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Autoradiographic localization of binding sites for arginine vasopressin and atrial natriuretic peptide on astrocytes and neurons of cultured rat central nervous system
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AUTORADIOGRAPHIC LOCALIZATION OF BINDING SITES FOR ARGININE VASOPRESSIN AND ATRIAL NATRIURETIC PEPTIDE ON ASTROCYTES AND NEURONS OF CULTURED RAT CENTRAL NERVOUS SYSTEM E. H%LI Department
of Physiology,
University
and
L. H~SLI*
of Basel, Vesalgasse
I, CH-4051
Basel. Switzerland
Abstract-The cellular localization of binding sites for [“‘Ilarginine vasopressin and [“‘I]atrial natriuretic pcptide was studied in explant cultures of rat spinal cord. brain stem and cerebellum by means of autoradiography. In brain stem cultures, especially in the nucleus of the solitary tract, a great number of neurons revealed binding sites for both peptides. In spinal cord cultures, many neurons of various sizes were labelled by [‘2il]arginine vasopressin. whereas only a small number of cells showed binding sites for [“‘I]atrial natriurctic peptide. Neurons in cerebellar cultures revealed little or no binding for the peptides. In addition to neurons, binding sites for [‘2iI]arginine vasopressin and [“51]atrial natriuretic peptide were also observed on glial cells. Simultaneous staining of the cultures with glial fibrillary acidic protein has shown that the labelled cells were glial fibrillary acidic protein-positive and could therefore be identified as astrocytes. Labelling of the cells by [“‘llarginine vasopressin and [“‘I]atrial natriuretic peptide was more intense in spinal cord and brain stem cultures than in cultures of cerebellum, providing evidence for a heterogeneity of astrocytes in different regions of the central nervous system. Binding of both [“‘Ilarginine vasopressm and [“‘I]atrial natriuretic peptide to neurons and astrocytes could be competed by the unlabelled peptides, suggesting specific binding of the radioligands. Our autoradiographic studies provide good evidence that in addition to neurons, astrocytes also express receptors for arginine vasopressin and atrial natriuretic peptide.
EXPERIMENTAL
The nonapeptide argininc vasopressin and the 3% amino acid pcptide atrial natriuretic peptidc (ANP) arc considered to act as neurotransmittersjneuromodulators in the mammalian CNS (cf. Refs 32 and 42). High affinity binding sites have been found for both kasoactive peptides on neurons in many regions of the CNS,h,“‘,‘7 3’)
PROCEDURES
Explant cultures were prepared from cerebellum of ncwborn rats and from brain stem (nucleus of the solitary tract. medulla oblongata) and from spinal cord (with or without attached dorsal root ganglia) of fetal rats ( I7 IX days in ~trwo OFA; Biological Research Laboratory, Fullinsdorf. SwitTcrland). Two explants were placed on collagen-coated AC‘LAR coverslips and grown in Roller tubes for I4 40 days (for details see Ref. 17). The cultures were rinsed m jcveral changes of Hank’s solution (37 C) before they were incubated in Hank’s solution containing [“‘I]arginine vasopressin or [“‘IIANP (IO-* M) for 45 min at 20 C. The incubation medium was supplemented by 0.X% bovine berum albumin and the peptidase inhibitors bacitracin (0.02’%) and trasylol (100 KIUjml). Specificity of binding was estimated by competing the radioligands with unlabelled arginine vasopressln or ANP at high concentrations (IO ’ to IO ‘M). After the incubation with [“il]arginine vasopressin or [“51]ANP, the cultures were carefully washed in 0.1% phosphate buffer (pH 7.3: IO times for I min) and lixed m glutaraldehyde vapour (50 C). After fixation. they were rinsed in ice-cold phosphate buffer and distilled water and then air-dried. The cultures were covered with Ilford L4 emulsion by means of the loop technique, exposed at 4 C for four weeks and developed with Kodak Dl9 developer (for details see Ref. 17).
Previous autoradiographic and electrophysiological studies from our laboratory indicate that not only neurons but also glial cells possess receptors for vasoactive peptides such as angiotensin II (Ang II), vasoactive intestinal peptide (VIP) and endothelin.lb,l”.‘:.?~ Furthermore, biochemical studies have shown that activation of glial peptide receptors influences second messenger systems such as CAMP, cGMP and inositoltriphosphate. suggesting the existence of receptors for various peptides on astrocytes (cf. Refs 18, 26 and 28). The aim of the present investigations was to study the cellular localization of binding sites for [“‘Ilarginine vasopressin and [“‘I]ANP in explant cultures from rat spinal cord, brain stem and cerebellum by means of autoradiography.
The radioligands [“‘Ilarginine vasopressin and [“‘I]ANP (specific activity 2200 Ci,‘mmol) were purchased from New England Nuclear Co. (NEN); bovine serum albumln and bacitracin were from Serva. trasylol from Bayer and the unlabelled peptides AVP and ANP from Bachcm.
*To Mhom correspondence should be addressed. ilhhreriations: Ang II. angiotensin II; ANP, atria1 natriurctic peptide; GFAP, glial fibrillary acidic protein; VIP, ~asoactive intestinal peptide. 159
HESULTS
In explant cultures of rat CNS, neurons and astrocytes were recognized by their morphological appearance or by their iocation in the cultures. Neurons usually remain in or at the edge of the expfant, whereas glial eeLIs migrate into the outgrowth zone where they form a dense network.“’ The various cell types have also been identified by specific staining properties: silver staining (after Bodian) for neurons (Fig. I A) or gtial fibrillary acidic protein (GFAP) for astrocytes {Figs 3A,C and 4D). Most of the cells in the outgrowth zone were GFAP-positive, and are thus considered to be astrocytes.
After incubation of brain stem cultures with [‘2SI]arginine vasopressin (10 * M), many large and medium-sized neurons were labelled. in cultures which were prepared from the area of the nucleus of the solitary tract, groups of neurons revealing binding sites for the peptide could be observed. Figure IA illustrates silver-staining of a group of neurons in the nucleus of the solitary tract which show binding sites for fn51]arginine vasopressin (Fig. 13). Labetled neurons of various shapes and sizes were also detected in cultures of the medulla oblongata (Fig. 1C). The silver grains were localized over the soma and dendrites of the neurons. Nerve fibres with varicosity-like
Fig. 1. Binding sites for [rz51]argininevasopressin on neurons. (A) Silver staining (after Bodian) of neurons lying in the m&us of the solitary tract (culture 21 days itr D&O). {S) Autora~o~a~b of the same c.uiture as in A. All neurons reveal binding sites for ~l*~i]ar~n~~e vasopressin (tOa M). (C) Neurons in an IS-day-ofd brain stem culture which are intensely labelied by [tz51]arginine vasopressin (fWRM). (D) Spinal cord culture after incubation witb [tzSI]arginine vasopressin (10-s M). One neuron is intensely labelled over the soma and processes, whereas two other neurons reveal only weak or no labehing (arrows: culture 25 days in vitro). Scale bars = 30 pm.
Ihl
Binding of vasopressin and ANP in CNS cultures structures growing out from the explant also revealed binding sites for [lZ51]arginine vasopressin. Neurons showing binding sites for [‘151]arginine vasopressin could also be detected in spinal cord cultures; the number of labelled cells was, however, smaller than in brain stem cultures. In spinal cord cultures with attached dorsal root ganglia, it was possible to localize the labelled neurons in both the ventral and dorsal horns. Figure ID illustrates a large spinal neuron in the ventral horn which is intensely labelled by l’~~I]arginine vasopressin, whereas adjacent neurons (arrows) show only little binding of the peptide. Labelling of spinal neurons by [‘ZSI]arginine vasopressin occurred over the soma and dendrites of the cells (Fig. 1D). In cerebellar cultures, however, we were unable to detect neurons which were labelled by [“‘I]arginine vasopressin, suggesting that cerebellar neurons do not express receptors for this peptide.
After incubation of brain stem cultures, a great number of neurons revealed binding sites for [“‘I]ANP (10 -* M). As was observed with arginine vasopressin. groups of intensely labelled neurons were especially observed in the nucleus of the solitary tract (Fig. 2A). In the medulla oblongata, many large, intensely labelled neurons could be detected (Fig. 28). The radioactivity was distributed over the soma and primary dendrites of the cells (Fig. 2A,B). In spinal cord cultures a smaller number of neurons revealed binding sites for [“51]ANP, the labelling usually being weaker than that of brain stem neurons. Binding of the peptide was mainly observed on large neurons in the ventral horns, probably motoneurons. Figure 2C and D illustrates such large neurons which show moderate binding for [“‘I]ANP over the cell bodies and processes. In cultures from cerebellum, only a few mediumsized neurons showed binding sites for [“51]ANP. the intensity of labelled neurons being weak to moderate. Binding of [‘251]arginine r~asopressin und [“SI]a&al natriuretic peptide to ustrocytes After incubation of the cultures with [“‘Ilarginine vasopressin and [“‘IJANP (IO-” M), a great number of astrocytes in both brain stem and spinal cord cultures were intensely labelled by the peptides. Silver grains were evenly distributed over the soma and processes of the cells (Figs 3B,D and 4A.C). No difference in the number of labelled astrocytes or in the intensity of labelling could be detected between brain stem and spinal cord cultures. Labelling of astrocytes in cerebellar cultures, however, was usually weaker than in spinal cord and brain stem (Fig. 4B). Simultaneous staining of the cultures with GFAP has revealed that the IabelIed cells were GFAP-positive. Figure 3 illustrates GFAP-positive astrocytes in a brain stem (A) and a spinal culture (C). The darkfield illumination micrographs of the same cells
demonstrate that all astrocytes are intensely labelled by [“‘I]arginine vasopressin (Fig. 3B,D). Figure 4D shows two GFAP-positive astrocytes with binding sites for [““I]ANP over the soma and processes. Binding of [‘Z51]arginine vasopressin and [“51]ANP to both neurons and astrocytes was markedly reduced or inhibited by adding excess unlabelled peptides (10 ’ to 10 ’ M) to the incubation medium, suggesting that labelling of the cells by the radioligands reflects “specific binding”. Figure 2E and F illustrates the com~tition of [‘?]ANP and ~‘~~l]arginine vasopressin (10 ’ M) by unlabelled ANP (E) and arginine vasopressin (F) both at IO’ 6M in cultures from the nucleus of the solitary tract and spinal cord. DISCUSSION
Binding sites on neurom Arginine vasopressin--a hormone of the hypothalamic/neurohypophyseal area involved in water nletabolism’~-as present in neurons and nerve fibres in various parts of the CNS.J”.4’ From neuroanatomical and immunohistochemical studies it has been suggested that arginine vasopressin-containing neurons in the hypothalamus send direct projections to the medulla oblongata and to the spinal cord (cf. Refs 5 and 53). Autoradiographic studies in sections of human and rat CNS have revealed binding sites for arginine vasopressin in the brain stem with especially of the solitary high densities in the nucleus tract.‘.‘.X.3”.‘7.4’High affinity binding sites for the peptide have also been described in slices of human and rat spinal cord.‘“.‘(’ Ontogenetic studies on fetal rats have shown that arginine vasopressin binding in the nucleus of the solitary tract and in the spinal cord was already present after 20 days in utero. whereas in other brain stem nuclei (e.g. nucleus accumbens. hypoglossal nuclei) and higher CNS structures (e.g. hippocampus), binding sites for the peptide occurred only after birth.‘” Our autoradiographic studies demonstrating that a great number of cultured brain stem neurons, especially those of the nucleus of the solitary tract as well as spinal neurons are labelled by [?]arginine vasopressin are thus consistent with data obtained in slices. The finding that cultured cerebellar neurons are not labelled by the peptide is supported by autoradiographic studies in slices of human and rat cerebellum, where no or only little specific binding was detected for arginine vasopressin.‘,s” Evidence for functional arginine vasopressin receptors in the brain stem has been obtained by electrophysiological studies demonstrating that facial motoneurons are excited by arginine vasopressin.j”.“’ In slices of rat spinal cord, the peptide was found to depolarize the majority of lateral horn cells.” Furthermore, biochemical investigations have shown that microinjection of arginine vasopressin into the nucleus of the solitary tract of rats results in the stimulation of inositol triphosphate.lJ
Fig. 2. Binding sites for [‘z51)atrial natriuretic peptide on neurons. (A) Large neurons in a 71-day-old culture of the nucleus of the solitary tract exhibiting binding sites for [‘“51]ANP (10 -s M). (8) Large brain stem neuron which is intensely labelled over the soma and processes by [‘L5i]ANP (1O.-x M. cuiture 21 dayc in t?irro). (C) Group of large spinal neurons in the ventral horn, presumably motoneurons which arc labellcd by [“‘I]ANP (IO-* M, spinal cord culture 18 days in vi/m). (D) Weakly labelled neuron in a spinal of binding cord culture after incubation with [‘“‘I]ANP (IO ’ M, culture 21 days in r:itro). (E) Competition sites for [“‘I]ANP on neurons and astrocytcs (IO ’ M) by addition of unlabelled ANP (10 -‘M) to the incubation medium (culture of the nucleus of the solitary tract. 21 days in t-&w). (F) After additron of unlabelled arginine vasopressin (IO- ’ M) to the incubation medium, binding of (‘2iljarginine vasopressin (IO-.’ M) to both neurons and astrocytes is markedly reduced or inhibited (spinal cord culture. I8 dnw ill ~.ifrt)). Scale bars -- 50 /urn (A.C F): ?Obim (B).
Binding
of vasopressin
and ANP in CNS cultures
163
Fig. 3. Binding sites for [“SI]arginine vasopressin on astrocytes. (A),(C) Staining of a brain stem (A) and a spinal cord culture (C) with glial fibrillary acidic protein (GFAP). All cells in the outgrowth zone of the cultures are GFAP-positive and are thus identified as astrocytes. (B),(D) Dark-field illumination of micrographs of the same cultures as shown in A and C. Ail astrocytes are intensely labelled over the cell bodies and processes by [‘Lsl]arginine vasopressin (10 -* M); brain stem culture I5 days in I&W: spinal cord culture
25 days in ri~o).
ANP, which was originally described to be synthesized and released from atria1 cardiocytes into the bloodstream, is known to exert potent diuretic and vasorelaxant actions (cf. Refs 1 and 42). By means of immunohistochemical studies, the presence 6f ANPimmunoreactive neurons and nerve fibres could be detected throughout the mammalian CNS including of brain stem and spinal cord. 14.35.36.38.44.52 By meanS biochemical and autoradiographic studies, binding sites were observed in rat brain stem, with especially high levels in the nucleus of the solitary tract.6~‘2.‘5.3” Our autoradiographic investigations, demonstrating that brain stem neurons were intensely labelled by [“‘I]ANP are thus in agreement with these findings. Evidence for ANP receptors in the brain stem has also been obtained by biochemical studies demon-
Scale bars = 30 pm.
strating that the peptide caused an increase in cGMP in this CNS region.Y.48 E~ectrophysioiogical studies in rats in viz~ have shown that electrical stimulation of the hypothalamic paraventricular nucleus caused an inhibition of a great number of neurons in the nucleus of the solitary tract, suggesting an ANP-mediated neural control of the cardiovascular system by the paraventricular nucleus.2s In our spinal cord cultures. only a few neurons were labelled by [“‘I]ANP and the intensity of labelling was usually weak. This is consistent with findings by other authors in slices of rat spinal cord, where only low densities of ANP sites were found, except for the pia mater and arachnoidea.j3 In contrast, cultured cerebeliar neurons showed little or no binding sites, in agreement with studies on cerebellar slices where little binding for
E.
164
H&LI
and L.H~SLI
Fig. 4. Binding sites for [‘?‘I]ANP on astrocytes. (A) Astrocytes in the outgrowth zone of a hram item culture. All cells reveal intense binding of [“‘I]ANP (IO- * M, culture 21 days in vitro). (B) Cerebella1 culture after incubation with [“‘I]ANP (IO s M). The astrocytes in the outgrowth zone are onl! moderately Iabelled by the peptide (culture 21 days in r*itro).(C) Astrocytes lying in the outgrowth zone of a spinal cord culture which are intensely labelled by [“51]ANP (IO ” M, culture 21 days tn r+rro).(Db GFAP-positive astrocytes which are labelled over the cell bodies and processes by [‘z51]ANP (IO *.M culture 25 days C ri~o). Scale bars = 50pm (A.B): 30 /urn (C,D).
ANP was detected on neurons.‘s.3’ Furthermore, in contrast to brain stem slices, no increase of cGMP induced by ANP was observed in the cerebellum.” Binding sites on astrocytes There is increasing evidence that glial cells also express receptors for many vasoactive peptides (cf. Refs 24, 26 and 28). The present study demonstrates that, in addition to neurons, many astrocytes in our explant cultures reveal binding for [‘*‘I]arginine vasopressin. Biochemical investigations on pure astroglial cultures from rat cortex and cerebellum have shown that arginine vasopressin induced a significant increase in inositol triphosphate, whereas the peptide
had little or no effect on inositol phospholipid turnover on astrocytes from spinal cords7 In contrast. our autoradiographic studies demonstrate that astrocytes in spinal cord cultures were also labelled by [‘*‘I]arginine vasopressin, suggesting the presence of glial arginine vasopressin receptors. This is supported by electrophysiological investigations showing that astrocytes in spinal cord and brain stem cultures were depolarized by arginine vasopressin.” These depolarizations were antagonized by an arginine vasopressin antagonist, indicating the existence of specific arginine vasopressin receptors on these cells2” The functional role of glial arginine vasopressin receptors is not yet known. Since the peptide causes a vasocon-
Binding
of vasopressin
striction in microvessels,4’ the question is raised whether brain vasculature is not only influenced by neuron? but also by astrocytes. In addition to neurons, [‘251]ANP was also bound to a great number of astrocytes. Studies in spinal cord cultures of mouse have shown that ANP binding sites were mainly confined to giial cells.43This is consistent with our findings demonstrating that a great number of astrocytes in spinal cord cultures are intensely labelled by [‘2SI]ANP, whereas less neurons reveal binding of the peptide. Stimulation of glial ANP receptors was found to produce a marked increase in the concentration of cGMP.~,““~” Furthermore, electrophysiological studies on cultured rat glioma cells have demonstrated that ANP produced a short hyperpo~arization followed by a long-lasting depolarization. Since the fluorescent signal measured by the calcium-indicator quin2 in these cells was not affected by the peptide, it was concluded that ANP does not change the cytosolic Ca*+ activity but induces an activation of K + channels.4’ Preliminary electrophysiological studies from our laboratory in explant
165
and ANP in CNS cultures
cultures from rat spinal cord and brain stem have shown that ANP has mainly a depolarizing effect on astrocytes. *’ From biochemical investigations demonstrating that ANP significantly decreased the incorporation of [3H]thymidine into cultured astrocytes it was suggested that the natriuretic peptide may function as an “antigrowth factor” for astrocytes in the CNS.29 CONCI.USlON
Our autoradiographic studies provide good evidence that in addition to neurons. astrocytes also express receptors for arginine vasopressin and ANP. There is relatively little information on the functional role of these receptors on astrocytes. Stimulation of both glial arginine vasopressin and ANP receptors influence second messengers such as inositoltriphosphate and cGMP.*.‘.~~“.” Evidence for the presence of receptors for second messengers on astrocytes has also been obtained by biochemical. automdiographi~ and immunohistochemical studies (cf. Ref. 18).
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(1989) Atria1 natriuretic factor-like immunoreactivity and in primary sensory neurons of spinal and trigeminal ganglia of guinea-pig: correlation with tachykinin immunoreactivity. Cell Tkr. Res. 258, 387.-392. 37.Phillips P. A.. Abrahams J. M., Kelly J., Paxinos G., Grzonka Z., Mendelsohn F. A. 0. and Johnston C. I. (1988) Localization of vasopressin binding sites in rat brain by in vitro autoradiography using a radioiodinated V, receptor antagonist. Neuroscience 27, 749-761. J.. Cantin M. and Genest J. (1984) Atria1 natriuretic factor (ANF) 38.Quirion R., DalpC M.. De Lean A., Gutkowska binding sites in brain and related structures. Peptides 5, 1167- 1172. M., Tribollet E. and Dreifuss J. J. (1987) Electrophysiological and autoradiographical evidence of V, 39.Raggenbass vasopressin receptors in the lateral septum of the rat brain. Proc. nufn. Acad. Sci. U.S.A. 84, 7778-7782. M., Goumaz M., Sermasi E., Tribollet E. and Dreifuss J. J. (1991) Vasopressin generates a persistent 40. Raggenbass voltage-dependent sodium current in a mammalian motoneuron. J. Neurosci. 11,1609-1616. 41. Reiser G., Hiipp H.-P. and Hamprecht B. (1987) Atria1 natriuretic polypeptide hormones induce membrane potential responses in cultured rat glioma cells. Bruin Res. 402, 164-167. 42. Samson W. K. and Quirion R. (1990) Atria1 Natriuretic Peptides. CRC Press, Boca Raton, FL. and specific localization of 43.Simonnet G., Allard M., Legendre P., Gahrion J. and Vincent J. D. (1989) Characteristics receptors for atrial natriuretic peptides at non-neuronal cells in cultured mouse spinal cord cells. Neuroscience 29, 189m199. D. M., Eskay R. L. and Zamir N. (1985) Distribution of atria1 natriuretic factor-like 44.Skofitsch G., Jacohowitz immunoreactive neurons in the rat brain. Neuroscience 16, 917-948. 45. Smock T., Cach R. and Topple A. (1987) Action of vasopressin on neurons and microvessels in the rat hippocampal slice. Expi Brain Res. 66, 401MO8. 46.Sofroniew M. V. (1980) Projections from vasopressin, oxytocin, and neurophysin neurons to neural targets in the rat and human. J. Histochem. Cytochem. 28, 475478. 47.Sofroniew M. V. and Schrell U. (1981) Evidence for a direct projection from oxytocin and vasopressin neurons in the hypothalamic paraventricular nucleus to the medulla oblongata: immunohistochemical visualization of both the horseradish peroxidase transported and the peptide produced by the same neurons. Neurosci. Len. 22, 211.-217. R., Grammer R. T. and Inagami T. (1986) Regional increase of cyclic GMP by atria1 natriuretic factor 48.Takayanagi in rat brain: markedly elevated response in spontaneously hypersensitive rats. Life Sci. 39, 573-580. M. and Dreifuss J. J. (1988) Localization and pharmacological 49.Trihollet E., Barheris C., Jard S., Dubois-Dauphin characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography. Brain Res. 442, IO5- 118. M. and Dreifuss J. J. (1991) Early appearance and transient 50.Trihollet E.. Goumaz M., Raggenbass M., Dubois-Dauphin expression of vasopressin receptors in the brain of rat fetus and infant. An autoradiographical and electrophysiological study. Detrl Brain Res. 58, 13 24. 51.Yeung V. T. F., Lai C. K.. Cockram C. S., Young J. D. and Nicholls M. G. (1991) Binding of brain and atria1 natriuretic peptides to cultured mouse astrocytes and effect on cyclic GMP. J. Neurochem. 56, 1684-1689. 52.Zamir N., Skofitsch G., Eskay R. L. and Jacobowitz D. M. (1986) Distribution of immunoreactive atria1 natriuretic peptides in the central nervous system of the rat. Brain Res. 365, 105-I 11. 53.Zimmerman E. A., Nilaver G., Hou-Yu A. and Silverman A. J. (1984) Vasopressinergic and oxytocinergic pathways in the central nervous system. Fedn Pro<. Fedn Am. Sots exp. Biol. 43, 91.-96. (Accepted 24 June 1992)
AUTORADIOGRAPHIC LOCALIZATION OF BINDING SITES FOR ARGININE VASOPRESSIN AND ATRIAL NATRIURETIC PEPTIDE ON ASTROCYTES AND NEURONS OF CULTURED RAT CENTRAL NERVOUS SYSTEM E. H%LI Department
of Physiology,
University
and
L. H~SLI*
of Basel, Vesalgasse
I, CH-4051
Basel. Switzerland
Abstract-The cellular localization of binding sites for [“‘Ilarginine vasopressin and [“‘I]atrial natriuretic pcptide was studied in explant cultures of rat spinal cord. brain stem and cerebellum by means of autoradiography. In brain stem cultures, especially in the nucleus of the solitary tract, a great number of neurons revealed binding sites for both peptides. In spinal cord cultures, many neurons of various sizes were labelled by [‘2il]arginine vasopressin. whereas only a small number of cells showed binding sites for [“‘I]atrial natriurctic peptide. Neurons in cerebellar cultures revealed little or no binding for the peptides. In addition to neurons, binding sites for [‘2iI]arginine vasopressin and [“51]atrial natriuretic peptide were also observed on glial cells. Simultaneous staining of the cultures with glial fibrillary acidic protein has shown that the labelled cells were glial fibrillary acidic protein-positive and could therefore be identified as astrocytes. Labelling of the cells by [“‘llarginine vasopressin and [“‘I]atrial natriuretic peptide was more intense in spinal cord and brain stem cultures than in cultures of cerebellum, providing evidence for a heterogeneity of astrocytes in different regions of the central nervous system. Binding of both [“‘Ilarginine vasopressm and [“‘I]atrial natriuretic peptide to neurons and astrocytes could be competed by the unlabelled peptides, suggesting specific binding of the radioligands. Our autoradiographic studies provide good evidence that in addition to neurons, astrocytes also express receptors for arginine vasopressin and atrial natriuretic peptide.
EXPERIMENTAL
The nonapeptide argininc vasopressin and the 3% amino acid pcptide atrial natriuretic peptidc (ANP) arc considered to act as neurotransmittersjneuromodulators in the mammalian CNS (cf. Refs 32 and 42). High affinity binding sites have been found for both kasoactive peptides on neurons in many regions of the CNS,h,“‘,‘7 3’)
PROCEDURES
Explant cultures were prepared from cerebellum of ncwborn rats and from brain stem (nucleus of the solitary tract. medulla oblongata) and from spinal cord (with or without attached dorsal root ganglia) of fetal rats ( I7 IX days in ~trwo OFA; Biological Research Laboratory, Fullinsdorf. SwitTcrland). Two explants were placed on collagen-coated AC‘LAR coverslips and grown in Roller tubes for I4 40 days (for details see Ref. 17). The cultures were rinsed m jcveral changes of Hank’s solution (37 C) before they were incubated in Hank’s solution containing [“‘I]arginine vasopressin or [“‘IIANP (IO-* M) for 45 min at 20 C. The incubation medium was supplemented by 0.X% bovine berum albumin and the peptidase inhibitors bacitracin (0.02’%) and trasylol (100 KIUjml). Specificity of binding was estimated by competing the radioligands with unlabelled arginine vasopressln or ANP at high concentrations (IO ’ to IO ‘M). After the incubation with [“il]arginine vasopressin or [“51]ANP, the cultures were carefully washed in 0.1% phosphate buffer (pH 7.3: IO times for I min) and lixed m glutaraldehyde vapour (50 C). After fixation. they were rinsed in ice-cold phosphate buffer and distilled water and then air-dried. The cultures were covered with Ilford L4 emulsion by means of the loop technique, exposed at 4 C for four weeks and developed with Kodak Dl9 developer (for details see Ref. 17).
Previous autoradiographic and electrophysiological studies from our laboratory indicate that not only neurons but also glial cells possess receptors for vasoactive peptides such as angiotensin II (Ang II), vasoactive intestinal peptide (VIP) and endothelin.lb,l”.‘:.?~ Furthermore, biochemical studies have shown that activation of glial peptide receptors influences second messenger systems such as CAMP, cGMP and inositoltriphosphate. suggesting the existence of receptors for various peptides on astrocytes (cf. Refs 18, 26 and 28). The aim of the present investigations was to study the cellular localization of binding sites for [“‘Ilarginine vasopressin and [“‘I]ANP in explant cultures from rat spinal cord, brain stem and cerebellum by means of autoradiography.
The radioligands [“‘Ilarginine vasopressin and [“‘I]ANP (specific activity 2200 Ci,‘mmol) were purchased from New England Nuclear Co. (NEN); bovine serum albumln and bacitracin were from Serva. trasylol from Bayer and the unlabelled peptides AVP and ANP from Bachcm.
*To Mhom correspondence should be addressed. ilhhreriations: Ang II. angiotensin II; ANP, atria1 natriurctic peptide; GFAP, glial fibrillary acidic protein; VIP, ~asoactive intestinal peptide. 159
HESULTS
In explant cultures of rat CNS, neurons and astrocytes were recognized by their morphological appearance or by their iocation in the cultures. Neurons usually remain in or at the edge of the expfant, whereas glial eeLIs migrate into the outgrowth zone where they form a dense network.“’ The various cell types have also been identified by specific staining properties: silver staining (after Bodian) for neurons (Fig. I A) or gtial fibrillary acidic protein (GFAP) for astrocytes {Figs 3A,C and 4D). Most of the cells in the outgrowth zone were GFAP-positive, and are thus considered to be astrocytes.
After incubation of brain stem cultures with [‘2SI]arginine vasopressin (10 * M), many large and medium-sized neurons were labelled. in cultures which were prepared from the area of the nucleus of the solitary tract, groups of neurons revealing binding sites for the peptide could be observed. Figure IA illustrates silver-staining of a group of neurons in the nucleus of the solitary tract which show binding sites for fn51]arginine vasopressin (Fig. 13). Labetled neurons of various shapes and sizes were also detected in cultures of the medulla oblongata (Fig. 1C). The silver grains were localized over the soma and dendrites of the neurons. Nerve fibres with varicosity-like
Fig. 1. Binding sites for [rz51]argininevasopressin on neurons. (A) Silver staining (after Bodian) of neurons lying in the m&us of the solitary tract (culture 21 days itr D&O). {S) Autora~o~a~b of the same c.uiture as in A. All neurons reveal binding sites for ~l*~i]ar~n~~e vasopressin (tOa M). (C) Neurons in an IS-day-ofd brain stem culture which are intensely labelied by [tz51]arginine vasopressin (fWRM). (D) Spinal cord culture after incubation witb [tzSI]arginine vasopressin (10-s M). One neuron is intensely labelled over the soma and processes, whereas two other neurons reveal only weak or no labehing (arrows: culture 25 days in vitro). Scale bars = 30 pm.
Ihl
Binding of vasopressin and ANP in CNS cultures structures growing out from the explant also revealed binding sites for [lZ51]arginine vasopressin. Neurons showing binding sites for [‘151]arginine vasopressin could also be detected in spinal cord cultures; the number of labelled cells was, however, smaller than in brain stem cultures. In spinal cord cultures with attached dorsal root ganglia, it was possible to localize the labelled neurons in both the ventral and dorsal horns. Figure ID illustrates a large spinal neuron in the ventral horn which is intensely labelled by l’~~I]arginine vasopressin, whereas adjacent neurons (arrows) show only little binding of the peptide. Labelling of spinal neurons by [‘ZSI]arginine vasopressin occurred over the soma and dendrites of the cells (Fig. 1D). In cerebellar cultures, however, we were unable to detect neurons which were labelled by [“‘I]arginine vasopressin, suggesting that cerebellar neurons do not express receptors for this peptide.
After incubation of brain stem cultures, a great number of neurons revealed binding sites for [“‘I]ANP (10 -* M). As was observed with arginine vasopressin. groups of intensely labelled neurons were especially observed in the nucleus of the solitary tract (Fig. 2A). In the medulla oblongata, many large, intensely labelled neurons could be detected (Fig. 28). The radioactivity was distributed over the soma and primary dendrites of the cells (Fig. 2A,B). In spinal cord cultures a smaller number of neurons revealed binding sites for [“51]ANP, the labelling usually being weaker than that of brain stem neurons. Binding of the peptide was mainly observed on large neurons in the ventral horns, probably motoneurons. Figure 2C and D illustrates such large neurons which show moderate binding for [“‘I]ANP over the cell bodies and processes. In cultures from cerebellum, only a few mediumsized neurons showed binding sites for [“51]ANP. the intensity of labelled neurons being weak to moderate. Binding of [‘251]arginine r~asopressin und [“SI]a&al natriuretic peptide to ustrocytes After incubation of the cultures with [“‘Ilarginine vasopressin and [“‘IJANP (IO-” M), a great number of astrocytes in both brain stem and spinal cord cultures were intensely labelled by the peptides. Silver grains were evenly distributed over the soma and processes of the cells (Figs 3B,D and 4A.C). No difference in the number of labelled astrocytes or in the intensity of labelling could be detected between brain stem and spinal cord cultures. Labelling of astrocytes in cerebellar cultures, however, was usually weaker than in spinal cord and brain stem (Fig. 4B). Simultaneous staining of the cultures with GFAP has revealed that the IabelIed cells were GFAP-positive. Figure 3 illustrates GFAP-positive astrocytes in a brain stem (A) and a spinal culture (C). The darkfield illumination micrographs of the same cells
demonstrate that all astrocytes are intensely labelled by [“‘I]arginine vasopressin (Fig. 3B,D). Figure 4D shows two GFAP-positive astrocytes with binding sites for [““I]ANP over the soma and processes. Binding of [‘Z51]arginine vasopressin and [“51]ANP to both neurons and astrocytes was markedly reduced or inhibited by adding excess unlabelled peptides (10 ’ to 10 ’ M) to the incubation medium, suggesting that labelling of the cells by the radioligands reflects “specific binding”. Figure 2E and F illustrates the com~tition of [‘?]ANP and ~‘~~l]arginine vasopressin (10 ’ M) by unlabelled ANP (E) and arginine vasopressin (F) both at IO’ 6M in cultures from the nucleus of the solitary tract and spinal cord. DISCUSSION
Binding sites on neurom Arginine vasopressin--a hormone of the hypothalamic/neurohypophyseal area involved in water nletabolism’~-as present in neurons and nerve fibres in various parts of the CNS.J”.4’ From neuroanatomical and immunohistochemical studies it has been suggested that arginine vasopressin-containing neurons in the hypothalamus send direct projections to the medulla oblongata and to the spinal cord (cf. Refs 5 and 53). Autoradiographic studies in sections of human and rat CNS have revealed binding sites for arginine vasopressin in the brain stem with especially of the solitary high densities in the nucleus tract.‘.‘.X.3”.‘7.4’High affinity binding sites for the peptide have also been described in slices of human and rat spinal cord.‘“.‘(’ Ontogenetic studies on fetal rats have shown that arginine vasopressin binding in the nucleus of the solitary tract and in the spinal cord was already present after 20 days in utero. whereas in other brain stem nuclei (e.g. nucleus accumbens. hypoglossal nuclei) and higher CNS structures (e.g. hippocampus), binding sites for the peptide occurred only after birth.‘” Our autoradiographic studies demonstrating that a great number of cultured brain stem neurons, especially those of the nucleus of the solitary tract as well as spinal neurons are labelled by [?]arginine vasopressin are thus consistent with data obtained in slices. The finding that cultured cerebellar neurons are not labelled by the peptide is supported by autoradiographic studies in slices of human and rat cerebellum, where no or only little specific binding was detected for arginine vasopressin.‘,s” Evidence for functional arginine vasopressin receptors in the brain stem has been obtained by electrophysiological studies demonstrating that facial motoneurons are excited by arginine vasopressin.j”.“’ In slices of rat spinal cord, the peptide was found to depolarize the majority of lateral horn cells.” Furthermore, biochemical investigations have shown that microinjection of arginine vasopressin into the nucleus of the solitary tract of rats results in the stimulation of inositol triphosphate.lJ
Fig. 2. Binding sites for [‘z51)atrial natriuretic peptide on neurons. (A) Large neurons in a 71-day-old culture of the nucleus of the solitary tract exhibiting binding sites for [‘“51]ANP (10 -s M). (8) Large brain stem neuron which is intensely labelled over the soma and processes by [‘L5i]ANP (1O.-x M. cuiture 21 dayc in t?irro). (C) Group of large spinal neurons in the ventral horn, presumably motoneurons which arc labellcd by [“‘I]ANP (IO-* M, spinal cord culture 18 days in vi/m). (D) Weakly labelled neuron in a spinal of binding cord culture after incubation with [‘“‘I]ANP (IO ’ M, culture 21 days in r:itro). (E) Competition sites for [“‘I]ANP on neurons and astrocytcs (IO ’ M) by addition of unlabelled ANP (10 -‘M) to the incubation medium (culture of the nucleus of the solitary tract. 21 days in t-&w). (F) After additron of unlabelled arginine vasopressin (IO- ’ M) to the incubation medium, binding of (‘2iljarginine vasopressin (IO-.’ M) to both neurons and astrocytes is markedly reduced or inhibited (spinal cord culture. I8 dnw ill ~.ifrt)). Scale bars -- 50 /urn (A.C F): ?Obim (B).
Binding
of vasopressin
and ANP in CNS cultures
163
Fig. 3. Binding sites for [“SI]arginine vasopressin on astrocytes. (A),(C) Staining of a brain stem (A) and a spinal cord culture (C) with glial fibrillary acidic protein (GFAP). All cells in the outgrowth zone of the cultures are GFAP-positive and are thus identified as astrocytes. (B),(D) Dark-field illumination of micrographs of the same cultures as shown in A and C. Ail astrocytes are intensely labelled over the cell bodies and processes by [‘Lsl]arginine vasopressin (10 -* M); brain stem culture I5 days in I&W: spinal cord culture
25 days in ri~o).
ANP, which was originally described to be synthesized and released from atria1 cardiocytes into the bloodstream, is known to exert potent diuretic and vasorelaxant actions (cf. Refs 1 and 42). By means of immunohistochemical studies, the presence 6f ANPimmunoreactive neurons and nerve fibres could be detected throughout the mammalian CNS including of brain stem and spinal cord. 14.35.36.38.44.52 By meanS biochemical and autoradiographic studies, binding sites were observed in rat brain stem, with especially high levels in the nucleus of the solitary tract.6~‘2.‘5.3” Our autoradiographic investigations, demonstrating that brain stem neurons were intensely labelled by [“‘I]ANP are thus in agreement with these findings. Evidence for ANP receptors in the brain stem has also been obtained by biochemical studies demon-
Scale bars = 30 pm.
strating that the peptide caused an increase in cGMP in this CNS region.Y.48 E~ectrophysioiogical studies in rats in viz~ have shown that electrical stimulation of the hypothalamic paraventricular nucleus caused an inhibition of a great number of neurons in the nucleus of the solitary tract, suggesting an ANP-mediated neural control of the cardiovascular system by the paraventricular nucleus.2s In our spinal cord cultures. only a few neurons were labelled by [“‘I]ANP and the intensity of labelling was usually weak. This is consistent with findings by other authors in slices of rat spinal cord, where only low densities of ANP sites were found, except for the pia mater and arachnoidea.j3 In contrast, cultured cerebeliar neurons showed little or no binding sites, in agreement with studies on cerebellar slices where little binding for
E.
164
H&LI
and L.H~SLI
Fig. 4. Binding sites for [‘?‘I]ANP on astrocytes. (A) Astrocytes in the outgrowth zone of a hram item culture. All cells reveal intense binding of [“‘I]ANP (IO- * M, culture 21 days in vitro). (B) Cerebella1 culture after incubation with [“‘I]ANP (IO s M). The astrocytes in the outgrowth zone are onl! moderately Iabelled by the peptide (culture 21 days in r*itro).(C) Astrocytes lying in the outgrowth zone of a spinal cord culture which are intensely labelled by [“51]ANP (IO ” M, culture 21 days tn r+rro).(Db GFAP-positive astrocytes which are labelled over the cell bodies and processes by [‘z51]ANP (IO *.M culture 25 days C ri~o). Scale bars = 50pm (A.B): 30 /urn (C,D).
ANP was detected on neurons.‘s.3’ Furthermore, in contrast to brain stem slices, no increase of cGMP induced by ANP was observed in the cerebellum.” Binding sites on astrocytes There is increasing evidence that glial cells also express receptors for many vasoactive peptides (cf. Refs 24, 26 and 28). The present study demonstrates that, in addition to neurons, many astrocytes in our explant cultures reveal binding for [‘*‘I]arginine vasopressin. Biochemical investigations on pure astroglial cultures from rat cortex and cerebellum have shown that arginine vasopressin induced a significant increase in inositol triphosphate, whereas the peptide
had little or no effect on inositol phospholipid turnover on astrocytes from spinal cords7 In contrast. our autoradiographic studies demonstrate that astrocytes in spinal cord cultures were also labelled by [‘*‘I]arginine vasopressin, suggesting the presence of glial arginine vasopressin receptors. This is supported by electrophysiological investigations showing that astrocytes in spinal cord and brain stem cultures were depolarized by arginine vasopressin.” These depolarizations were antagonized by an arginine vasopressin antagonist, indicating the existence of specific arginine vasopressin receptors on these cells2” The functional role of glial arginine vasopressin receptors is not yet known. Since the peptide causes a vasocon-
Binding
of vasopressin
striction in microvessels,4’ the question is raised whether brain vasculature is not only influenced by neuron? but also by astrocytes. In addition to neurons, [‘251]ANP was also bound to a great number of astrocytes. Studies in spinal cord cultures of mouse have shown that ANP binding sites were mainly confined to giial cells.43This is consistent with our findings demonstrating that a great number of astrocytes in spinal cord cultures are intensely labelled by [‘2SI]ANP, whereas less neurons reveal binding of the peptide. Stimulation of glial ANP receptors was found to produce a marked increase in the concentration of cGMP.~,““~” Furthermore, electrophysiological studies on cultured rat glioma cells have demonstrated that ANP produced a short hyperpo~arization followed by a long-lasting depolarization. Since the fluorescent signal measured by the calcium-indicator quin2 in these cells was not affected by the peptide, it was concluded that ANP does not change the cytosolic Ca*+ activity but induces an activation of K + channels.4’ Preliminary electrophysiological studies from our laboratory in explant
165
and ANP in CNS cultures
cultures from rat spinal cord and brain stem have shown that ANP has mainly a depolarizing effect on astrocytes. *’ From biochemical investigations demonstrating that ANP significantly decreased the incorporation of [3H]thymidine into cultured astrocytes it was suggested that the natriuretic peptide may function as an “antigrowth factor” for astrocytes in the CNS.29 CONCI.USlON
Our autoradiographic studies provide good evidence that in addition to neurons. astrocytes also express receptors for arginine vasopressin and ANP. There is relatively little information on the functional role of these receptors on astrocytes. Stimulation of both glial arginine vasopressin and ANP receptors influence second messengers such as inositoltriphosphate and cGMP.*.‘.~~“.” Evidence for the presence of receptors for second messengers on astrocytes has also been obtained by biochemical. automdiographi~ and immunohistochemical studies (cf. Ref. 18).
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