Arginine vasopressin induces the expression of c-fos in the mouse septum and hippocampus

Molecular Brain Research, 7 (1990) 131-137 Elsevier

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BRESM 70183

Arginine vasopressin induces the expression of c-fos in the mouse septum and hippocampus R Rathna Giri, Jitendra R. Dave, Boris Tabakoff and Paula L. Hoffman Division of Intramural Clinical and Biological Research, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892 (U.S.A.) (Accepted 22 August 1989)

Key words: Arginine vasopressin; c-los mRNA level; Septal V t receptor; Nerve growth factor; Oxytocin; Hippocampal V 1 receptor

Arginine vasopressin is a neuropeptide that has been shown to modulate functional ethanol tolerance and memory processes. These actions of vasopressin in the CNS have been shown by us and others to be mediated by V 1 receptors. Intracerebroventricular injection of vasopressin in mice resulted in a substantial increase in mRNA for the proto-oncogene c-los in septum and hippocampus, but no increase in cerebral cortex. A Vl-selective agonist also increased septal c-los mRNA levels, while a VE-selective agonist was less effective. Similarly, the response to vasopressin was more effectively blocked by a V 1- than a V:selective antagonist. These results indicate that vasopressin acts specifically at V 1 receptors in mouse septum and hippocampus to increase c-los mRNA. The vasopressin metabolite, AVP(4-9), also increased c-los mRNA levels in septum and hippocampus, while the response to oxytocin, which has different effects from vasopressin on memory and tolerance, was greater in hippocampus than in septum. Nerve growth factor, in contrast to the other peptides, had a more pronounced effect on c-fos mRNA levels in cerebral cortex than in the other brain areas. Increased c-los expression has been hypothesized to play a role in neuroadaptation, and these results suggest that modulation of septal c-los expression could be important for vasopressin effects on ethanol tolerance and/or memory. INTRODUCTION T h e n e u r o p e p t i d e arginine vasopressin (AVP) has been shown to maintain functional tolerance to ethanol, once that tolerance has been established 17"35. This effect of A V P is m e d i a t e d by V l - t y p e vasopressin receptors in the CNS 35. The ability of A V P to facilitate m e m o r y consolidation 39 also a p p e a r s to be m e d i a t e d by CNS V1 vasopressin receptors 9'13. A l t h o u g h the anatomical localization of receptors involved in the effects of A V P on n e u r o a d a p t i v e processes such as tolerance and m e m o r y has not been definitively d e t e r m i n e d , a high density of V : t y p e vasopressin receptors has been d e m o n s t r a t e d , by ourselves 18 and others 28'38, in the lateral septum. Such receptors are also present in the h i p p o c a m p u s 28'3s. The biochemical mechanism by which A V P can effect long-term n e u r o a d a p t i v e changes is an intriguing question. O n e possible means is suggested by a n o t h e r r e p o r t e d action of the peptide: A V P was found to p r o d u c e kindling when administered intracerebroventricularly (i.c.v.). A l t h o u g h this finding is somewhat controversial 5, several studies have shown that, while the first injection of the p e p t i d e had no discernible behavioral effect, A V P induced seizures after r e p e a t e d injections 4'2°. Kindling-induced seizures 11, as well as o t h e r types of seizure 19'26, have b e e n d e m o n s t r a t e d to be

associated with increased expression of the proto-oncogene, c-los, in the adult CNS. It has also b e e n suggested that c-los expression m a y be i m p o r t a n t for the synaptic differentiation that is p r e s u m a b l y involved in n e u r o a d a p tive processes such as learning, m e m o r y or functional tolerance 14'27. A biochemical factor involved in enhanced c-los expression in certain cells is an increase in intracellular calcium levels 27, and the action of A V P at the V 1 vasopressin r e c e p t o r is characterized by increases in phosphatidylinositol t u r n o v e r and subsequent increases in intracellular calcium levels 24'37. These d a t a suggested that vasopressin might affect the expression of c-los in the CNS, an action that could play a role in the ability of A V P to m o d u l a t e n e u r o a d a p t i v e processes. In the present study, we have investigated the effect of A V P on c-los expression when the p e p t i d e is injected in vivo, under conditions similar to those used to d e m o n s t r a t e the maintenance of ethanol tolerance by A V P 35.

MATERIALS AND METHODS Arginine vasopressin (AVP) and [1-(fl-mercapto-fl,fl-cyclopentamethylenepropionic acid), 2-(OEt)Tyr, 4-Val]arginine vasopressin [d(CH2)sTyr(Et)AVP ] (a V:selective antagonist) were obtained from Bachem, Inc. (Torrance, CA). The V:selective agonist, [2-Phe,3-Ile,8-Orn]vasopressin ([2-Phe,8-Orn]VT), the V2-selective agonist, [1-fl-mercaptopropionic acid, 4-Val,8-D-Arg]vasopressin

Correspondence: P.L. Hoffman, National Institute on Alcohol Abuse and Alcoholism, 12501 Washington Avenue, Rockville, MD 20852, U.S.A. 0169-328X/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

132 (dVDAVP) and a V2-selective antagonist, [1-(fl-mercapto-fl,flcyclopentamethylenepropionic acid), 2-D-Ile,4-Ile]arginine vasopressin (d(CH2)5[2-D-IIe,4-Ile]AVP), were generously supplied by Dr. Maurice Manning (Dept. of Biochemistry, Medical College of Ohio, Toledo, OH). Oxytocin was from the laboratory of the late Dr. Roderich Walter (University of Illinois at Chicago, Chicago, IL). The AVP metabolite [4-pGlu,6 Cyt]AVP(4-9) (AVP(4-9)) was purchased from Peninsula Labs (Belmont, CA). Nerve growth factor (NGF) was purchased from Collaborative Research, Inc. (Bedford, MA). All other chemicals were of the highest purity available.

Peptide treatment Male C57BL/6NCR mice (20-25 g) were obtained from NCI, Frederick, MD and were housed 4 per cage under conditions of controlled temperature and lighting (12 h light-dark cycle) for at least 1 week prior to being used in experiments. Mice were implanted with i.c.v, cannulae under pentobarbital anesthesia as described previously35, and were then housed individually for a 3-day postoperative recovery period. Animals were handled daily to reduce stress during the experimental procedure. Randomly selected animals were checked for the placement of cannulae by injection of 10/~1 of Methylene blue dye and, after sacrifice, by investigation of the spread of the dye in the ventricles. Each experiment included groups (4 mice/group) of naive mice (uncannulated, untreated), control mice (i.c.v. injections of artificial CSF vehicle)35 and peptide-treated mice. The injection volume was 2/~1, and peptides were administered at the doses indicated in the Results section. When peptide antagonists were used to block the response to AVP, both peptides were administered simultaneously in the 2 ktl injection volume. Mice were sacrificed by decapitation 15 min after drug or vehicle injection (with the exception of the time course experiments) and naive mice were also sacrificed by decapitation. Brains were rapidly frozen in isopentane on dry ice. After partial thawing, brain areas were dissected using a brain slicer. The septal area was dissected bilaterally from a 1.2 mm-thick brain slice (coordinates: bregma to 1.2 mm anterior to bregma) 32. This area, comprising primarily lateral septum, was delineated laterally by the lateral ventricles, and ventrally by a cut just above the anterior commissure2s'32. The other brain areas (cortex and hippocampus) were dissected bilaterally from a 1.5-mm-thick brain slice (coordinates: 1.0 to 2.5 mm posterior to bregma) 32. All tissues were kept frozen at -70 °C, and brain areas from 4 mice in each group were pooled for RNA extraction.

M NaCI and 0.015 M trisodium citrate, pH 7), 40 mM NaHzPO4, pH 6.5, 200/~g/ml of tRNA (Boehringer-Mannheim, Indianapolis, IN), 50 ktg/ml of herring sperm DNA (Boehringer-Mannheim), 0.8x Denhardt's solution, 10 mM EDTA and 0.1% SDS. Hybridization was carried out overnight at 42 °C in a solution of prehybridization buffer and dextran sulfate (4:1), containing 106 cpm/ml of a 4.8 kb HindIII/BamHI fragment of the mouse c-los gene labeled with 3Zp by nick translation (Lofstrand Labs, Gaithersburg, MD). After hybridization the blots were washed twice with 2× SSC for 10 min at room temperature and twice with 0.1 x SSC containing 0.1% SDS for 30 min at 65 °C. Blots were then exposed for 24 h to Kodak XAR-2 X-ray film. After removal of the c-los probe by washing in 96% formamide solution, the blots were subjected to hybridization with a 2.2 kb EcoRV/HindIII fragment of c-myc and, following a second wash, with a 4.6 kb XhoI/Sphl fragment of c-ras, both labeled with 32p by nick translation (both obtained from Lofstrand Labs). The autoradiograms were quantitated by densitometric scanning on a Gilford spectrophotometer or with an image array processing system (Sierra Scientific High Resolution CCD camera; Macintosh MAC II computer equipped with Data Translation quick-capture frame-grabber board; IMAGE 1.06 software written by W. Rasband, NIMH). Both of these methods gave qualitatively similar results. The integrity of the RNA was assessed and the RNA was quantitated densitometrically from ethidium bromide-stained gels7, and mRNA levels are expressed on the basis of 28S RNA levels. The percent change in c-los mRNA levels in experimental animals was calculated based on the level of c-los mRNA in naive or CSF-treated mice. RESULTS I n t r a c e r e b r o v e n t r i c u l a r a d m i n i s t r a t i o n o f 1 ng o f A V P r e s u l t e d in a s i g n i f i c a n t i n c r e a s e in c-los m R N A levels in septum and h i p p o c a m p u s of C 5 7 B L / 6 N C R mice (percent increase over untreated mice, mean + S.E.M., septum: 466 + 117 (n = 4 e x p e r i m e n t s ) ; h i p p o c a m p u s , 603 + 131 (n = 5 e x p e r i m e n t s ) ; P < 0.02 a n d P < 0.01 (t-test),

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Northern blot analysis RNA was extracted according to the procedure of Cathala et al. 6, with minor modifications. Tissue was homogenized in 5 vol of 5 M guanidine monothiocyanate in 50 mM Tris-HCl, containing 10 mM EDTA and 8% (v/v) fl-mercaptoethanol, pH 7.5. RNA was precipitated with 4 M LiC1 at 4 °C overnight and reprecipitated with 2 M LiCI containing 4 M urea. The precipitate was collected by centrifugation at 11,000 g for 90 rain at 4 °C. RNA was solubilized in 10 mM Tris-HCl containing 1% sodium dodecyl sulphate (SDS) and 1 mM EDTA, pH 7.5, and, after phenol and chloroform extractions, was precipitated with ethanol. The precipitate was suspended in denaturing buffer ( l x gel running buffer (20 mM MOPS, pH 7.0, 5 mM sodium acetate, pH 6.0 and 1 mM EDTA), 50% (v/v) formamide and 2.2 M formaldehyde) and heated at 65 °C for 5 rain. The RNA was then size-fractionated on 1% formaldehyde-agarose gels7'22 containing l x gel running buffer and 2.2 M formaldehyde, run at 70 V for 0.5 h and at 100 V for 4.5 h. After electrophoresis, gels were stained for 15 min in l x gel running buffer containing 2 ~g/ml ethidium bromide, then destained overnight in 1 x gel running buffer and transferred to Gene Screen nylon membranes (NEN-Dupont, Boston, MA) by electroblotting in 25 mM NaHzPO 4, pH 6.5 (15 V, overnight at 4 °C). The filters were rinsed in phosphate buffer and baked at 80 °C for 2 h in a vacuum oven. The blots were incubated for 2 h at 42 °C in a prehybridization buffer consisting of 50% (v/v) formamide, 2x SSC (1 x SSC = 0.15

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Fig. 1. Autoradiogram of Northern blot of mouse brain RNA hybridized with 32p-labelled c-fos probe. C57BL/6 mice were injected with vehicle (CSF, "V') or AVP ('A', 1 ng) and sacrificed 15 rain later. Tissue was also obtained from naive (N), untreated mice. RNA was extracted from the indicated brain areas and subjected to Northern blot analysis as described in the text. Tissue from 4 mice per group was pooled for each brain area. Equal amounts of RNA (10/~g) were applied in each lane.

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log AVP (rig) Fig. 2. Time course (A) and dose-response curve (B) for AVP stimulation of c-los mRNA in mouse septum. C57BL/6 mice were injected i.c.v, with AVP (open circle) or vehicle (artificial CSF (closed circle)) as described in the text. Mice were sacrificed at the indicated times after injection of 1 ng of AVP (A) or at 15 min after injection of AVP (B), and c-los mRNA in the septum was quantitated following Northern blot analysis. Results are presented as the percent increase in c-fos mRNA over the level in naive mice, and represent the mean values from 2 experiments, in which pooled tissue from 4 mice was used to generate each point. In B, the range of the values in the two experiments is indicated by the bars. In the experiments depicted in A, CSF injection produced a mean 53% increase (range, 18-87), measured at 15 min after injection.

respectively, compared to response to CSF in each area (Figs. 1 and 4). AVP had no measurable effect on the level of c-fos m R N A in the cerebral cortex (Fig. 1). Brain levels of c-fos m R N A were low in naive mice, and injection of artificial CSF vehicle had only a slight effect, compared to AVP (percent increase over untreated mice, m e a n + S . E . M . , septum: 111 + 25; hippocampus: 87 + 15 (n = 7 experiments)) (Fig. 1). The characteristics of the AVP-induced increase in c-fos m R N A were further examined in the septal area. Time course studies showed that the response to AVP (1 ng) was transient. In one experiment, there was a

Treatment Fig. 3. Effect of selective V]- and Vz-agonists (A) and antagonists (B) on c-los mRNA in mouse septum. C57BL/6 mice were injected with AVP, agonists, or AVP plus antagonist simultaneously, as described in the text. Mice were sacrificed at 15 min after injection, and c-fos mRNA in the septum was quantitated following Northern blot analysis. Results represent mean values from two experiments (range: CSF, 64-169; AVP, 625-706; [2-Phe,8-Orn]VT, 668-931; dVDAVP, 64-381) (A) or values from one experiment (B), in which tissue from 4 mice was pooled for each group. maximal increase at 15 min after injection, and a rapid return to baseline levels by 30 min. In a second experiment, levels were maximal at 15 and 30 min, and returned more gradually to baseline over 2 - 4 h. The average of the results from the two experiments is shown in Fig. 2A. The reason for the difference in time course in the two experiments is not k n o w n , but may reflect differences in the rate of distribution or removal of AVP in the brain following i.c.v, injection. In any case, the transient nature of the effect is similar to that reported for the effects of other neurotransmitters and peptides in vitro 14'a5'36. Dose-response studies indicated that once the dose of AVP exceeded 10 pg (10 fmol), a maximal increase in c-fos m R N A was evidenced (Fig. 2B). As can be seen in Figs. 2 and 3, the increase in septal c-fos expression in response to AVP ranged from approximately 2.5- to 7-fold in different experiments. Injection of a V~-selective agonist significantly increased c-los m R N A levels in the septum, similar to AVP (percent increase over untreated mice, m e a n + S.E.M., CSF, 99 + 20 (n = 5 experiments); V 1 agonist, 628 + 188 (n = 3 experiments); P < 0.05, t-test) (Fig. 3A). In

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neously, a Vl-selective antagonist blocked the effect of AVP on c-fos m R N A , while a Ve-selective antagonist was less effective (Fig. 3B). The effect of AVP in hippocampus appeared similar to the effect in the septal area in several respects. First, the Vl-selective agonist was more effective than the V 2selective agonist in increasing hippocampal c-los m R N A levels (Fig. 4) (percent increase over untreated mice, range in two experiments, CSF, 0-183; V t agonist (0.1 ng), 671-883; V 2 agonist (1.0 ng), 128-375). Furthermore, the effect of AVP was much more efficiently blocked by the Vt-selective antagonist; this difference was more pronounced than in the septum (Fig. 5). The time course and dose-response curves for AVP stimula-

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Fig. 4. Autoradiogram of Northern blot of hippocampal RNA hybridized with 32p-labeled c-los probe. C57BL/6 mice were injected i.c.v, with the peptides indicated below, sacrificed 15 min later, and R N A was isolated as described in the text. Tissue from 4 mice per group was pooled for each brain area. Equal amounts (10 ug) of R N A were applied in each lane. Treatments: lane 1, naive; lane 2, CSF; lane 3, AVP (1 ng); lane 4, d V D A V P (V 2 agonist) (1 ng); lane 5, [2-Phe,8-Orn]VT(V 1 agonist) (0.1 ng). Integrity and quantity of R N A were determined by ethidium bromide staining as described in the text.

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Fig. 5. Effect of V 1- and V2-selective AVP antagonists on the AVP-induced increase of c-los m R N A in mouse hippocampus. C57BL/6 mice were injected with AVP and antagonist simultaneously, as described in the text. Mice were sacrificed at 15 rain after injection, and c-los m R N A in the hippocampus was quantitated following Northern blot analysis. Results are presented as percent increase in c-fos m R N A over the level in naive mice, and represent values from one experiment in which tissue from 4 mice was pooled for each group.

Fig. 6. Time course (A) and dose-response curve (B) for AVP stimulation of c-fos m R N A in mouse hippocampus. C57BL/6 mice were injected i.c.v, with AVP (open circles) or vehicle (artificial CSF (closed circles)) as described in the text. Mice were sacrificed at the indicated time after injection of 1 ng of AVP (A) or at 15 min after injection of AVP (B), and c-los m R N A in the hippocampus was quantitated following Northern blot analysis. Results are presented as the percent increase in c-los m R N A over the level in naive mice, and represent the mean values from 2 experiments (A) or values from a single experiment in which pooled tissue from 4 mice was used to generate each point (B) (except for 1 ng AVP, which represents the mean from 3 groups of mice). In the experiments depicted in A, CSF injection produced a mean 106% increase (range 96-116), measured at 15 rain after injection.

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Fig. 7. Effect of various peptides on c-fos expression in mouse brain. C57BL/6 mice were injected i.c.v, with AVP(1 ng), AVP(4-9) (1 ng), oxytocin (1 ng) or nerve growth factor NGF (10 ng) as described in the text. Mice were sacrificed 15 min after injection of all peptides except NGF (30 min after injection), and c-fos mRNA in the indicated brain areas was quantitated following Northern blot analysis. Results represent mean _+S.E.M. from 3 experiments with AVP, and means of 2 experiments with AVP(4-9) and oxytocin. The range of values was: septum: AVP(4-9), 195-824; oxytocin, 0-77; hippocampus: AVP(4-9), 256-554; oxytocin, 171-474. Levels of c-fos mRNA in cerebral cortex were not measurable (n.d.) after injection of AVP, AVP(4-9) or oxytocin. The results with NGF are from a single experiment in which tissue from 4 mice was pooled for each brain area. In this figure, the percent increase in c-fos mRNA is based on the amount of message in tissue of CSF-treated mice, rather than naive mice. tion of c-los m R N A in hippocampus (Fig. 6) were also similar to those in the septum, although an effect was observed in hippocampus only after the dose of AVP exceeded 100 pg (Fig. 6). We further tested the specificity of the response to AVP by injecting mice with oxytocin (1 ng), the AVP metabolite peptide, AVP(4-9) (1 ng) or nerve growth factor (NGF, 10 ng). Like AVP, neither oxytocin nor AVP(4-9) had an appreciable effect on c-fos m R N A in the cerebral cortex. Oxytocin had a greater effect in hippocampus, where AVP and oxytocin induced similar increases in c-los m R N A , than in septum, while AVP(49) consistently increased c-los m R N A levels in both septum and hippocampus (Fig. 7). N G F differed from the other peptides in that its administration resulted in a large increase in c-fos m R N A in cerebral cortex, with a smaller increase in hippocampus and essentially no effect in the septum (Fig. 7). The effects of N G F were measured at 30 min after injection; there was no effect of N G F in any brain area tested at 15 min after injection. Low levels of expression of c-myc and c-ras m R N A in were found in all brain areas tested in naive or vehicletreated mice. Vasopressin (up to 4 h after injection) had no effect on the m R N A levels for these two protooncogenes (data not shown).

DISCUSSION Our results show that AVP, administered in vivo, can transiently increase the levels of c-los m R N A in circumscribed areas of brain. This action appears to be mediated by Vl-vasopressin receptors in both the septum and the hippocampus, areas which have been shown to have a relatively high density of these receptors 28'38. Thus, the V2-selective agonist, dVDAVP, even when administered at a dose 10-fold higher than the Vl-selective agonist, had a relatively small and inconsistent effect on c-los m R N A levels, both in septum (see legend to Fig. 3) and hippocampus, in contrast to the effects of AVP or the Vl-selective agonist. Furthermore, the Vl-selective antagonist was a more effective blocker of the response to AVP than the V2-selective antagonist. The finding that the V2-selective antagonist, although less effective than the Vl-selective antagonist, did reduce the effect of AVP to some degree, particularly in the septum, may reflect the fact that AVP antagonists are in general less selective than agonists 23'35. In the periphery, V1 vasopressin receptors mediate stimulatory effects of vasopressin on phosphatidylinositol metabolism and increases in intracellular calcium 24'37, and similar findings have been reported in hippocampal and septal slices 1°'34. These data are consistent with the suggestion that AVP could increase c-los m R N A levels in brain by acting at postsynaptic V1 receptors. On the other hand, we have recently found that treatment of mice with 6-hydroxydopamine decreases the number of vasopressin receptors in the lateral septum is, suggesting that a portion of vasopressin receptors may be localized presynaptically on catecholaminergic neurons. It is possible, therefore, that the in vivo effect of AVP on c-los expression in brain could be in part indirect, i.e., mediated by changes in neurotransmitter release. It has been reported that injections of glutamate or noradrenaline directly into rat hippocampus produce a "non-specific" increase in c-los m R N A in that brain area, since saline injection caused a similar increase (60-80% increase over baseline values) 19. Such an increase may in fact result from injury caused by the direct injections into brain, since increases in c-los protein have been observed both in neurons and glial cells following cortical injury ]2' 31. In addition, in preliminary studies, in which i.c.v. injections were performed under ether anesthesia, we found that ether alone produced an apparently nonspecific increase in c-los m R N A levels in brain. Thus, it is necessary to carefully control the conditions of the experiment, including stress to the animals, when c-los m R N A expression is measured in vivo. However, under the conditions used in our study, AVP appeared to produce a specific effect on c-los m R N A . Thus, while

136 CSF vehicle injection did increase c-los m R N A in septum and hippocampus, by an amount similar to that reported in other studies 19, we found that AVP administration consistently resulted in much larger increases in c-los mRNA, and the response to AVP was blocked by a V~ receptor antagonist. The regional specificity of the effect of AVP, and in particular the lack of effect in cerebral cortex, also argues against a non-specific injury- or stress-induced response. The finding that the increased c-los m R N A response to AVP, AVP(4-9), oxytocin and nerve growth factor (NGF) was differentially distributed among brain regions further supports the contention that the observed increases are specific receptor-mediated events. The pattern of effects of vasopressin and oxytocin on c-los m R N A levels parallels the distribution of receptors for these peptides in the brain. For example, high levels of vasopressin receptors are found in lateral septum and hippocampus 28'3s, while oxytocin receptors are lower in the septum 29 and higher in the ventral hippocampus 2s. There is little binding of either peptide in the cerebral cortex. While AVP(4-9) and AVP binding sites have been reported to be differentially distributed in brain, AVP(4-9) does bind to areas of the hippocampus 1, and our data suggest that the AVP metabolite peptide can also interact with binding sites in the septal region. The effects of NGF on c-los expression in brain are of particular interest. This protein has been reported to increase c-fos m R N A in PC12 cells in culture, presumably via interaction with receptors on the cell surface s . NGF receptors in brain, however, have been localized to cholinergic neuron terminals in the hippocampus and cerebral cortex 33. When NGF interacts with these receptors, the NGF-receptor complex that forms is believed to be carried by retrograde transport to cell bodies in the medial septum/vertical and horizontal limbs of the diagonal band, and the nucleus basalis 33, where NGF exerts its physiological effects. According to this scheme, an NGF-induced increase in c-los m R N A levels in the brain areas that contain NGF receptors (i.e. terminal areas in the hippocampus and cortex), which was observed in the present study, is unexpected. However, in agreement with these results, it has recently been reported that injections of NGF into rat cortex result in induction of c-los protein in several areas of the ipsilaterai cortex 3~. As suggested by these authors, the effects of NGF in the brain could be indirect, and further work will be necessary to clarify this action of N G E The characteristics of the effects of AVP on c-los m R N A levels in the septal area and in the hippocampus, are, for the most part, consistent with the pattern of peptide effects on maintenance of ethanol tolerance 35. In addition to the greater efficacy of Vl-selective agonists

and antagonists for both actions, the doses of AVP that increase c-los m R N A are comparable to the daily doses necessary for maintenance of tolerance. Interestingly, the effects of AVP on maintenance of ethanol tolerance 16, as well as on both septal and hippocampal c-fos m R N A levels, appeared to have a very steep dose-response curve. However, comparison of the effects of AVP and oxytocin on c-los m R N A in septum and hippocampus suggests that the increase in c-fos expression in the septum may be a key factor in mediating peptide effects on tolerance. Thus, oxytocin, which did not maintain ethanol tolerance 17, had only a small and inconsistent effect on c-los m R N A levels in the septum, in comparison to AVE In contrast, in the hippocampus, oxytocin produced an increase in c-los m R N A that was similar to that produced by vasopressin. NGF also had no measurable effect on c-los m R N A in the septum, and, in preliminary studies, was much less effective than AVP in maintaining ethanol tolerance (Szab6, G., Tabakoff, B. and Hoffman, P.L., unpublished observation). NGF, like AVP and oxytocin, did increase hippocampal c-fos mRNA. The metabolite peptide AVP(4-9) has not been tested for its efficacy in maintaining ethanol tolerance, but was reported to be very potent at influencing memory consolidation 3. The structure-activity relationships for the effects of vasopressin-related peptides on maintenance of ethanol tolerance and on memory consolidation were alike 15, suggesting that the peptides might influence similar underlying mechanisms for these neuroadaptive phenomena. The present data suggest that increases in septal c-los expression, which were comparable in response to AVP and AVP(4-9), could represent such a mechanism. In this regard, it is of interest to note that oxytocin, which, as discussed, had little effect on septal c-fos m R N A , has been reported to have effects opposite to AVP on memory consolidation (see refs. 15, 39). Based on the presented results, one may hypothesize that the increase in c-los m R N A levels, and presumably the subsequent gene activation ~°, produced by vasopressin and its analogs could serve to translate short-term peptide-mediated signals to long-term changes in synaptic efficacy. One possible mechanism for this effect could involve the reported neurotrophic action of AVP, which has been suggested 2 as a means by which AVP could modulate morphological changes that could, in turn, promote the neuroadaptive changes involved in certain aspects of memory or ethanol tolerance.

Acknowledgement. We thank Drs. Hideaki Ishizawa and Gyula Szab6 for assistance with the experiments, and Ms. Robin DuBeau for secretarial assistance. RR.G. is a Fogarty International Visiting Associate. We are grateful to Dr. M. Manning for the gifts of V land V2-selective agonists and antagonists.

137

REFERENCES 1 Brinton, R.E., Gehlert, D.R., Wamsley, J.K., Wan, Y.P. and Yamamura, H.I., Vasopressin metabolite, AVP(4-9), binding sites in brain: distribution distinct from that of parent peptide, Life Sci., 38 (1986) 443-452. 2 Brinton, R.E. and Gruener, R., Vasopressin promotes neurite growth in cultured embryonic neurons, Synapse, 1 (1987) 329-334. 3 Burbach, J.P.H., Kov~ics, G.L., de Wied, D., van Nispen, J.W. and Greven, H.M., A major metabolite of arginine vasopressin in the brain is a highly potent neuropeptide, Science, 221 (1983) 1310-1311, 4 Burnard, D.M., Pittman, Q.J. and Veale, W.L., Increased motor disturbances in response to arginine vasopressin following hemorrhage or hypertonic saline: evidence for central AVP release in rats, Brain Res., 273 (1983) 59-65. 5 Cain, D.P., Plant, J., Rouleau, S. and Corcoran, M.E., Failure to kindle seizures after repeated intracerebral administration of arginine vasopressin, Life Sci., 38 (1986) 985-989. 6 Cathala, G., Savouret, J.F., Mendez, B., West, B.L., Karin, M., Martial, J.A. and Baxter, J.D., A method for isolation of intact translationally active ribonucleic acid, DNA, 2 (1983) 329-335. 7 Chen-Kiang, S., Wolgemuth, D.J., Hsu, M.-T. and Darnell, J.E. Jr., Transcription and accurate polyadenylation in vitro of RNA from the major late adenovirus 2 transcription unit, Cell, 28 (1982) 575-584. 8 Curran, T. and Morgan, J.I., Superinduction of c-los by nerve growth factor in the presence of peripherally active benzodiazepines, Science, 229 (1985) 1265-1268. 9 de Wied, D., Gaffori, O., van Ree, J.M. and de Jong W., Central target for the behavioral effects of vasopressin peptides, Nature (Lond.), 308 (1984) 276-278. 10 Dorsa, D.M., Brot, M.D., Shewey, L.M., Meyers, K.M., Szot, P. and Miller, M.A., Interaction of a vasopressin antagonist with vasopressin receptors in the septum of the rat brain, Synapse, 2 (1988) 205-211. 11 Dragunow, M. and Robertson, H.A., Kindling stimulation induces c-los proteins(s) in granule ceils of the rat dentate gyrus, Nature (Lond.), 329 (1987) 441-442. 12 Dragunow, M. and Robertson, H.A., Brain injury induces c-los protein(s) in nerve and glial-like cells in adult mammalian brain, Brain Res., 455 (1988) 295-299. 13 Ettenberg, A., Intracerebroventricular application of a vasopressin antagonist peptide prevents the behavioral actions of vasopressin, Behav. Brain Res., 14 (1984) 201-211. 14 Greenberg, M.E., Ziff, E.B. and Greene, L.A., Stimulation of neuronal acetylcholine receptors induces rapid gene transcription, Science, 234 (1986) 80-83. 15 Hoffman, P.L., Central nervous system effects of neurohypophyseal peptides. In C.W. Smith (Ed.), The Peptides, Academic Press, New York, 1987, pp. 239-295. 16 Hoffman, P.L., Ritzmann, R.E and Tabakoff, B., The influence of arginine vasopressin and oxytocin on ethanol dependence and tolerance. In M. Galanter (Ed.), Currents in Alcoholism, Vol. 5, Grune and Stratton, New York, 1979, pp. 5-16. 17 Hoffman, P.L., Ritzmann, R.F., Walter, R. and Tabakoff, B., Arginine vasopressin maintains ethanol tolerance, Nature (Lond.), 276 (1978) 614-616. 18 Ishizawa, H., Tabakoff, B., Mefford, I.N. and Hoffman, P.L., Reduction of arginine vasopressin binding sites in mouse lateral septum by treatment with 6-hydroxydopamine, Brain Res., in press. 19 Kaczmarek, L., Siedlecki, J.A. and Danysz, W., Proto-oncogene c-los induction in rat hippocampus, Mol. Brain Res., 3 (1988) 183-186. 20 Kasting, N.W., Veale, W.L. and Cooper, K.E., Convulsive and

hypothermic effects of vasopressin in the brain of rats, Can. J. Physiol. Pharmacol., 58 (1980) 316-319. 21 Le Gal La Salle, G., Long-lasting and sequential increase of c-los oncoprotein in kainic acid-induced status epilepticus, Neurosci. Lett., 88 (1988) 127-130. 22 Lehrach, H., Diamond, D., Wozney, J.M. and Boedtker, H., RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination, Biochemistry, 16 (1977) 4743-4751. 23 Manning, M. and Sawyer, W.H., Development of selective agonists and antagonists of vasopressin and oxytocin. In R.W. Schrier (Ed.), Vasopressin, Raven, New York, 1985, pp. 131-144. 24 Michell, R.H., Kirk, C.J. and Billah, M.M., Hormonal stimulation of phosphatidylinositol breakdown with particular reference to the hepatic effects of vasopressin, Biochem. Soc. Trans., 7 (1979) 861-865. 25 Milbrandt, J., Nerve growth factor rapidly induces c-.fos mRNA in PC12 rat pheochromocytoma cells, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4789-4793. 26 Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-los expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 27 Morgan, J.I. and Curran, T., Role of ion flux in the control of c-los expression, Nature (Lond.), 322 (1986) 552-555. 28 Poulin, P., Lederis, K. and Pittman, Q.J., Subcellular localization and characterization of vasopressin binding sites in the central septal area, lateral septum, and hippocampus of the rat brain, J. Neurochem., 50 (1988) 889-898. 29 Raggenbass, M., Dubois-Dauphin, M., Tribollet, E. and Dreifuss, J.J., Direct excitatory action of vasopressin in the lateral septum of the rat brain, Brain Res., 459 (1988) 60-69. 30 Rauscher, F.J. III, Cohen, D.R., Curran, T., Bos, T.J., Vogt, P.K., Bohman, D., Tijan, R. and Franza, B.R. Jr., Fosassociated protein p39 is the product of the jun proto-oncogene, Science, 240 (1988) 1010-1016. 31 Sharp, ER., Gonzales, M.F., Hisanaga, K., Mobley, W.C. and Sagar, S.M., Induction of the c-los gene product in rat forebrain following cortical lesions and NGF injections, Neurosci. Lett., 100 (1989) 117-122. 32 Slotnick, B.M. and Leonard, C.M., A Stereotoxic Atlas of the Albino Mouse Forebrain, DHEW Publication No. (ADM) 75-100, U.S. Gov't Printing Office, Washington, DC, 1975. 33 Springer, J.E., Nerve growth factor receptors in the central nervous system, Exp. Neurol., 102 (1988) 354-365. 34 Stephens, L.R. and Logan, S., Vasopressin stimulates inositol lipid metabolism in rat hippocampus, J. Neurochem., 46 (1986) 649-651. 35 Szab6, G., Tabakoff, B. and Hoffman, P.L., Receptors with V 1 characteristics mediate the maintenance of ethanol tolerance by vasopressin, J. Pharmacol. Exp. Ther., 247 (1988) 536-541. 36 Taubman, M.B., Berk, B.C., Izumo, S., Tsuda, T., Alexander, R.W. and Nadal-Ginard, B., Angiotensin II induces c-fos mRNA in aortic smooth muscle, J. Biol. Chem., 264 (1989) 526-530. 37 Thomas, A.P., Alexander, J. and Williamson, J.R., Relationship between inositol polyphosphate production and the increase of cytosolic free Ca2÷ induced by vasopressin in isolated hepatocytes, J. Biol. Chem., 259 (1984) 5574-5584. 38 Tribollet, E., Barberis, C., Jard, S., Dubois-Dauphin, M. and Dreifuss, J.J., Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in rat brain by light microscopic autoradiography, Brain Res., 442 (1988) 105-118. 39 van Wimersma Greidanus, Tj. B., van Roe, J.M. and de Wied, D., Vasopressin and memory, Pharmacol. Ther., 20 (1983) 437-458.