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Effects of lesions in the paraventricular nucleus of the hypothalamus on vasopressin and oxytocin contents in brainstem and spinal cord of rat
326
Brain Research, 260 (1983) 326-329 Elsevier Biomedical Press
Effects of lesions in ~ e paraventr~ular nucleus of the h ~ a l a m u s on vas,.,'q~essin a ~ o x ~ i n c ~ m t s in brainstem ~ ~ 1 cord of rat R. E. LANG, J. HELL, D. GANTEN, K. HERMANN, W. RASCHER and Th. U N G E R
German Institute for High Blood Pressure Research and Department of Pharmacology, University+of Heidelberg, lm Neuenheimer Feld 366, D-6900 Heidelberg (F. R. G.) (Accepted October 18th, 1982)
Key-words: paraventricular nucleus hypothalami - lesions - vasopressin - oxytocin - brainstem - spinal cord high performance liquid chromatography
The effects of lesions of the paraventricular nucleus in rat hypothalamus (PVN) on the vasopressin (AVP) and oxytocin (OT) contents of the brainstem and spinal cord, as measured by radioimmunoassay, were studied. AVP decreased by 50% and 80% in brainstem and spinal cord of lesioned animals, whereas OT disappeared almost completely. Therefore, in contrast to OT, the PVN is not the only site of origin of AVP-containing nerve fibers projecting to the brainstem.
The neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), along with their associated neurophysins, are produced by separate hypothalamic magnocellular neurons most of which are concentrated in two distinct cell groups of the hypothalamus, the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). In addition, a group of parvocellular neurons has been described in the suprachiasmatic nucleus (SCN) which contains exclusively AVP and its associated neurophysin ~-68'~2. The AVP and OT neurons of the PVN and SON, but not of the SCN, send nerve fibers to the posterior pituitary, the site where AVP and OT are stored for release into the blood. In recent years it has become evident, from morphological studies, that axons containing the classic neurohypophysial peptides are found not only within the hypothalamo-neurohypophysial tract but also in a number of extrahypothalamic sites. Of particular interest was the observation that axons staining for AVP and OT innervate distinct preganglionic parasympathetic and sympathetic cell groups in the brainstem and spinal cord, indicating a possible role of these projections in integrating autonomic functions. The PVN has been considered as the main source of nerve fibers to this region ~,5,~°-~.The SCN 0006-8993/83/0000-0000/$03.00 ~'~1983 Elsevier Biomedical Press
has been suggested as an additional source, whereas the SON has been claimed to project exclusively to the posterior pituitary~. Most of these findings have been obtained by means of immunocytochemical techniques without establishing the hormone specificity of staining by biochemical methods. Moreover, no attempts have been made to determine the relative contribution of different hypothalamic nuclei to the hormone content in the brainstem and spinal cord. We present here the results of experiments designed to characterize, by biochemical methods, the nature of the AVP- and OT-like immunoreactivity contained in the brainstem and spinal cord of the rat by biochemical methods and to determine quantitatively, with the aid of selective lesions, the relative contribution of the PVN to these areas. Male Wistar rats (Thomae, Biberach) 260 280 g in weight, were anesthetized with halothane and the head secured in a stereotactic holder. Following exposure of the brain lesions were placed bilaterally in the PVN with a tungsten electrode of 0.2 mm tip diameter. The electrode was located 0.4 m m laterally from midline and 1.0 mm posterior to the bregma with the tip lowered to a depth of 7.9 mm into the brain. Lesions
327 were made using a radiofrequency lesion generator (Radionics Inc. Model RFG-4), 10 mA, 500 kHz, 40 s). In control animals electrodes were lowered into the brain 1 mm dorsal to the electrode position for the experimental groups but no current was passed. Twelve days after lesion or sham-lesion animals were decapitated by means of a guillotin e and the brains, including brainstem, the thoracic part of the spinal cord and the posterior pituitary gland, rapidly removed. The brainstem was excised by two transverse sections rostrally crossing between the caudal end of the colliculi on the dorsal surface and the rostral end of the decussatio pyramidum. The spinal cord was removed to the level of Th 12. Tissues were weighed and frozen. The remaining brain, including hypothalamus, was fixed in formalin and sectioned at 10 t~m in the frontal plane. Every fifth section was stained with hematoxyline- eosin and examined microscopically. Extraction of tissues and radioimmunoassay of AVP and OT were performed as previously described3.4. Recovery of AVP was 64 _+ 4.5% (mean + S.D., n = 10) and of O T 7 2 +_ 6%. Cross-reactivity of the AVP antibody with OTwas less than 0.1%, and the OT antibody showed less than 0.5% cross-reactivity with AVP. The measurements were corrected for losses during extraction and expressed as mol per mg wet weight. Before application to high performance liquid chromatography (HPLC) brain extracts were purified on octadecasilyl-silica (ODS) cartridges (Sep-Pak C 18, Waters Assoc.). Separations were performed on ODS-silica reversed phase columns (Bondapak C18,300 x 4 mm, Waters Assoc.) using a linear 30-80% methanol gradient in ammonium acetate buffer (pH 5.4) for elution. The eluted fractions were lyophilized and analyzed for their AVP and OT contents by radioimmunoassay. Radioimmunoassay of tissue extracts revealed that OT concentrations are about (~9-fold higher than those of AVP in brainstem and spinal cord (Fig. 2). On separation of brainstem extracts by HPLC, distinct single peaks of AVP and OT immunoreactivity were detected eluting
~0
L fmol I ~ct~
"/¢,iV~hanoi
~.00 r---'l AVP
mill OT 80
/
300 / /
60
/ 20C
/
/
z,O
/ / I0C
/ /
20
III 20
30
'tO
fraction
Fig. 1. Characterization of AVP- and OT-like immunoreactive material from brainstem extracts by HPLC. The immunoreactivity elutes at the position of synthetic AVP and OT standards (arrows). For technical details see text.
exactly at the positions where synthetic AVP and OT appeared (Fig. 1). The same picture was obtained on separation of extracts from spinal cord. In 12 out of 20 animals which underwent lesioning, the whole region of the PVN was found to be damaged in both lateral and rostro-caudal directions. The lesions extended from the plane of the SCN to the dorsomedial hypothalamic nucleus and involved parts of the anterior hypothalamic nucleus and the dorsomedial nucleus. The SCN and SON appeared in all cases completely intact. In those animals bearing complete bilateral lesions of PVN the content of OT in brainstem and cervico-thoracic part of spinal cord was reduced by more than 90% (brainstem: 12.8 _+ 1.7 fmol/mg in sham controls versus 0.18 _+ 0.003 fmol/mg in lesioned animals: spinal cord: 3.7 _+ 1.2 fmol/mg versus 0.08 __ 0.02 fmol/mg, means _+ S.E.M.: n = 12) (Fig. 2b). In contrast, AVP was diminished by only about 50%. in brainstem (1.9 + 0.09 fmol/mg in controls versus 0.9 fmol __ 0.08 fmol/mg in lesioned animals) and more than 80~ in spinal cord (0.44 _+ 0.05 fmol/mg versus 0.06 _+ 0.01 fmol/mg) as compared to sham-operated controls (Fig. 2a). Lesions resulted in an about 50% reduction of both AVP and OT in the pituitary gland.
328 a)
EFFECTS OF BfLATERAL PVN LESIONS ON THE VASORRESS~N C O N T E N T OF RAT BRAIN STEM AND SPINAL CORD [ f m o l / m g wet weight]
AV P
J~]
1.5
CONTROL
m
PVN LES.
* : n :
P
0.5'
brain stem
I))
OT fmol/m
spinal cord
EFFECTS OF BILATERAL PVN LESIONS ON THE OXYTOCIN CONTENT OF RAT BRAIN STEM AND SPINAL CORO[fmol/mg wet weight]
CONTROL
10
5,
Fig. 2. A V P (a) and OT (b) contents o f brainstem and spinal cord o f lesioned (dark columns and sham-lesioned rats. Means _+ S . E M . n = 12.
Our results based on t~ssue extraction and subsequent analysis by radioimmunoassay and HPLC demonstrate clearly the presence of OT as well as AVP in the brainstem and spinal cord of the rat. There is a large difference in the concentrations of the hormones in that AVP amounts to only about one-fifth to one-tenth of the concentrations of OT. This is in good agreement with the findings of Nilaver et al. who estimated, by immunocytochemical techniques, the amount of OT fibers in this region to be about 80% of the total number of neurophysin-containing fibers ~. There have been few other studies on the direct measurements of AVP and OT in regions outside the hypothalamo-hypophysial system. Our data are similar to those recently published by Dogterom et al. 2 as far as the ratio of OT to AVP in the brainstem is concerned. No attempt was made by these authors to assay AVP and OT
in the spinal cord. Moreover, the identity of brainstem AVP and OT with that of the pituitary peptides was not estimated in these experiments. In the present study such identity with the genuine peptides has been demonstrated for both peptides by HPLC, which currently represents the most powerful technique for separating small peptides. A number of immunocytochemical studies point to the PVN as the major source of AVP and OT found in brainstem and spinal cord. Nerve fibers stained with antibodies to neurophysins, AVP and OT have been traced from this site down to various nuclei of the brainstem and of the intermedialateral gray or the substantia gelatinosa of the spinal cord ~-5.~.~t. In our hands bilateral lesions of the PVN, shown to be complete by subsequent histological examination, did not abolish the entire AVP content of the brainstem and spinal cord, but caused a reduction of 50 and 80~. This was in contrast to OT, which almost completely disappeared following such lesions. It is very unlikely that the residual AVP in the brainstem and spinal cord of animals bearing bilateral PVN lesions represents blood-borne hormones which have been trapped in the brain since pilot studies revealed that perfusion of the brain with saline does not significantly affect the AVP and OT levels in the brainstem. In addition, 10-fold changes in the plasma hormone levels, induced by water deprivation, failed to significantly alter the hormone content of the brainstem (Lang et al., unpublished). Therefore. our lesion experiments suggest that the area of the PVN may not be the only site of origin of AVP-containing nerve fibers projecting to the brainstem. There are some other hypothalamic and even extrahypothalamic regions containing magnocellular or parvocellular neurons capable of synthesizing the classic neurohypophysial peptides. Thus, magnoceUular AVP and OT producing cells have been located within the hypothalamus but outside of the PVN and SON. Some are concentrated in the anterior commissural nucleus, which has been reported to have about half the number of OT-containing neurons as PVN; others are scattered through
329 the preoptic area, the lateral hypothalamic area and the zona incerta6-7. Magnocellular neurophysin-containing perikarya have also been demonstrated outside of the hypothalamus, above the anterior commissure between the fornix columns and the triangular nucleus of the septum 6.7. The projections of these neurons are still ill defined and their possible contribution to the AVP and OT found in the caudal parts of the brain remains to be elucidated. The efferent projections of parvocellular neurons within the SCN which produce exclusively AVP and its associated neurophysin have been described in detail in a recent immunocytochemical study~. The fibers of this cell group form pathways to various limbic structures. In addition, some of them, by coursing along the dorsal hypothalamus and through the central gray of the mesencephalon, reach the area of the nucleus of the solitary tract in the brainstem. A major part of the latter pathway may have remained intact following PVN destruction in our studies and could have contributed to the relatively high amount of AVP found in the brainstem of lesioned animals as compared to the OT content. While the neurohypophysial hormones stored in the posterior pituitary have been known for a long time to be involved in processes such as
1 Buijs, R. M., Intra- and extrahypothalamic vasopressin and ocytocin pathways in the rat, Cell Tiss. Res., 192 (1978) 423-435. 2 Dogterom, J., Snijdewint, F. G. M. and Buijs, R. M., The distribution of vasopressin and oxytocin in the rat brain, Neurosci. Left., 9 (1978) 341-346. 3 Lang, R. E., Rascher, W., Unger, Th. and Ganten, D., Reduced content of vasopressin in the brain of spontaneously hypertensive as compared to normotensive rats, NeuroscL Lett., 23 ( 1981) 199-202. 4 Lang, R. E., Rascher, W., Heil, J., Unger, Th., Wiedemann, G. and Ganten, D., Angiotensin stimulates oxytocin release, Life Sci., 29 (1981) 1425-1428. 5 Nilaver, G., Zimmerman, E. A., Wilkins, J., Michaels, J., Hoffman, D. and Silvbrman, A. J., Magnocellular hypothalamic projections to the lower brain stem and spinal cord of the rat, Neuroendocrinology, 30 (1980) 150-158. 6 Rhodes, C. H., Morrell, J. I. and Pfaff, D. W., Immunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and numbers of cells containing neurophysin, oxytocin, and vasopressin, J. comp. Neurol., 198 (1981 ) 45-64. 7 Sofroniew, M. V. and Weindl, A., Extrahypothalamic neurophysin-containing perikarya, fiber pathways and
salt-water homeostasis, milk ejection and uterine contractility, the physiological significance of these hormones found in the lower parts of the brain and the spinal cord is still not understood. The innervation of the nucleus tractus solitarii of the brainstem by vasopressinergic nerve terminals suggests that AVP may play a role in cardiovascular control at this level since this area receives inputs from pressor receptors of the aortic arch and the carotid sinus LS~~t. The presence of AVP and OT in the substantia gelatinosa of the spinal cord provides evidence for some functions of both hormones in pain perception. In addition, the detection of OT-staining nerve endings at sympathetic cell groups in various levels of the spinal cord suggests that OT is implicated in the control of the sympathetic innervation of some visceral organs such as kidney or adrenal gland ~ . The present study demonstrates that the PVN is the major, but not the only, site from which the AVP-containing nerve fibers in the brainstem and spinal cord originate. This site of origin of the descending fibers is of importance for future studies concerned with the role of neurohypophysial hormones in these parts of the nervous system.
8
9
10
11
12
fiber clusters in the rat brain, Endocrinology, 102 (1978) 334. Sofroniew, M. V. and Weindk A., Projections from the parvocellular vasopressin- and neurophysin-containing neurons of th~ suprachiasmatic nucleus, Amer. J. Anat., 153 (1978) 391-430. Sterba, G., Hoffmann, E., Solecki, R., Naumann, W., Hoheisel, G. and Schober, F., The neurosecretory hypothalamo-hindbrain pathway and its possible significance for the regulation of blood pressure and the milk-ejection reflex, Cell Tiss. Res., 196 (1979) 321-336. Swanson, L. W., Immunohistochemical evidence for a neurophysin-containing autonomic pathway arising in the paraventricular nucleus of the hypothalamus, Brain Research, 128 (1977) 346-353. Swanson, L. W. and Sawchenko, P. E., Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms, Neuroendocrinology, 31 (1980) 410-417. Vandesande, F., Dierickx, K. and De Mey, J., Identification of the vasopressin-neurophysin producing neurons of the rat suprachiasmatic nuclei, Cell Tiss. Res., 156 (1975b) 377-380.
Brain Research, 260 (1983) 326-329 Elsevier Biomedical Press
Effects of lesions in ~ e paraventr~ular nucleus of the h ~ a l a m u s on vas,.,'q~essin a ~ o x ~ i n c ~ m t s in brainstem ~ ~ 1 cord of rat R. E. LANG, J. HELL, D. GANTEN, K. HERMANN, W. RASCHER and Th. U N G E R
German Institute for High Blood Pressure Research and Department of Pharmacology, University+of Heidelberg, lm Neuenheimer Feld 366, D-6900 Heidelberg (F. R. G.) (Accepted October 18th, 1982)
Key-words: paraventricular nucleus hypothalami - lesions - vasopressin - oxytocin - brainstem - spinal cord high performance liquid chromatography
The effects of lesions of the paraventricular nucleus in rat hypothalamus (PVN) on the vasopressin (AVP) and oxytocin (OT) contents of the brainstem and spinal cord, as measured by radioimmunoassay, were studied. AVP decreased by 50% and 80% in brainstem and spinal cord of lesioned animals, whereas OT disappeared almost completely. Therefore, in contrast to OT, the PVN is not the only site of origin of AVP-containing nerve fibers projecting to the brainstem.
The neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), along with their associated neurophysins, are produced by separate hypothalamic magnocellular neurons most of which are concentrated in two distinct cell groups of the hypothalamus, the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). In addition, a group of parvocellular neurons has been described in the suprachiasmatic nucleus (SCN) which contains exclusively AVP and its associated neurophysin ~-68'~2. The AVP and OT neurons of the PVN and SON, but not of the SCN, send nerve fibers to the posterior pituitary, the site where AVP and OT are stored for release into the blood. In recent years it has become evident, from morphological studies, that axons containing the classic neurohypophysial peptides are found not only within the hypothalamo-neurohypophysial tract but also in a number of extrahypothalamic sites. Of particular interest was the observation that axons staining for AVP and OT innervate distinct preganglionic parasympathetic and sympathetic cell groups in the brainstem and spinal cord, indicating a possible role of these projections in integrating autonomic functions. The PVN has been considered as the main source of nerve fibers to this region ~,5,~°-~.The SCN 0006-8993/83/0000-0000/$03.00 ~'~1983 Elsevier Biomedical Press
has been suggested as an additional source, whereas the SON has been claimed to project exclusively to the posterior pituitary~. Most of these findings have been obtained by means of immunocytochemical techniques without establishing the hormone specificity of staining by biochemical methods. Moreover, no attempts have been made to determine the relative contribution of different hypothalamic nuclei to the hormone content in the brainstem and spinal cord. We present here the results of experiments designed to characterize, by biochemical methods, the nature of the AVP- and OT-like immunoreactivity contained in the brainstem and spinal cord of the rat by biochemical methods and to determine quantitatively, with the aid of selective lesions, the relative contribution of the PVN to these areas. Male Wistar rats (Thomae, Biberach) 260 280 g in weight, were anesthetized with halothane and the head secured in a stereotactic holder. Following exposure of the brain lesions were placed bilaterally in the PVN with a tungsten electrode of 0.2 mm tip diameter. The electrode was located 0.4 m m laterally from midline and 1.0 mm posterior to the bregma with the tip lowered to a depth of 7.9 mm into the brain. Lesions
327 were made using a radiofrequency lesion generator (Radionics Inc. Model RFG-4), 10 mA, 500 kHz, 40 s). In control animals electrodes were lowered into the brain 1 mm dorsal to the electrode position for the experimental groups but no current was passed. Twelve days after lesion or sham-lesion animals were decapitated by means of a guillotin e and the brains, including brainstem, the thoracic part of the spinal cord and the posterior pituitary gland, rapidly removed. The brainstem was excised by two transverse sections rostrally crossing between the caudal end of the colliculi on the dorsal surface and the rostral end of the decussatio pyramidum. The spinal cord was removed to the level of Th 12. Tissues were weighed and frozen. The remaining brain, including hypothalamus, was fixed in formalin and sectioned at 10 t~m in the frontal plane. Every fifth section was stained with hematoxyline- eosin and examined microscopically. Extraction of tissues and radioimmunoassay of AVP and OT were performed as previously described3.4. Recovery of AVP was 64 _+ 4.5% (mean + S.D., n = 10) and of O T 7 2 +_ 6%. Cross-reactivity of the AVP antibody with OTwas less than 0.1%, and the OT antibody showed less than 0.5% cross-reactivity with AVP. The measurements were corrected for losses during extraction and expressed as mol per mg wet weight. Before application to high performance liquid chromatography (HPLC) brain extracts were purified on octadecasilyl-silica (ODS) cartridges (Sep-Pak C 18, Waters Assoc.). Separations were performed on ODS-silica reversed phase columns (Bondapak C18,300 x 4 mm, Waters Assoc.) using a linear 30-80% methanol gradient in ammonium acetate buffer (pH 5.4) for elution. The eluted fractions were lyophilized and analyzed for their AVP and OT contents by radioimmunoassay. Radioimmunoassay of tissue extracts revealed that OT concentrations are about (~9-fold higher than those of AVP in brainstem and spinal cord (Fig. 2). On separation of brainstem extracts by HPLC, distinct single peaks of AVP and OT immunoreactivity were detected eluting
~0
L fmol I ~ct~
"/¢,iV~hanoi
~.00 r---'l AVP
mill OT 80
/
300 / /
60
/ 20C
/
/
z,O
/ / I0C
/ /
20
III 20
30
'tO
fraction
Fig. 1. Characterization of AVP- and OT-like immunoreactive material from brainstem extracts by HPLC. The immunoreactivity elutes at the position of synthetic AVP and OT standards (arrows). For technical details see text.
exactly at the positions where synthetic AVP and OT appeared (Fig. 1). The same picture was obtained on separation of extracts from spinal cord. In 12 out of 20 animals which underwent lesioning, the whole region of the PVN was found to be damaged in both lateral and rostro-caudal directions. The lesions extended from the plane of the SCN to the dorsomedial hypothalamic nucleus and involved parts of the anterior hypothalamic nucleus and the dorsomedial nucleus. The SCN and SON appeared in all cases completely intact. In those animals bearing complete bilateral lesions of PVN the content of OT in brainstem and cervico-thoracic part of spinal cord was reduced by more than 90% (brainstem: 12.8 _+ 1.7 fmol/mg in sham controls versus 0.18 _+ 0.003 fmol/mg in lesioned animals: spinal cord: 3.7 _+ 1.2 fmol/mg versus 0.08 __ 0.02 fmol/mg, means _+ S.E.M.: n = 12) (Fig. 2b). In contrast, AVP was diminished by only about 50%. in brainstem (1.9 + 0.09 fmol/mg in controls versus 0.9 fmol __ 0.08 fmol/mg in lesioned animals) and more than 80~ in spinal cord (0.44 _+ 0.05 fmol/mg versus 0.06 _+ 0.01 fmol/mg) as compared to sham-operated controls (Fig. 2a). Lesions resulted in an about 50% reduction of both AVP and OT in the pituitary gland.
328 a)
EFFECTS OF BfLATERAL PVN LESIONS ON THE VASORRESS~N C O N T E N T OF RAT BRAIN STEM AND SPINAL CORD [ f m o l / m g wet weight]
AV P
J~]
1.5
CONTROL
m
PVN LES.
* : n :
P
0.5'
brain stem
I))
OT fmol/m
spinal cord
EFFECTS OF BILATERAL PVN LESIONS ON THE OXYTOCIN CONTENT OF RAT BRAIN STEM AND SPINAL CORO[fmol/mg wet weight]
CONTROL
10
5,
Fig. 2. A V P (a) and OT (b) contents o f brainstem and spinal cord o f lesioned (dark columns and sham-lesioned rats. Means _+ S . E M . n = 12.
Our results based on t~ssue extraction and subsequent analysis by radioimmunoassay and HPLC demonstrate clearly the presence of OT as well as AVP in the brainstem and spinal cord of the rat. There is a large difference in the concentrations of the hormones in that AVP amounts to only about one-fifth to one-tenth of the concentrations of OT. This is in good agreement with the findings of Nilaver et al. who estimated, by immunocytochemical techniques, the amount of OT fibers in this region to be about 80% of the total number of neurophysin-containing fibers ~. There have been few other studies on the direct measurements of AVP and OT in regions outside the hypothalamo-hypophysial system. Our data are similar to those recently published by Dogterom et al. 2 as far as the ratio of OT to AVP in the brainstem is concerned. No attempt was made by these authors to assay AVP and OT
in the spinal cord. Moreover, the identity of brainstem AVP and OT with that of the pituitary peptides was not estimated in these experiments. In the present study such identity with the genuine peptides has been demonstrated for both peptides by HPLC, which currently represents the most powerful technique for separating small peptides. A number of immunocytochemical studies point to the PVN as the major source of AVP and OT found in brainstem and spinal cord. Nerve fibers stained with antibodies to neurophysins, AVP and OT have been traced from this site down to various nuclei of the brainstem and of the intermedialateral gray or the substantia gelatinosa of the spinal cord ~-5.~.~t. In our hands bilateral lesions of the PVN, shown to be complete by subsequent histological examination, did not abolish the entire AVP content of the brainstem and spinal cord, but caused a reduction of 50 and 80~. This was in contrast to OT, which almost completely disappeared following such lesions. It is very unlikely that the residual AVP in the brainstem and spinal cord of animals bearing bilateral PVN lesions represents blood-borne hormones which have been trapped in the brain since pilot studies revealed that perfusion of the brain with saline does not significantly affect the AVP and OT levels in the brainstem. In addition, 10-fold changes in the plasma hormone levels, induced by water deprivation, failed to significantly alter the hormone content of the brainstem (Lang et al., unpublished). Therefore. our lesion experiments suggest that the area of the PVN may not be the only site of origin of AVP-containing nerve fibers projecting to the brainstem. There are some other hypothalamic and even extrahypothalamic regions containing magnocellular or parvocellular neurons capable of synthesizing the classic neurohypophysial peptides. Thus, magnoceUular AVP and OT producing cells have been located within the hypothalamus but outside of the PVN and SON. Some are concentrated in the anterior commissural nucleus, which has been reported to have about half the number of OT-containing neurons as PVN; others are scattered through
329 the preoptic area, the lateral hypothalamic area and the zona incerta6-7. Magnocellular neurophysin-containing perikarya have also been demonstrated outside of the hypothalamus, above the anterior commissure between the fornix columns and the triangular nucleus of the septum 6.7. The projections of these neurons are still ill defined and their possible contribution to the AVP and OT found in the caudal parts of the brain remains to be elucidated. The efferent projections of parvocellular neurons within the SCN which produce exclusively AVP and its associated neurophysin have been described in detail in a recent immunocytochemical study~. The fibers of this cell group form pathways to various limbic structures. In addition, some of them, by coursing along the dorsal hypothalamus and through the central gray of the mesencephalon, reach the area of the nucleus of the solitary tract in the brainstem. A major part of the latter pathway may have remained intact following PVN destruction in our studies and could have contributed to the relatively high amount of AVP found in the brainstem of lesioned animals as compared to the OT content. While the neurohypophysial hormones stored in the posterior pituitary have been known for a long time to be involved in processes such as
1 Buijs, R. M., Intra- and extrahypothalamic vasopressin and ocytocin pathways in the rat, Cell Tiss. Res., 192 (1978) 423-435. 2 Dogterom, J., Snijdewint, F. G. M. and Buijs, R. M., The distribution of vasopressin and oxytocin in the rat brain, Neurosci. Left., 9 (1978) 341-346. 3 Lang, R. E., Rascher, W., Unger, Th. and Ganten, D., Reduced content of vasopressin in the brain of spontaneously hypertensive as compared to normotensive rats, NeuroscL Lett., 23 ( 1981) 199-202. 4 Lang, R. E., Rascher, W., Heil, J., Unger, Th., Wiedemann, G. and Ganten, D., Angiotensin stimulates oxytocin release, Life Sci., 29 (1981) 1425-1428. 5 Nilaver, G., Zimmerman, E. A., Wilkins, J., Michaels, J., Hoffman, D. and Silvbrman, A. J., Magnocellular hypothalamic projections to the lower brain stem and spinal cord of the rat, Neuroendocrinology, 30 (1980) 150-158. 6 Rhodes, C. H., Morrell, J. I. and Pfaff, D. W., Immunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and numbers of cells containing neurophysin, oxytocin, and vasopressin, J. comp. Neurol., 198 (1981 ) 45-64. 7 Sofroniew, M. V. and Weindl, A., Extrahypothalamic neurophysin-containing perikarya, fiber pathways and
salt-water homeostasis, milk ejection and uterine contractility, the physiological significance of these hormones found in the lower parts of the brain and the spinal cord is still not understood. The innervation of the nucleus tractus solitarii of the brainstem by vasopressinergic nerve terminals suggests that AVP may play a role in cardiovascular control at this level since this area receives inputs from pressor receptors of the aortic arch and the carotid sinus LS~~t. The presence of AVP and OT in the substantia gelatinosa of the spinal cord provides evidence for some functions of both hormones in pain perception. In addition, the detection of OT-staining nerve endings at sympathetic cell groups in various levels of the spinal cord suggests that OT is implicated in the control of the sympathetic innervation of some visceral organs such as kidney or adrenal gland ~ . The present study demonstrates that the PVN is the major, but not the only, site from which the AVP-containing nerve fibers in the brainstem and spinal cord originate. This site of origin of the descending fibers is of importance for future studies concerned with the role of neurohypophysial hormones in these parts of the nervous system.
8
9
10
11
12
fiber clusters in the rat brain, Endocrinology, 102 (1978) 334. Sofroniew, M. V. and Weindk A., Projections from the parvocellular vasopressin- and neurophysin-containing neurons of th~ suprachiasmatic nucleus, Amer. J. Anat., 153 (1978) 391-430. Sterba, G., Hoffmann, E., Solecki, R., Naumann, W., Hoheisel, G. and Schober, F., The neurosecretory hypothalamo-hindbrain pathway and its possible significance for the regulation of blood pressure and the milk-ejection reflex, Cell Tiss. Res., 196 (1979) 321-336. Swanson, L. W., Immunohistochemical evidence for a neurophysin-containing autonomic pathway arising in the paraventricular nucleus of the hypothalamus, Brain Research, 128 (1977) 346-353. Swanson, L. W. and Sawchenko, P. E., Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms, Neuroendocrinology, 31 (1980) 410-417. Vandesande, F., Dierickx, K. and De Mey, J., Identification of the vasopressin-neurophysin producing neurons of the rat suprachiasmatic nuclei, Cell Tiss. Res., 156 (1975b) 377-380.