesion placements were verified in 100/am brain sections cut on a vibratome. Blocks of tissue from 5 rostrocaudal levels through each supraoptic nucleus were postfixed in osmium tetroxide and embedded in Epon. Ultrathin sections of supraoptic nucleus and the tissue immediately dorsal to the nucleus were cut with a Reichert ultra-microtome, stained with uranyl acetate and lead citrate and examined for evidence of degenerating terminals. Three non-lesioned animals served as controls. Suitably placed SFO lesions that included minimal encroachment on the adjacent hippocampal commissure were obtained from 8 of 54 lesioned animals. In each animal, terminal degeneration of the electron dense type was present throughout the rostrocaudal extent of the supraoptic nucleus proper, and in the area immediately dorsal to the nucleus (Figs. 1 and
366 2). The earliest detectible degeneration was seen 18 h after the lesion, at which time certain terminals appeared swollen and contained centrally clumped synaptic vesicles. After 24 h, degenerating terminals were readily recognized by their electron opacity, shrunken profiles, granulated dense matrix containing swollen and crenated mitochondria and synaptic vesicles of varying size. A post-lesion survival period of 24-36 h appeared to be optimal for visualization of terminals still attached to their postsynaptic elements. After longer survival periods, degenerating terminals became progressively ensheathed by glial processes that separated them from any discernible postsynaptic structure (Fig. 2B). Although it is difficult to generalize on the basis of degenerating material, it would appear that the contents of axon terminals of SFO origin include both small spherical synaptic vesicles and larger dense
core vesicles (Figs, 1C and 2A) and form symmetrical synaptic contacts since there is little or no sub-synaptic specialization in the post-synaptic membrane. As for their location within the supraoptic nucleus proper, one-third of degenerating terminals were observed in contact with the somata of cells that contained neurosecretory granules (Fig. 1B), thereby confirming that at least part of the projection from SFO to the supraoptic nucleus is monosynaptic to neurosecretory cells. The remaining degenerating terminals were observed to contact one, and occasionally two, small dendritic profiles (Fig. 2A). However, the number of synaptic contacts within the nucleus was sparse. A survey of 254 grid squares, each containing an average of 150 synapses, and comprising an area of 1.799 mme, yielded a total of 42 degenerating synapses, i.e. approximately 0.1% of the synapses in the sample. A sparse number of degenerat-
Fig. 1. A: schematic illustration of a portion of the rat brain in coronal section to illustrate the approximate dimensions of the largest lesion at the site of the subfornical organ that resulted in terminal degeneration in the supraoptic nucleus. B: low power (x 12,000) view illustrates neuropile with the partial profiles of two supraoptic somata (S) containing neurosecretory granules (arrowheads) in their cytoplasm. There are two synaptic terminals (arrows) on the upper soma; one of these displays electron dense degeneration. C: in a higher power (× 45,000) view, the degenerating terminal shown in B is seen to contain both small clear and larger dense core vesicles. and crenated mitochondria. Note the apparent absence of any postsynaptic membrane specialization.
367
Fig. 2. A: in the upper part of the figure, a degenerating terminal is in apparent contact with two dendrites. For comparison, a normal terminal is seen in the lower part of the figure. B: the darkened profile of an axon terminal within the supraoptic nucleus, in a later stage of degeneration, surrounded by layers of glial membrane. Magnification x 17,000. ing terminals was also identified in the neuropil immediately dorsal to the supraoptic nucleus; some were in contact with small dendrites, but most were ensheathed in glial processes. The present observations complement recent light microscopic studies6,8,9,15 and to our knowledge provide the first ultrastructural description of SFO afferents to supraoptic nucleus neurosecretory neurons. Both axosomatic and axodendritic degenerating terminals have been previously observed within the supraoptic nucleus following AV3V lesions 1 which would almost certainly have disrupted fibers in passage from the SFO. The finding of both axosomatic and axodendritic connections from SFO is of interest since it may suggest that there is more than one functional pathway from SFO to supraoptic neurons. Such an arrangement might explain apparent differences in synaptic sign, i.e. excitation vs inhibition observed from supraoptic neurons following SFO stimulation~:. Recent anterograde autoradiographic tracer studies also suggest that SFO afferents to the
supraoptic nucleus are distributed to areas containing both oxytocinergic and vasopressinergic neurons, in sharp contrast to the more restricted topographical distribution of other afferent pathways, notably the A1 noradrenergic projection that appears to be localized to areas of the supraoptic nucleus that contain a predominance of vasopressinergic cell bodieslL While preliminary electrophysiological data suggest that SFO fibers innervate both oxytocinergic and vasopressinergic supraoptic neurons 12, a definite answer will require ultrastructural visualization of afferent terminals in monosynaptic contact with postsynaptic profiles that demonstrate immunoreactivity for vasopressin and/or oxytocin. Complementary data that the SFO influences not only vasopressin release5 but also oxytocin release would support any claim in this direction. We are grateful to the Medical Research Council of Canada for financial support, and to Gwen Peard for typing the manuscript.
368 1 Carithers, J., Bealer, S. L., Brody, M. J. and Johnson, A. K., Fine structural evidence of degeneration in supraoptic nucleus and subfornical organ of rats with lesions in the anteroventral third ventricle, Brain Research, 201 (1980) 1-12. 2 Dellmann, H.-D. and Simpson, J. B., The subfornical organ, Int. Rev. Cytol., 58 (1979) 333-421. 3 Douglas, W. W., Mechanism of release of neurohypophysial hormones: stimulus-secretion coupling. In R. O. Greep and E. B. Astwood (Eds.), Handbook of Physiology, Vol. IV, American Physiological Society, Washington, 1974, pp. 191-224. 4 Dutton, A. and Dyball, R. E. J., Phasic firing enhances vasopressin release from the rat neurohypophysis, J. Physiol. (Lond.), 290 (1979) 433-440. 5 Knepel, W., Nutto, D. and Meyer, D. K., Effect of transection of subfornical organ efferent projections on vasopressin release induced by angiotensin or isoprenaline in the rat, Brain Research, 248 (1982) 180-184. 6 Lind, R. W., Van Hoesen, G. W. and Johnson, A. K., An HRP study of the connections of the subfornical organ of the rat, J. comp. Neurol., 210 (1982) 265-277. 7 Mangiapane, M. L. and Simpson, J. B., Subfornical organ: forebrain site of pressor and dipsogenic action of angiotensin If, Amer. J. Physiol., 239 (1980) P382-P389. 8 Miselis, R., The efferent projections of the subfornical organ of the rat; a cir~umventricular organ within a neural network subserving water balance, Brain Research, 230 (1981) 1-23.
9 Miselis, R., The subfornical organ's neural connections and their role in water balance, Peptides, 3 (1982) 501-502. 10 Poulain, D. A. and Wakerley, J. B., Electrophysiology of hypothalamic magnocellular neurones secreting oxytocin and vasopressin, Neuroscience, 7 (1982) 773-808. 11 Sawchenko, P. E. and Swanson, L. W., The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev., 4 (1982) 275-325. 12 Sgro, S., Siatitsas, I. S. and Renaud, L. P., Connections of rat supraoptic nucleus (SON) neurosecretory neurons with the subfornical organ (SFO): an electrophysiological study, Soc. Neurosci. Abstr., 8 (1982) 423. 13 Simpson, J. B., Epstein, A. N. and Camardo, J. S., Jr., Localization of receptors for the dipsogenic action of angiotensin II in the subfornical organ of the rat, J. comp. physiol. Psychol., 92 (1978) 581-608. 14 Simpson, J. B. and Routtenberg, A., Subfornical organ: site of drinking elicitation by angiotensin If, Science, 181 (1973) 1172-1174. 15 Swanson, L. W. and Sawchenko, P. E., Hypothalamic integration: organization of the paraventricular and supraoptic nuclei, Ann. Rev. Neurosci., 6 (1983) 269-324. 16 Thrasher, T. N., Simpson, J. B. and Ramsay, D. J., Lesions of the subfornical organ block angiotensin-induced drinking in the dog, Neuroendocrinology, 35 (1982) 68-72.
365
Elsevier
Terminal degeneration in supraoptic nucleus following subfornical organ lesions: ultrastructural observations in the rat L. P. RENAUD, J. ROGERS and S. SGRO Neurosciences Unit, Montreal General Hospital and McGill University, 1650 Cedar Avenue, Montreal, Que. H3G 1A4 (Canada)
(Accepted May 17th, 1983) Key words: subfornical organ - - supraoptic nucleus connections - - terminal degeneration - - neurosecretory neurons
A projection from the subfornical organ (SFO) to the supraoptic nucleus, recently identified in light microscopicstudies, was examined at the ultrastruetural level following lesions in SFO. After 18-36 h, axon terminal degeneration was identified in axosomatic contacts with supraoptic neurosecretory neurons, and in axodendritic contacts within and around the supraoptic nucleus. These observations confirm a monosynaptic pathway from SFO to supraoptic neurosecretory neurons that may participate in the release of vasopressin followingactivation of angiotensin II receptors in SFO. The supraoptic nucleus performs a vital role in the maintenance of body fluid balance, achieved through controlled secretion of antidiuretic hormone ( A D H ) from posterior pituitary axon terminals of its vasopressinergic neurons. Since neurohypophyseal hormone release is governed by the state of excitability and impulse frequency in neurosecretory neurons 3,4,10, it is important to identify and characterize the actions of both general factors, such as plasma osmotic pressure, and neural influences on supraoptic neuronal excitability. There is also a need for further study of structural and functional characteristics of specific neural connections to supraoptic neurons that relay information from other regions known to participate in body fluid balance. Recently, attention has been focused on a circumventricular structure, the subfornical organ (SFO), shown to undergo structural and biochemical alterations during dehydration and identified as a receptor site for intravenous or intracerebroventricular angiotensin II in the induction of drinking behaviourg,13A4,16, a centrally mediated pressor response 7, and release of pituitary vasopressinS. Relevant to the last of these items are light microscopy studies that demonstrate a projection from SFO to the supraoptic nucleus6,8,15, and electrophysiological observations that SFO stimulation influences the excitability of supraoptic neurosecretory neurons12. In order to confirm that these latter events may be mediated through a monosynaptic 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
pathway, we utilized degeneration techniques and electron microscopy to identify ultrastructural features of SFO axon terminals in the supraoptic nucleus. In male Sprague-Dawley rats under pentobarbital anesthesia, small electrolytic lesions were placed in the SFO using anodal currents of 0.5 m A delivered for 5-7 s through a glass-insulated platinum electrode with a tip exposure of 50/~m. After survival periods ranging from 18 to 96 h animals were deeply anesthetized with pentobarbital and perfused transcardially with 1% glutaraldehyde-l% paraformaldehyde in 0.1 M phosphate buffer. SFO lesion placements were verified in 100/am brain sections cut on a vibratome. Blocks of tissue from 5 rostrocaudal levels through each supraoptic nucleus were postfixed in osmium tetroxide and embedded in Epon. Ultrathin sections of supraoptic nucleus and the tissue immediately dorsal to the nucleus were cut with a Reichert ultra-microtome, stained with uranyl acetate and lead citrate and examined for evidence of degenerating terminals. Three non-lesioned animals served as controls. Suitably placed SFO lesions that included minimal encroachment on the adjacent hippocampal commissure were obtained from 8 of 54 lesioned animals. In each animal, terminal degeneration of the electron dense type was present throughout the rostrocaudal extent of the supraoptic nucleus proper, and in the area immediately dorsal to the nucleus (Figs. 1 and
366 2). The earliest detectible degeneration was seen 18 h after the lesion, at which time certain terminals appeared swollen and contained centrally clumped synaptic vesicles. After 24 h, degenerating terminals were readily recognized by their electron opacity, shrunken profiles, granulated dense matrix containing swollen and crenated mitochondria and synaptic vesicles of varying size. A post-lesion survival period of 24-36 h appeared to be optimal for visualization of terminals still attached to their postsynaptic elements. After longer survival periods, degenerating terminals became progressively ensheathed by glial processes that separated them from any discernible postsynaptic structure (Fig. 2B). Although it is difficult to generalize on the basis of degenerating material, it would appear that the contents of axon terminals of SFO origin include both small spherical synaptic vesicles and larger dense
core vesicles (Figs, 1C and 2A) and form symmetrical synaptic contacts since there is little or no sub-synaptic specialization in the post-synaptic membrane. As for their location within the supraoptic nucleus proper, one-third of degenerating terminals were observed in contact with the somata of cells that contained neurosecretory granules (Fig. 1B), thereby confirming that at least part of the projection from SFO to the supraoptic nucleus is monosynaptic to neurosecretory cells. The remaining degenerating terminals were observed to contact one, and occasionally two, small dendritic profiles (Fig. 2A). However, the number of synaptic contacts within the nucleus was sparse. A survey of 254 grid squares, each containing an average of 150 synapses, and comprising an area of 1.799 mme, yielded a total of 42 degenerating synapses, i.e. approximately 0.1% of the synapses in the sample. A sparse number of degenerat-
Fig. 1. A: schematic illustration of a portion of the rat brain in coronal section to illustrate the approximate dimensions of the largest lesion at the site of the subfornical organ that resulted in terminal degeneration in the supraoptic nucleus. B: low power (x 12,000) view illustrates neuropile with the partial profiles of two supraoptic somata (S) containing neurosecretory granules (arrowheads) in their cytoplasm. There are two synaptic terminals (arrows) on the upper soma; one of these displays electron dense degeneration. C: in a higher power (× 45,000) view, the degenerating terminal shown in B is seen to contain both small clear and larger dense core vesicles. and crenated mitochondria. Note the apparent absence of any postsynaptic membrane specialization.
367
Fig. 2. A: in the upper part of the figure, a degenerating terminal is in apparent contact with two dendrites. For comparison, a normal terminal is seen in the lower part of the figure. B: the darkened profile of an axon terminal within the supraoptic nucleus, in a later stage of degeneration, surrounded by layers of glial membrane. Magnification x 17,000. ing terminals was also identified in the neuropil immediately dorsal to the supraoptic nucleus; some were in contact with small dendrites, but most were ensheathed in glial processes. The present observations complement recent light microscopic studies6,8,9,15 and to our knowledge provide the first ultrastructural description of SFO afferents to supraoptic nucleus neurosecretory neurons. Both axosomatic and axodendritic degenerating terminals have been previously observed within the supraoptic nucleus following AV3V lesions 1 which would almost certainly have disrupted fibers in passage from the SFO. The finding of both axosomatic and axodendritic connections from SFO is of interest since it may suggest that there is more than one functional pathway from SFO to supraoptic neurons. Such an arrangement might explain apparent differences in synaptic sign, i.e. excitation vs inhibition observed from supraoptic neurons following SFO stimulation~:. Recent anterograde autoradiographic tracer studies also suggest that SFO afferents to the
supraoptic nucleus are distributed to areas containing both oxytocinergic and vasopressinergic neurons, in sharp contrast to the more restricted topographical distribution of other afferent pathways, notably the A1 noradrenergic projection that appears to be localized to areas of the supraoptic nucleus that contain a predominance of vasopressinergic cell bodieslL While preliminary electrophysiological data suggest that SFO fibers innervate both oxytocinergic and vasopressinergic supraoptic neurons 12, a definite answer will require ultrastructural visualization of afferent terminals in monosynaptic contact with postsynaptic profiles that demonstrate immunoreactivity for vasopressin and/or oxytocin. Complementary data that the SFO influences not only vasopressin release5 but also oxytocin release would support any claim in this direction. We are grateful to the Medical Research Council of Canada for financial support, and to Gwen Peard for typing the manuscript.
368 1 Carithers, J., Bealer, S. L., Brody, M. J. and Johnson, A. K., Fine structural evidence of degeneration in supraoptic nucleus and subfornical organ of rats with lesions in the anteroventral third ventricle, Brain Research, 201 (1980) 1-12. 2 Dellmann, H.-D. and Simpson, J. B., The subfornical organ, Int. Rev. Cytol., 58 (1979) 333-421. 3 Douglas, W. W., Mechanism of release of neurohypophysial hormones: stimulus-secretion coupling. In R. O. Greep and E. B. Astwood (Eds.), Handbook of Physiology, Vol. IV, American Physiological Society, Washington, 1974, pp. 191-224. 4 Dutton, A. and Dyball, R. E. J., Phasic firing enhances vasopressin release from the rat neurohypophysis, J. Physiol. (Lond.), 290 (1979) 433-440. 5 Knepel, W., Nutto, D. and Meyer, D. K., Effect of transection of subfornical organ efferent projections on vasopressin release induced by angiotensin or isoprenaline in the rat, Brain Research, 248 (1982) 180-184. 6 Lind, R. W., Van Hoesen, G. W. and Johnson, A. K., An HRP study of the connections of the subfornical organ of the rat, J. comp. Neurol., 210 (1982) 265-277. 7 Mangiapane, M. L. and Simpson, J. B., Subfornical organ: forebrain site of pressor and dipsogenic action of angiotensin If, Amer. J. Physiol., 239 (1980) P382-P389. 8 Miselis, R., The efferent projections of the subfornical organ of the rat; a cir~umventricular organ within a neural network subserving water balance, Brain Research, 230 (1981) 1-23.
9 Miselis, R., The subfornical organ's neural connections and their role in water balance, Peptides, 3 (1982) 501-502. 10 Poulain, D. A. and Wakerley, J. B., Electrophysiology of hypothalamic magnocellular neurones secreting oxytocin and vasopressin, Neuroscience, 7 (1982) 773-808. 11 Sawchenko, P. E. and Swanson, L. W., The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev., 4 (1982) 275-325. 12 Sgro, S., Siatitsas, I. S. and Renaud, L. P., Connections of rat supraoptic nucleus (SON) neurosecretory neurons with the subfornical organ (SFO): an electrophysiological study, Soc. Neurosci. Abstr., 8 (1982) 423. 13 Simpson, J. B., Epstein, A. N. and Camardo, J. S., Jr., Localization of receptors for the dipsogenic action of angiotensin II in the subfornical organ of the rat, J. comp. physiol. Psychol., 92 (1978) 581-608. 14 Simpson, J. B. and Routtenberg, A., Subfornical organ: site of drinking elicitation by angiotensin If, Science, 181 (1973) 1172-1174. 15 Swanson, L. W. and Sawchenko, P. E., Hypothalamic integration: organization of the paraventricular and supraoptic nuclei, Ann. Rev. Neurosci., 6 (1983) 269-324. 16 Thrasher, T. N., Simpson, J. B. and Ramsay, D. J., Lesions of the subfornical organ block angiotensin-induced drinking in the dog, Neuroendocrinology, 35 (1982) 68-72.