Vasopressin Neuromodulation in the Hippocampus

Home , Hippocampal formation, Neuroendocrinology, Slice preparation

The Journal of Neuroscience, March 1989, g(3): 752-759

Roberta E. Brinton and Bruce S. McEwen Laboratory of Neuroendocrinology, The Rockefeller University, New York, New York 10021

This study explored an effector mechanism associated with b). Two decadesago these investigators began to explore the the arginine vasopressin (AVP) recognition site in the hip- behavioral effectsof vasopressinand other peptides upon tasks pocampus, namely, potentiation of norepinephrine (NE)-in- thought to be indicators of mnemonic function (for review, see duced CAMP accumulation in the surviving hippocampal slice. Strupp and Levitsky, 1985). Resultsof their studieshave chal- The biochemical mechanisms that underlie the AVP poten- lenged us and others to explore the biochemical aspectsof va- tiation were investigated as follows: First, the actions of AVP sopressinaction in brain. upon NE-induced accumulation of CAMP in hippocampal Putative receptorsfor vasopressinhave been detected by au- slices from rat brain were specific to AVP and not shared toradiography in each of the developmental subdivisionsof the by other closely related peptides, namely, oxytocin and brain (Biegon et al., 1984; Brinton et al., 1984; DeKloet et al., AVP,-,. Second, the AVP-induced neuromodulation involved 1985). Of particular interest with respect to the present work beta-adrenergic receptors, with AVP having no effect on are the arginine vasopressin(AVP) recognition siteswithin the CAMP levels in the absence of NE. Third, the potentiation by hippocampalformation. In Ammon’s horn, AVP receptorswere AVP was biphasic, with lower AVP concentrations poten- observed in the pyramidal cell layer (Biegonet al., 1984; Brinton tiating NE-induced CAMP accumulation, while higher con- et al., 1984) and in the strati oriens, radiatum, and lacunosum centrations did not potentiate. Fourth, an antagonist of V,- moleculare (Brinton et al., 1984). Within the dentate gyrus, AVP type AVP receptors blocked AVP potentiation. Fifth, AVP receptorswere specifically localized to the molecular layer with potentiation was dependent upon extracellular calcium con- no detectable recognition sitesin the hilus (Biegon et al., 1984; centrations. Sixth, AVP potentiation was blocked by 50 I.IM Brinton et al., 1984). Receptor binding studies using synaptic trifluoperazine, which is consistent with a calcium-calmo- membranes suggestthat the recognition site for AVP in the dulin involvement but which might also implicate protein ki- hippocampus is a high-affinity binding site with a Kd of ap- nase C. These alternatives and the nature of the calcium proximately 3 nM (Audigier and Barberis,1985). involvement are discussed. AVP actions thus appear to in- Electrophysiological evidence supports the postulate of func- volve interactions between several second-messenger sys- tionally coupled AVP receptors in the hippocampus.Intracel- tems and suggest a biochemical mechanism by which AVP lular recordings from CA, pyramidal neurons showed that va- exerts its centrally mediated behavioral effects. sopressininduced a slow increase in spike dischargethat was gradually reversible (Mizuno et al., 1984). Extracellular record- The emergingknowledge of peptide actions in brain constitutes ings showeda similar pattern of excitation with AVP inducing a major influence upon the growing understanding of neuronal an increasein neuronal firing in the Ammon’s horn of the hip- responseproperties. The study of one such substance, vaso- pocampus (Muhlenthaler et al., 1982). To date, the electro- pressin, has contributed insights into peptide effectson behav- physiological effectsof AVP in the dentate gyrus are not known, ioral, biophysical, and biochemicalaspects of neuronal function. However, recent data from patch-clamp analysisof sympathetic Vasopressinis a 9 amino acid peptide which is biologically ganglion cells suggestthe interesting possibility that AVP acts active in the periphery and in the CNS. Peripherally, vasopressin as a potent regulator of channel opening (Matsumoto et al., receptors and their associatedeffector mechanismshave been 1987). extensively characterized (for review, seeJard, 1983). In con- Biochemical investigations of AVP action in hippocampus trast, the exploration of CNS vasopressinreceptors and effector stem, in part, from the behavioral studies of Kovacs and co- mechanismsis still in an early stageof development (Brinton, workers (1979a, b). These investigators found that lesioningthe 1987). Attention to the possiblerole of vasopressinas a neurally dorsal noradrenergicbundle, which innervates the hippocampus active peptide was initiated by the pioneering work of DeWied (Moore and Bloom, 1979) abolished the behavioral effects of and his colleagues(DeWied, 1971, 1984; Kovacs et al., 1979a, vasopressin(Kovacs et al., 1979a).This study provided the first evidence that AVP interacted with the monoamine norepinephrine (NE) to produce its behavioral effects. Exploring this Received Sept. 1, 1987; revised July 5, 1988; accepted Aug. 15, 1988. We wish to thank Drs. Peter Davies, Jack Grebb, and Robert Spencer for interaction, Church (1983) and later we (Brinton and McEwen, thoughtful criticisms of this manuscript and Dr. Thomas Vickroy for suggestions 1985, 1986) and others (Newman, 1985) showedthat AVP po- regarding CAMP determinations. We are especially grateful to Dr. Maurice Man- tentiated NE-induced accumulation of cyclic AMP in the hip- ning for his very generous gifts of vasopressin and vasopressin analogs. This work was supported by NIMH 41256 to B. S. McEwen and by NIMH Postdoctoral pocampus. Fellowship, MH b9224 to R. E. Brinton. The present experiments were undertaken to explore the Correspondence should be addressed to Dr. Roberta E. Brinton at her present mechanismby which AVP modulates NE-induced accumula- address: Division of Biological Sciences, School of Pharmacy, University of South- ern California, 1985 Zonal Avenue, Los Angeles, CA 90033. tion of CAMP. The data show that in the hippocampal slice, Copyright 0 1989 Society for Neuroscience 0270-6474/89/030752-08$02.00/O vasopressinmodulates NE-induced accumulation of CAMP in The Journal of Neuroscience, March 1989, 9(3) 753 a dose-dependent and peptide-specific manner. The data further suggest that the neuromodulatory effect of AVP in the hippocampus involves a calcium-dependent process that is inhibited by a V,-type vasopressin receptor antagonist and by a calmodulin and protein kinase C antagonist, trifluoperazine. These findings further our understanding of vasopressin action in the CNS and may also serve to provide insights into the biochemical basis of the behavioral effects attributed to vasopressin. Prelim- inary forms of some of the results presented in this paper have 1 been presented previously (Brinton and McEwen, 1985, 1986).

Materials and Methods Experiments to investigate the effect of AVP upon NE-induced accumulation of CAMP were performed in hippocampal slices from male Sprague-Dawley rats (150-l 75 g; Charles River, Kingston, New York). I Hippocampal tissue was cut using a McIlwain tissue chopper into 325 NE micron slices then turned 90” and sliced again. (In our experiments there was no difference between results obtained in singly or doubly cut slices.) Figure I. AVP neuromodulation of NE-induced accumulation (If CAMP Slices were then incubated in constantly oxygenated (95% OJ5% CO,) in slices from rat hippocampus. AVP (250 nM) alone has no effect upon modified Na bicarbonate Krebs Ringer for 35 min in a 37°C shaking CAMP accumulation while- significantly potentiating that induced by water bath. The medium was then exchanged with one containing 30 NE. Data were analyzed by Student’s t test, * = p < 0.05 vs. NE alone, ,UMRolipram (a phosphodiesterase inhibitor that does not inhibit aden- n = at least 6 for each condition. osine-stimulated adenylate cyclase; gift of N. Sprzgala, Schering Phar- maceuticals AG, FRG) and the incubation continued for another 20 min. Another phosphodiesterase inhibitor, 3-isobutyl- 1-methylxan- competitive protein binding assay (modified from Gilman, 1970). Ex- thine (Sigma Chemical Company, St. Louis), was also tested, and while perimental samples and CAMP standards dissolved in 7% TCA were lower basal levels were observed, there was no difference in the mag- extracted 3 times in 6 volumes of diethyl ether. Extracted samples (50 nitude of the potentiating effect of AVP. Following the incubation in ~1) and standards (50 ~1) were incubated in an ice-water bath in the the presence of Rolipram, samples for basal levels of CAMP accumu- presence of 150 ~1 [5,8-)H]-adenosine 3’,5’ cyclic phosphate (Amer- lation were taken. At this point incubation in the presence of the phos- sham) containing approximately 40,000 cpm/l50 ~1 and 100 ~1 protein phodiesterase inhibitor and the test substances was initiated and the kinase (7.5 &tube; Sigma) for 90 min. The binding reaction was ter- incubation continued for an additional 12 min at 37°C in constantly minated by the addition of 100 ~1 of activated charcoal/BSA solution; oxygenated Krebs Ringer. Following both the 20 min preincubation the samples were then vortexed and centrifuged at 15,000 x g for 10 period in the presence of the phosphodiesterase inhibitor and in the min. Aliquots of supematant (250 ~1) were counted for radioactivity in incubation in the presence of test substances, 500 ~1 samples containing a scintillation counter. Cyclic AMP content was determined by linear- approximately 500 rg of wet weight tissue were aliquoted into 1.7 ml regression analysis based on a log/logit transformation. Data were ana- conical capped microfuge tubes resting in a bath of ice water. Basal lyzed either by a Student’s t test or by 1-way analysis of variance followed CAMP accumulation was measured in single samples from each test by a Newman-Keuls test for multiple comparisons. vial, and basal CAMP accumulation was averaged across all conditions within an individual experiment. Each experimental condition was mea- Results sured in triplicate within each experiment. Aliquoted slices quickly settled to the bottom of the tube and the Krebs Ringer was vacuumed Dose-responsefor vasopressinpotentiation of NE-induced off followed bv the immediate addition of 250 rrl of ice-cold 7% tri- accumulation of CAMP chloroacetic acid (TCA) to terminate the accumulation of CAMP. Sam- In the hippocampal slice preparation, in vitro addition of va- ples were then sonicated and centrifuged at 15,000 x g for 15 min. The sopressindid not increase CAMP accumulation above basal suuematant was removed and 250 ul of 0.2 N NaOH added to the pellet for protein determination by the method of Bradford (1976). TCA su- levels, whereasaddition of 10 PM NE resultedin a 3-fold increase pematants were processed as described below. over basallevels (Fig. 1). The simultaneouspresence of 250 nM All experimental conditions contained 0.01% ascorbic acid and 2 /LM AVP and NE resulted in approximately a 5-fold increase in bacitracin (Sigma). For experiments performed in the absence of added CAMP accumulation over baselineand a 40% increasein CAMP calcium, the osmblarity was adjusted by increasing MgCl, to 2.6 mM. above that which accumulated in responseto NE alone (Fig. 1). Otherwise. the modified Krebs Ringer CDH 7.4) consisted of the following (in mh): 124 NaCl, 5 KCl, 26 NaHCOi,, ‘1.3 MgCl,, 1.4 KH,PO,, Thus, while 250 nM AVP had no effect alone upon CAMP ac- 0.8 CaCl,, 10 dextrose. NE [(-)arterenol; Sigma] at a concentration of cumulation, AVP significantly [F(5,44) = 5.1, p < O.OOl] po- 10 PM was used in all experiments. Oxytocin and AVP,, were obtained tentiated the effect of NE upon CAMP accumulation. The mod- from Peninsula. AVP and the V, receptor antagonist [ 1-(beta-mercapto- ulatory effect of AVP showsan inverted U-shape relationship beta, beta’-cyclopentamethylene propionic acid), 2-CO-(methyl) tyrosine] arg,-vasopressin [d(CH,),Try(Me)AVP] were a generous gift of Dr. within a concentration range of 10-1000 nM. Lower concentra- Maurice Manning (Medical College of Ohio, Toledo, OH). These pep- tions (50-250 nM) of AVP potentiated NE-induced accumula- tides, as well as the‘ calmodulin antagonist trifluoperazine (Sigma), were tion of CAMP, while the highest concentration (1000 nM) did diluted into Krebs Rinaer immediatelv nrior to use. BAY K 8644 (gift not potentiate (Table 1). of Dr. A. Scriabine, Miles Institute, New Haven, CT) and forskohn (Sigma) were diluted into 95% ethanol, and 10 ~1 of concentrated stock Peptide specljicity solution was added to 10 ml of Krebs Ringer for use in the experiments. Exneriments utilizing BAY K 8644 were performed in greatly subdued Becausethe metabolite peptides of vasopressin, AVP,, and lighting. Phorbol 12,-l 3-dibutyrate (PDBu; LC Services Corp.; Wobum, AVP,, (Burbach and Lebouille, 1983), have been shown to be MA) was dissolved in 100% dimethvl sulfoxide (DMSO) and diluted behaviorally active (Burbach et al., 1983; DeWied et al., 1984), to a final DMSO concentration of 0.61% for experimental use. Control we tested whether the metabolite, AVP,, also modulated NE- experiments in which 10 ~1 of 95% ethanol was diluted into 10 mol Krebs Ringer or Krebs solution containing 0.01% DMSO showed no induced accumulation of CAMP. Additionally, we testedwheth- effect upon NE-induced accumulation of CAMP. er oxytocin, a peptide closelyrelated in amino acid composition Cyclic AMP accumulation was determined using a protein kinase to vasopressinthat has been shown to have behavioral effects 754 Brinton and McEwen l Vasopressin Neuromodulation in Hippocampus

Table 1. Effect of AVP concentration on NEinduced accumulation Table 2. Effect of vasopressin metabolite peptide, AVP 4-9, and of CAMP in the hippocampus oxytocin (OXY) upon NE-induced accumulation of CAMP in the hippocampus Percentof cAMP/mg proteina NE cAMP/mg protein Percent Condition CDrnOlj control Condition (pmol) of NE A. Basal 79 + 9 (46) 33 Basal 62 IIT6 22 B. NE, 101~ 236 + 16(16) 100 NE, 10 /.tM 280 f 20 100 C. NE+ 10nMAVP 222 + 24(8) 94 NE + AVP, 100nM 455 + 45" 163 D. NE + 50 nM AVP 267 + ll(3) 113 NE + AVP,,, 10nM 290 f 15 104 E. NE + 100nMAVP 342 + 39 (6)b 144 NE + AVP,,, 100nM 262 f 25 94 F. NE + 250nM AVP 350 + 27 (12)b 148 NE + OXY, 10nM 239 1- 16 84 G. NE + 1000nM AVP 260 + 37(S) 110 NE + OXY, 100nM 217 & 12 77 NE concentration = 10 MM in all cases. Mean values + SEMs are given (8). The NE + OXY, 1000nM 224 ic 47 80 data were analyzed by l-way ANOVA followed by a Newman-Keuls test. NE concentration was 10 PM in all cases. Values are means + SEMs from at least * n given in parentheses. 3 separate determinations. Data were analyzed by l-way ANOVA followed by “p i 0.05 vs. conditions B and C. Newman-Keuls test. * p < 0.05 vs. NE alone. oppositeto thoseof AVP (Kovacs et al., 1979b), would influence NE-induced accumulation of CAMP. As seenin Table 2, neither experiments showed that a higher concentration (2.5 mM) of 10 or 100 nM AVP,, nor 1O-1000 nM oxytocin significantly calcium no longerenhanced the AVP-induced potentiation (Fig. affected NE-induced accumulation of CAMP. 3). The accumulation of CAMP during basal conditions and in responseto NE wasnot significantly affected by the presenceor Beta-adrenergic receptor antagonist and forskolin activity absenceof extracellular calcium. Thus, it appearsthat it is the Propranolol (10 PM), a beta-adrenergicreceptor antagonist, ef- AVP neuromodulatory influence that is selectively sensitive to fectively blocked [F (2,15) = 28.9, p < O.OOOl]both the NE- concentrations of extracellular calcium. induced accumulation of CAMP and the potentiating effect of The dependency upon extracellular calcium suggestedthat AVP (Table 3). This finding supports the hypothesis that va- calcium accesscould be a. factor in the mechanism by which sopressinacts to modulate beta-adrenergicreceptor-coupled ad- AVP modulatedNE-induced accumulation of CAMP. We there- enylate cyclase. This hypothesis was further supported by ex- fore tested whether the calcium channel activator BAY K 8644 periments carried out using the adenylate cyclase activator (Schramm et al., 1983) would influence the neuromodulatory forskolin (Seamonand Daly, 1986).At 10 PM, forskolin induced effect of AVP. In the absenceof extracellular calcium (Fig. 4) a greater than 2-fold increasein CAMP accumulation. In the or at a low concentration (0.4 mM Ca2+;data not shown),neither presenceof the same concentration of forskolin, vasopressin AVP nor BAY K 8644 had any potentiating effect upon NE- neither potentiated nor inhibited CAMP accumulation (Table induced accumulation of CAMP. In the presenceof 0.8 mM 3). This result provides evidence that vasopressinis not mod- calcium, the potentiating effect of AVP wasrestored, while BAY ulating adenylate cyclase directly but is more likely acting to K 8644 alone had no effect. However, BAY K 8644 + AVP in modulate a receptor-coupled event. combination significantly [F(2,12) 12.2, p < 0.00ll’potentiated the effect of NE (Fig. 4). While the potentiation observed in the Vasopressin V, receptor antagonist activity presenceof AVP + BAY K 8644 was not significantly greater Behavioral studieshad shownthat the memory-enhancingeffect than that seenin the presenceof AVP alone, the potentiation of AVP was abolished with the V, receptor antagonist observed wth AVP and BAY K 8644 neverthelessrepresented d(CH,),Tyr(Me)AVP (LeMoal et al., 1981; DeWied et al., 1984). a total increaseof 83% over the CAMP produced by NE alone This antagonist is specific for the V, vasopressin receptor in and a 23% increaseover that produced in the presenceof NE liver and blood vessels(Kruszynski et al., 1980). Becauseof the + AVP (Fig. 4). antagonism of the behavioral effect, we tested whether The biphasic nature of both the AVP dosedependency and d(CH,),Tyr(Me)AVP would antagonize the AVP-induced neu- the calcium sensitivity of the AVP-induced neuromodulation romodulation in the hippocampus.As shown in Figure 2, this was reminiscent of calcium/calmodulin regulation of adenylate antagonist significantly [F (2,6) = 9.2, p < 0.011 blocked the cyclase(Brostrom et al., 1975, 1978; Cheupg, 1980; Treisman AVP potentiation of NE-induced accumulation of CAMP. Va- et al., 1983). We hypothesized that if AVP-induced neuromo- sopressinantagonists specific for the V, type of vasopressin dulation involved a calmodulin-dependent process, then tri- receptor did not antagonize the AVP potentiation (Brinton and fluoperazine (TFP), a calmodulin antagonist (Levin and Weiss, McEwen, 1986). 1977), should block the potentiating effect of AVP. Data presented in Table 4 show that TFP, while having no effect upon Calcium pathway experiments CAMP accumulation in responseto NE alone, significantly Becausethe peripheral V, receptor is linked to calcium mobi- blocked the potentiating effect of AVP [F (3,16) = 10.0, p < lization (Kirk et al., 1981) and becausethe V, receptorantagonist O.OOl]. blocked the potentiating effect of AVP, we tested the calcium Although TFP is a potent calmodulin antagonist, it can also dependency of the AVP-induced neuromodulation. When ex- antagonize protein kinase C activity (Schatzman et al., 1981; tracellular calcium was absent, the potentiating effect of AVP Wrenn et al., 1981; Nishizuka, 1984). We therefore undertook was lost (Fig. 3). In contrast, replacement of extracellular cal- experiments using the phorbol ester PDBu to test the effect of cium restored the AVP-induced potentiation (Fig. 3). Further protein kinase C activation upon NE-induced CAMP accumu- The Journal of Neuroscience, March 1989, 9(3) 755

no.0 mN ca++ N 0.E 111 cat+ IO - l- m2.5 IN Ca’*

IO -

iO- - NE NE + AW NE NE + AW NE NE + AW NE t AVP NE t AWt ! 1d ICbl .TVr IMel AVP Figure 3. Calcium dependency of AVP neuromodulation of NE-induced accumulation of CAMP in the hippocampus. In the absence of Figure 2. V,-receptor antagonist peptide d(CH,),Tyr(Me)AVP antag- extracellular calcium, NE-induced accumulation of CAMP is unaffected onizes the potentiating effect of AVP upon NE-induced accumulation while the potentiating effect of AVP is lost. In the presence of 0.8 mM of CAMP in the hippocampus. NE concentration, 10 PM; AVP concen- calcium, the neuromodulatory action of AVP is restored. In contrast, tration, 250 nM; d(CH,),Tyr(Me)AVP concentration, 100 nM. Data were in the presence of 2.5 mM calcium, AVP modulation is inhibitory upon analyzed by 1-way ANOVA followed by a Newman-Keuls test, ** = p NE-induced accumulation of CAMP. NE concentration, 10 PM; AVP i 0.01 vs. NE alone; * = p < 0.05 vs. NE + AVP. concentration, 250 nM. Data were analyzed using a paired Student’s t test, * = p < 0.05. lation. In the presenceof 100nM PDBu and without NE, CAMP antagonistof V, receptors,which implicatesthe phosphoinositol accumulation increasedapproximately 2-fold over basallevels pathway. A calcium-related mechanismwas suggestedby a de- [CAMP in pmol/mg protein + SEM (n); basal = 39 + 5 (3); pendency of the AVP potentiation upon extracellular calcium. PDBu = 88 + 17 (3)]. AT 1 PM PDBu, CAMP accumulation Moreover, the potentiation induced by AVP wasblocked by the increased3-fold over basal levels (Fig. 5). Finally, CAMP ac- calmodulin and protein kinase C antagonist TFP. The main cumulation induced by the simultaneouspresence of both PDBu findings described in the present report in the hippocampus, and NE was essentiallyequal to the summation of CAMP ac- that of AVP potentiation and the calcium dependencyof AVP- cumulation in responseto NE alone plus PDBu alone (Fig. 5). induced neuromodulation, are consistent with those recently Thus, PDBu doesnot mimic the effects of AVP, in that it ac- reported for AVP modulation of NE-induced CAMP accumu- tivates the CAMP responseboth in the absenceof and in the lation in isolated rat superior cervical ganglion (Petit et al., presenceof NE. 1988). Related data and major issuesraised by these findings are discussedbelow. Discussion

The presentseries of experimentsdemonstrates that vasopressin 206 1 exerts a modulatory effect upon beta-adrenergic receptor-in- r-Jo.0 a Cl’+ duced accumulation of CAMP in hippocampal slicesfrom rat Ea0.B BN ca++ brain. In addition, the potentiation by AVP was biphasic and specific to vasopressin,as neither oxytocin nor the metabolite P 1% peptide AVP, mimicked the effect of AVP in the 1O-l 000 nM 5 concentration range. The AVP potentiation was blocked by an Y 7s aJ E Table 3. Effect of propranolol on AVP-induced neuromodulation and : 10( effect of AVP upon forskolin stimulated adenylate cyclase in the IF hippocampus

cAMP/mg protein Condition above basal (pmol) 5( AW BAY K AW + AW BAV K AW + A. NE 127 * 17 (5) BAY K BAY K B. NE + AVP 187 * 15” (5) Figure 4. Effect of AVP and BAY K 8644 upon NE-induced accu- C. NE + AVP + propranolol 30 * lob (5) mulation of CAMP in 0.0 and 0.8 mM calcium. All conditions occurred D. Forskolin 157 * 11 (3) in the presence of 10 IBM NE to stimulate adenylate cyclase activity. In E. Forskolin + AVP 168 & 21 (3) the presence of 0.0 mM Ca2+, 2.6 mM MgCl, neither 250 nM AVP nor 5 NM BAY K 8644 singly or in combination affected CAMP accumulation NE concentration, 10 PM; AVP concentration, 250 no; propanolol concentration, induced by NE. In the presence of 0.8 mM Cal+, AVP potentiation was 10 ELM; forskolin, 10 pm Values are means * SEM (n). Basal CAMP levels were restored. BAY K 8644 was again ineffective when present singly but 79 t 6 (5) for propranolol experiments and 76 i 16 (3) for forskolin experiments. significantly enhanced the potentiating effect of AVP when both were Statistical analysis was by l-way ANOVA, followed by a Newman-Keuls test. present in the incubation medium. Data were analyzed by l-way AN- “p 4 0.05 vs. NE alone. OVA followed by a Newman-Keuls test, * = p < 0.05 vs. NE alone, n “P < 0.05 vs. NE + AVP. = 5 in each condition. 756 Brinton and McEwen * Vasopressin Neuromodulation in Hippocampus

350 Table 4. Effect of trifluoperazine (TFP) upon AVP-induced neuromodulation in the hippocampus l- cAMP/mg protein Percent Condition (pm4 of NE A. Basal 56 k 7 (10) 27 B. NE 205 k 19 (5) 100 C. NE + AVP 291 -t 6 (5) 142 D. NE + TFP 220 k 12 (5) 107 E. NE + AVP + TFP 229 -t 11 (5)” 112 NE concentration, 10 PM; AVP concentration, 250 tm; TFP concentration, 50 wc~. Values represent means + SEMs (n). Statistical analysis was by 1-way ANOVA followed by a Newman-Keuls test. a p < 0.01 vs. NE alone. “p < 0.01 vs. NE + AVP. bSl1 NE I POW NEtkwJ

Figure 5. Effect of phorbol 12, 13-dibutyrate, PDBu, upon CAMP accumulation in the hippocampus. PDBu alone induces a 2-fold increase A VP modulation of beta-adrenergic-stimulatedadenylate over basal accumulation of CAMP. When NE and PDBu were present in combination, NE/PDBu, CAMP accumulation was additive to the cyclase sum of NE alone plus PDBu alone minus basal (since basal CAMP Propranolol significantly blocked the CAMP accumulationwhich accumulation has contributed twice under the arithmetic addition con- occurred in responseto both NE and NE + AVP, suggesting dition and onlv once to the condition where NE and PDBu were both the involvement of beta-adrenergicreceptors. This hypothesis present during-the incubation). NE concentration, 10 PM; PDBu concentration, 1 FM. is supported by the findings of Petit et al. (1988), who found that in the superior cervical ganglion AVP significantly potentiated the CAMP accumulation in responseto the specificbeta- agonistisoproteronol. Theseinvestigators also found that phen- Dose-responseand peptide specijicity tolamine, an alpha-adrenergicantagonist, had no effect upon The biphasic character of vasopressinaction is not unique to CAMP accumulation induced by NE nor upon the AVP poten- its neuromodulatory effect. An inverted-U shapedose relation- tiation of NE-induced CAMP accumulation. Taken together, ship has also been observed in the ability of AVP to induce thesedata support the hypothesisthat AVP modulatesthe CAMP neurite outgrowth in embryonic neurons growing in culture accumulation induced by beta-receptor activation. (Brinton and Gruener, 1987). Lower concentrations of AVP enhancedneurite outgrowth, while higher concentrationsmark- Possiblesynaptic localization of A VP neuromodulation within edly inhibited neuritic extension in culture. the hippocampus The potentiating effect of AVP on NE-induced accumulation Within the hippocampus, AVP neuromodulation of NE-in- of CAMP was specificto vasopressinand not mimicked by oxy- duced CAMP accumulation may occur most prominently in the tocin or by the metabolite peptide AVP,. Theseresults do not dentate gyrus. This postulate is basedon the observation that preclude oxytocin or AVP, from having AVP-like effects at binding sitesfor the V, antagonist which blocked the AVP po- higher concentration. The inability of the metabolite peptide tentiation are localized to the dentate gyrus and do not occur AVP,,, which has been shownto be behaviorally more potent in other regionsof the hippocampus(van Leeuwenet al., 1986; than AVP (Burbach et al., 1983; DeWied et al., 1984) to mod- R. E. Brinton, H. I. Yamamura, and B. S. McEwen, unpublished ulate NE-induced CAMP accumulation in a concentration range observation). The localization of antagonist binding sites, to- at which AVP itself is effective impliesthat modulation of CAMP gether with the presenceof AVP binding sitesin Ammon’s horn accumulation may not be the basisfor theseeffects which are and the dentate gyrus, implies heterogeneity of putative AVP common to both peptides.There is evidencethat AVP,_, binding receptors in the hippocampal formation. sitesand putative AVP receptors are found in different places; Is the effect of AVP on NE-induced accumulation of CAMP i.e., putative AVP receptorsare found in pyramidal cell layers a pre- or postsynaptic event? The initial studiesof Kovacs et and dentate gyrus of the hippocampus (Biegon et al., 1984; al. (1979a) demonstrated for the first time that the behavioral Brinton et al., 1984), where specificbinding for the metabolite actions of AVP dependedon the presenceof NE by showing peptide is absent. Instead, recognition sites for AVP,_, are de- that lesions of the dorsal noradrenergic bundle abolished the tected only in the hilus of the dentate gyrus (Brinton et al., 1986). behavioral effectsof AVP. These studiesdid not, however, es- Thus, the similarity in the behavioral effects of AVP and its tablish a pre- or postsynaptic site of action. Subsequentwork, metabolite AVP,-, may not be due to a convergence upon a including the present studies, has supported a postsynaptic common biochemical processbut rather to a convergenceupon mechanism.First and foremost, AVP doesnot by itself increase the sameanatomical substrate.Such a casecould be envisaged CAMP accumulation. A presynaptic regulation of NE release where AVP acted directly to influence dentate granule cell ac- would be evidenced by an increasein CAMP accumulation in tivity, whereas AVP,_, acted indirectly upon dentate granule responseto AVP alone. The lack of effect of AVP alone upon cells via a feedforward circuit between the polymorph cells of CAMP accumulation has been reported in hippocampal slices the hilar region and the granule cells of the dentate gyrus (Bayer, (this study; Church, 1983; Newman, 1985), as well as in the 1985). Such a postulate is consistent with the different distri- superior cervical ganglion (Petit et al., 1988). Moreover, direct bution of recognition sitesfor AVP and its metabolite peptide, measurementsindicate that AVP doesnot alter uptake or release AVP,-,. in hippocampal slices (Hagan and Balfour, 1983). Supportive The Journal of Neuroscience, March 1989, 9(3) 757 evidence from autoradiography indicates that 3H-AVP binding and that TFP inhibited AVP-induced potentiation, favors the sites are not lost or diminished following 6-hydroxydopamine involvement of a calcium/calmodulin-dependent mechanism. lesions of the dorsal noradrenergic bundle which markedly re- The other limb of the PI pathway involves generation of the duce NE levels in the hippocampus (R. E. Brinton, Th. A. Voor- metabolite, IP, (Berridge and Irvine, 1984). If V,-receptor ac- huis, and E. R. DeKloet, unpublished observations). tivation of PI metabolism is involved in AVP potentiation in the hippocampal slice, then how might this mechanism operate Involvement of V,-type receptors in A VP potentiation in relation to the dependency on external calcium? One possi- Experiments with a peptide antagonist of the V, receptor, bility is that the primary actions of PI metabolites such as IP,, d(CH,),Tyr(Me)AVP (Kruszynski et al., 1980) which has also which cause mobilization of internal CaZ+ stores, is dependent been found to block the behavioral effects of AVP (LeMoal et upon or regulated by external Ca*+ levels. Thus, a reduction in al., 198 1; DeWied et al., 1984) that are dependent on NE (Ko- external Ca2+ could reduce the amount of mobilized Ca2+ avail- vats et al., 1979a), indicate that a V,-type of receptor is involved able to internal compartments, where, for example, adenylate in the AVP effects on NE-induced CAMP accumulation. Because cyclase activation occurs. This possibility is supported by the V, receptors involve activation of phosphoinositol (PI) metab- data of Kirk et al. (1981) and LeBoff et al. (1986) who found olism in the periphery (Bone et al., 1984; Hanley et al., 1984; that mobilization of internal stores of Ca*+ was influenced by Kiraly et al., 1986) and in the hippocampus (Stephens and Lo- CaZ+ levels in the external medium. However, in these systems gan, 1986) it is likely that potentiating effects of AVP on NE- AVP-induced PI turnover does not decline to zero in the absence induced CAMP accumulation may involve all or part of this of external Ca*+. This is in contrast to AVP-induced neuromodu- pathway. A similar inference has been made for the ability of lation in the hippocampus (this report) and in the superior cer- AVP in the anterior pituitary to activate PI metabolism through vical ganglion (Petit et al., 1988) and is also in contrast to AVP V,-like receptors (Raymond et al., 1985) and to potentiate the activation of glycogen phosphorylase in liver (Blackmore et al., CAMP response to corticotrophin-releasing factor (Giguere and 1978). In each of these latter instances, the absence of Ca2+ in Labrie, 1982). the external medium results in a loss of AVP-induced effects. Thus, there appear to be events dependent on V,-receptor ac- Possible mechanisms of calcium dependency of A VP-induced tivation in both the periphery and the CNS which are distin- neuromodulation guished by their relative versus absolute requirement upon ex- The AVP modulation of NE-induced CAMP accumulation is ternal calcium. dependent on Ca2+ ions in the external medium, not only in The dependency on calcium ions in the hippocampal slice surviving hippocampal slices (this study) but also in the isolated was biphasic, in that concentrations up to 1 mM facilitated the surviving rat superior cervical ganglion (Petit et al., 1988). The AVP potentiation effect, whereas a concentration of 2.5 mM no basis for this calcium dependency may be due to an interaction longer facilitated the potentiation. These effects were observed with a pool of calcium/calmodulin-dependent adenylate cyclase in the presence of a phosphodiesterase inhibitor and might (Brostrom et al., 1975). This hypothesis is supported by the therefore represent a direct effect of calcium to inhibit adenylate inhibition of AVP-induced potentiation by the calmodulin an- cyclase allosterically, as has been described (Hanski et al., 1977). tagonist TFP. While the data supports this hypothesis, it is not In order for calcium to have this effect in the presence of AVP, proved by the TFP effect since TFP has been found to antagonize whereas it did not inhibit CAMP accumulation with NE alone, protein kinase C (Schatzman et al., 1981; Wrenn et al., 1981). it is necessary to postulate an effect of AVP that increases access However, the present experiments revealed an important dis- of internal pools to external calcium concentrations. tinction between the effects of AVP and those of protein kinase These findings collectively raise questions regarding possible C stimulation by the phorbol ester PDBu, namely, that AVP additional factors that determine Ca2+ flux between the external alone did not induce CAMP accumulation, whereas PDBu did. environment and internal pools. In the present study, experi- This result might be explained by the observations of Nichols ments in which Ca2+ flux was manipulated by the calcium chan- et al. (1986) who found that PDBu stimulates the release of NE nel activator BAY K 8644 showed that this compound alone and other neurotransmitters. In light of these findings and the had no effect upon NE-induced CAMP accumulation. However, present data, the additivity of PDBu and NE upon CAMP ac- a trend to enhance the AVP potentiation was observed in the cumulation suggests that PDBu action may be due to the release presence of BAY K 8644. It should be noted that BAY K 8644 of neurotransmitters other than NE which stimulate CAMP for- activates Ca2+ flux only when the cell is depolarized (Nowycky mation. Alternatively, PDBu might have an effect via protein et al., 1985). Petit et al. (1988) observed a similar trend to kinase C activation upon adenylate cyclase activity, as has been potentiate NE-induced CAMP accumulation in the superior cer- demonstrated in some other systems (Cronin and Canonico, vical ganglion using the calcium ionophore A23 187. Thus, we 1985; Nabika et al., 1985; Sugden et al., 1985; Abou-Samra et conclude, albeit cautiously, that the weight of evidence favors al., 1987). Regardless of which possibility may apply, the ad- an AVP-stimulated calcium entry mechanism, potentially in- ditivity of PDBu and NE upon CAMP accumulation in hippo- volving AVP activation of calcium channels. More work on this campal slices suggests an activation of 2 separate compartments topic is clearly required and might best be carried out in cultured of adenylate cyclase, whereas the synergy of AVP with NE sug- cells in which survival is not as great a problem as in the hip- gests a convergence upon the same compartment. In the superior pocampal slice. Such a postulate is supported by recent studies cervical ganglion, where AVP also potentiates NE-induced CAMP in sympathetic ganglion cultures showing that AVP increases accumulation in a calcium-dependent fashion, the addition of channel opening (Matsumoto et al., 1987). a different protein kinase C activator, TPA, had no effect upon In summary, in the hippocampal slice, vasopressin poten- NE-induced CAMP accumulation (Petit et al., 1988). Therefore, tiates the accumulation of CAMP in response to NE in a manner the weight of the evidence based upon the fact that activation that is dependent on calcium in the external medium and that of protein kinase C by PDBu did not mimic the effect of AVP, is blocked by the calmodulin antagonist TFP under conditions 758 Brinton and McEwen * Vasopressin Neuromodulation in Hippocampus in which protein kinase C involvement is a less likely alternative. Browning, M., R. Huganir, and P. Greengard (1985) Protein phos- The weight of evidence supports a postsynaptic V, receptor, phorylation and neuronal function. J. Neurochem. 45: 1 l-23. which is probably involved in activating phosphoinositol me- Burbach, J. P., and J. L. Lebouille (1983) Proteolytic conversion of arginine-vasopressin and oxytocin by brain synaptic membranes. J. tabolism. At this stage, in order to account for the observed Biol. Chem. 258: 1487-1494. dependence on external CaZ+, it can only be speculated that V,- Burbach, J. P., G. Kovacs, D. DeWied, J. W. van Nispen, and H. M. receptor activation somehow leads to enhanced Ca2+ access and Greven (1983) A major metabolite of arginine vasopressin in the entry, as well as stimulation of PI turnover. Modulating CAMP brain is a highly potent neuropeptide. Science 221: 13 10-l 3 12. Cheung, W. Y. (1980) Calmodulin plays a pivotal role in cellular accumulation via a calcium dependent step provides a pathway regulation. Science 207: 19-27. whereby it is possible to influence both CAMP-dependent and Church, A. C. (1983) Vasopressin potentiates the stimulation of cyclic calcium/calmodulin-dependent protein kinases (see Browning AMP accumulation by norepinephrine. Peptides 4: 262-263. et al., 1985; Cohen, 1985; McGuinness et al., 1985). The ca- Cohen, P. (1985) The role of protein phosphorylation in the hormonal control of enzyme activity. Eur. J. Biochem. 151: 439448. pacity to potentially influence both classes of protein kinases, Cronin. M.. and P. L. Canonico (1985) Tumor nromoters enhance and thus their associated substrates, could impact significantly basal and growth hormone releasing factor stimulated cyclic AMP upon the functional correlates of the hippocampus, including levels in anterior pituitary cell. Biochem. Biophys. Res. Commun. those involved with mnemonic processes. 129: 404410. DeKloet, E. R., F. Rotteveel, Th. A. Voorhuis, and M. Terlou (1985) Topography of binding sites for neurohypophyseal hormones in rat References brain. Eur. J. Pharmacol. 110: 113-l 19. Abou-Samra, A.-B., J. P. Harwood, V. C. Manganiello, K. J. Catt, and DeWied, D. (197 1) Long term effect of vasopressin on the maintenance G. Aguilera (1987) Phorbol 12-myristate 13-acetate and vasopressin of a conditioned avoidance response in rats. Nature 232: 58-60. potentiate the effect of corticotropin-releasing factor on cyclic AMP DeWied, D. (1984) Neurohypophyseal hormone influences on learning production in rat anterior pituitary cells. J. Biol. Chem. 262: 1129- and memory processes. In Neurobiology of Learning and Memory, 1136. G. Lynch, J. McGaugh, and N. M. Weinberger, eds., pp. 289-312, Audigier, S., and C. Barberis (1985) Pharmacological characterization Guildford, New York. of two specific binding sites for neurohypophyseel hormones in hip- DeWied, D., 0. Gaffori, J. M. van Ree, and W. de Jong (1984) Central pocampal synaptic plasma membranes of the rat. EMBO J. 4: 1407- target for the behavioural effects of vasopressin neuropeptides. Nature 1412. 308: 276-278. Bayer, S. A. (1985) Hippocampal region. In The Rat Nervous System, Giguere, V., and F. Labrie (1982) Vasopressin potentiates cyclic AMP G. Paxinos, ed., pp. 335-3521 Academic, New York. accumulation and ACTH release induced by corticotropin-releasing Berridae. M. J.. and R. F. Irvine (1984) Inositol trisnhosnhate. a novel factor (CRF) in rat anterior pituitary cells in culture. Endocrinology secoid messenger in cellular signal transduction. Nature 3i2: 3 15- 111: 1752-1754. 321. Gilman, A. G. (1970) A protein binding assay for adenosine 3’5’- Biegon, A., M. Terlou, Th. D. Voorhuis, and E. R. DeKloet (1984) cyclic monophosphate. Proc. Natl. Acad. Sci. USA 67: 305-3 12. Arginine-vasopressin binding sites in rat brain: A quantitative au- Hagan, J. J., and D. J. Balfour (1983) Lysine vasopressin fails to alter toradiographic study. Neurosci. Lett. 44: 229-234. ^ (‘H)-noradrenaline uptake or release from hippocampal tissue in vi- Blackmore, P. F., F. F. Brumley, J. L. Marks, and J. H. Exton (1978) tro. Life Sci. 32: 25 17-2522. Studies on alpha adrenergic activation of hepatic glucose output. J. Hanley, M. R., H. P. Benton, S. L. Lightman, K. Todd, E. A. Bone, P. Biol. Chem. 253: 4851-4858. Fretten, S. Palmer, C. J. Kirk, and R. H. Mitchell (1984) A vaso- Bone. E. A.. P. Fretten. S. Palmer. C. J. Kirk. and R. H. Mitchell (1984) pressin-like peptide in the mammalian sympathetic nervous system. Rapid accumulation of inositol phosphates in isolated rat superior Nature 309: 258-26 1. cervical sympathetic ganglia exposed to V,-vasopressin and muscarin- Hanski, E., N. Sevilla, and A. Levitzki (1977) The allosteric inhibition ic cholinergic stimuli. Biochem. J. 221: 803-8 11. by calcium of soluble and partially purified adenylate cyclase from Bradford, M. (1976) A rapid and sensitive method for the quantitation turkey erythrocytes. ?? 76: 5 13-520. of microgram quantities of protein utilizing the principle of protein- Jard, S. (1983) Vasopressin isoreceptors in mammals: Relation to dry binding. Anal. Biochem. 72: 248-254. cyclic AMP-dependent and cyclic AMP-independent transduction Brinton, R. E. (1987) Common origins of cellular communication in mechanisms. Current Topics Membr. Transp. 18: 255-285. the immune, endocrine and nervous systems. In The Neuro-lmmune- Kiraly, M., S. Audigier, E. Tribollet, C. Barberis, M. Dolivo, and J. J. Endocrine Connection, C. Cotman et al., eds., pp. 49-57, Raven, New Dreifuss (1986) Biochemical and electrophysiological evidence of York. functional vasopressin receptors in the rat superior cervical ganglion. Brinton, R. E., and R. P. Gruener (1987) Vasopressin promotes neurite Proc. Natl. Acad. Sci. USA 83: 5335-5339. growth in cultured embryonic neurons. Synapse 1: 329-334. Brinton, R. E., and B. S. McEwen (1985) Vasopressin-induced neu- Kirk, C., R. Mitchell, and D. Hems (198 1) Phosphatidylinositol me- ronal mechanisms in the hippocampus. Sot. Neurosci. Abstr. 11: tabolism in rat hepatocytes stimulated by vasopressin. Biochem. J. 104.2. 194: 155-165. Brinton, R. E., and B. S. McEwen (1986) Vasopressin neuromodu- Kovacs, G., B.. Bohus, and D. H. Versteeg (1979a) Facilitation of lation in the hippocampus: Calcium-calmodulin or protein kinase C? memory consolidation by vasopressin: Mediation by terminals of the Sot. Neurosci. Abstr. 12: 223.13. dorsal noradrenergic bundle? Brain Res. 172: 73-85. Brinton, R. E., K. W. Gee, J. K. Wamsley, T. P. Davis, and H. I. Kovacs, G., B. Bohus, D. H. Versteeg, E. R. DeKloet, and D. DeWied Yamamura (1984) Regional distribution of putative vasopressin (1979b) Effect ofoxytocin and vasopressin on memory consolidation: receptors in rat brain and pituitary by quantitative autoradiography. Sites of action and catecholaminergic correlates after local microin- Proc. Natl. Acad. Sci. USA 81: 7248-7252. jection into limbic midbrain structures. Brain Res. 175: 303-3 14. Brinton, R. E., D. R. Gehlert, J. K. Wamsley, Y. P. Wan, and H. I. Kruszynski, M., B. Lammek, and M. Manning (1980) [l(Beta-mer- Yamamura (1986) Vasopressin metabolite, AVP,, binding sites in capto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-meth- brain: Distribution distinct from that of parent peptide. Life Sci. 38: yl)tyrosine]arginine-vasopressin and [ l-(beta-mercapto-beta,beta-cy- 443452. clopentamethylenepropionic acid)]arginine-vasopressin, two highly Brostrom, C., Y.-C. Huang, B. Breckenridge, and D. J. Wolff (1975) potent antagonists of the vasopressor response to arginine-vasopres- Identification of a calcium-binding protein as a calcium-dependent sin. J. Med. Chem. 23: 364-368. regulator of brain adenylate cyclase. Proc. Natl. Acad. Sci. USA 72: LeBoff, M. S., J. Chang, M. Henry, D. Beaudoin, L. Swiston, and E. 64-68. Brown (1986) Role of cytosolic calcium in the control of CAMP Brostrom, M., C. Brostrom, B. Breckenridge, and D. J. Wolff (1978) content by calcium in bovine parathyroid cells. Mol. Cell. Endocrinol. Calcium-dependent regulation of brain adenylate cyclase. Adv. Cyclic 45: 127-135. Nucleotide Res. 9: 85-99. LeMoal, M., G. F. Koob, L. Y. Koda, F. E. Bloom, M. Manning, W. The Journal of Neuroscience, March 1989, 9(3) 759

H. Sawyer, and J. Rivier (1981) Vasopressin receptor antagonist Petit, P., C. Barberis, and S. Jard (1988) Vasopressin potentiates the prevents behavioral effects of vasopressin. Nature 291: 49 l-493. noradrenaline-induced accumulation ofcyclic AMP in the rat superior Levin, R., and B. Weiss (1977) Binding of trifluoperazine to the cal- cervical ganglion. Brain Res. 440: 299-304. cium-dependent activator of cyclic nucleotide phosphodiesterase. Mol. Raymond, V., P. K. Leung, R. Veilleux, and F. Labrie (1985) Vaso- Pharmacol. 13: 690-697. pressin rapidly stimulates phosphatidic acid-phosphatidyl inositol Matsumoto, S., D. L. Kreulen, and R. Gruener (1987) Dissociated turnover in rat anterior pituitary cells. FEBS Lett. 18: 196-200. cell cultures of guinea pig prevertebral ganglia: Physiological and cy- Schatzman, R., B. C. Wise, and J. F. Kuo (1981) Phospholipid-sen- tochemical characteristics. Sot. Neurosci. Abstr. 13: 19 1.3. sitive calcium-dependent protein kinase: Inhibition by anti-psychotic McGuinness, T., Y. Lai, C. Ouimet, and P. Greengard (1985) Calcium/ drugs. Biochem. Biophys. Res. Commun. 98: 669-676. calmodulin-dependent protein phosphorylation in the nervous sys- Schramm, M., C. Tomas, R. Toward, and C. Franckowiak (1983) tem. In Calcium in Biological Systems, R. Rubin, G. Weiss, and J. Novel dihydropyridines with positive inotropic action through acti- Putney, eds., pp. 291-305, Plenum, New York. vation of Ca channels. Nature 303: 535-537. Mizuno, Y., Y. Oomura, N. Hoei, and D. Carpenter (1984) Action of Seamon, K. B., and J. W. Daly (1986) Forskolin: Its biological and vasopressin on CA1 pyramidal neurons in rat hippocampal slices. chemical properties. Adv. Cyclic Nucleotide Protein Phosphorylation Brain Res. 309: 241-246. Res. 20: 2-150. Moore, R. Y., and F. E. Bloom (1979) Central catecholamine neuron Stephens, L. R., and S. D. Logan (1986) Arginine-vasopressin stim- systems: Anatomy and physiology of the norepinephrine and epi- ulates inositol phospholipid metabolism in rat hippocampus. J. Neu- nephrine systems. Annu. Rev. Neurosci. 2: 113-168. rochem. 46: 649-65 1. Muhlenthaler, M., J. Dreifuss, and B. Gahwiler (1982) Vasopressin Strupp, B. J., and D. Levitsky (1985) A mnemonic role for vasopressin: excites hippocampal neurons. Nature 296: 749-75 1. The evidence for and against. Neurosci. Biobehav. Rev. 9: 399-4 11. Nabika, T., Y. Nara, Y. Yamori, W. Lovenberg, and J. Endo (1985) Sugden, D., J. Vanecek, D. Klein, T. Thomas, and W. Anderson (1985) Angiotensin II and phorbol ester enhance isoproterenol- and vaso- Activation of protein kinase C potentiates isoprenaline-induced cyclic active intestinal peptide (VIP)-induced cyclic AMP accumulation in AMP accumulation in rat pinealocytes. Nature 314: 359-363. vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 131: Treisman. G. J.. S. Baalev. and M. E. Gneav (1983) Calmodulin- 30-36. sensitive and calmoduiin~insensitive components of adenylate cyclase Newman, M. (1985) Vasopressin inhibits cyclic AMP accumulation activity in rat striatum have differential responsiveness to guanyl and adenylate cyclase activity in cerebral preparation. FEBS Lett. nucleotides. J. Neurochem. 41: 1398-1406. 181: 203-206. van Leeuwen, F., E. v. d. Beek, J. van Heerikhiuze, and Y. Wan (1986) Nichols, R., J. W. Haycock, J. K. Wang, and P. Greengard (1986) Light microscopic autoradiographic localization of vasopressin pres- Phorbol ester enhancement of neurotransmitter release from rat brain sor antagonist binding sites in the rat brain, pituitary and kidney. Sot. synaptosomes. J. Neurochem. 48: 6 15-62 1. Neurosci. Abstr. 12: 229.7. Nishizuka, Y. (1984) The role of protein kinase C in cell surface signal Wrenn, R., N. Katoh, R. Schatzman, and J. F. Kuo (1981) Inhibition transduction and tumour promotion. Nature 308: 693-698. by phenothiazine antipsychotic drugs of calcium-dependent phos- Nowycky, M. C., A. P. Fox, and R. W. Tsien (1985) Three types of phorylation of cerebral cortex proteins regulated by phospholipid or neuronal calcium channel with different calcium agonist sensitivity. calmodulin. Life Sci. 29: 725-733. Nature 316: 440-446.