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Dipsogenic effect of pituitary adenylate cyclase activating polypeptide (PACAP38) injected into the lateral hypothalamus
BRAIN RESEARCH Brain Research696 (1995) 254-257
ELSEVIER
Short communication
Dipsogenic effect of pituitary adenylate cyclase activating polypeptide (PACAP38) injected into the lateral hypothalamus M. Puig de Parada *, M.A. Parada, L. Hernfindez Los Andes University, Department of Physiology, School of Medicine, Apartado postal 109, M~rida 5101-A, Venezuela
Accepted 13 June 1995
Abstract
PACAP38 bilaterally injected in the vicinity of the perifornical lateral hypothalamus (pfLH) induced drinking behavior in rats. The animals (n = 12) drank 19.7 + 4.1 ml of water during the hour following PACAP38 microinjections (1 nmol/0.5 txl). In the same rat sulpiride microinjections (45 nmol/0.5 /.d) had relatively mild effects (7.8 + 1.4 ml/h). The dipsogenic effects of sulpiride and PACAP38 were well correlated suggesting that both substances trigger drinking behavior activating the same hypothalamic mechanisms. Neither sulpride nor PACAP38 promoted drinking when injected just 1.3 mm behind the effective zone. This negative result is an evidence of the neuroanatomical specificity of the dipsogenic effects of both substances. These preliminary results suggest that PACAP38 in the pfLH could be a neuropeptide regulating drinking behavior and perhaps body fluid volume and osmolarity and arterial blood pressure. Keywords: PACAP; Microinjection;DA; Lateral hypothalamus;Sulpride; D2 receptor;Drinking; Osmolarity;Blood pressure
Pituitary adenylate cyclase activating polypeptide 38 (PACAP38) is a bioactive neuropeptide recently isolated from ovine hypothalamic tissues [11]. PACAP38 stimulates adenylate cyclase and increases cAMP levels in rat anterior pituitary cell cultures [11]. The 27 N-terminal amino acids of PACAP38 termed PACAP27 [12] has similar potency to activate pituitary adenylate cyclase. PACAP is widely distributed in peripheral tissues [1] as well as in the central nervous system (CNS) [1,5,10] in rats. It has been suggested that PACAP38 is a neurotransmitter/neuromodulator [7] that acts through two types of receptors [6,13]. Type I PACAP-binding site binds PACAP27 and PACAP38 with high affinity, is not displaceable by vasoactive intestinal polypeptide (VIP) and it is said to be PACAP specific. Type II PACAP-binding site is not specific for PACAP and binds also VIP [21]. The highest concentration of PACAP has been detected in the hypothalamus [1,10]. Immunohistochemistry and sandwich-enzyme immunoassay have shown high concentrations of PACAP in the lateral hypothalamus (LH) [10] and the perifornical nuclei [5]. Moreover, the LH is the
* Corresponding author. Fax: (58) (74) 63-4587; E-maih [email protected] 0006-8993/95/$09.50 © 1995 ElsevierScienceB.V. All rights reserved SSDI 0006-8993(95)00824-1
hypothalamic area with the highest density of type I PACAP-binding sites [8]. These facts suggest that PACAP could be involved in the regulation of one or more of the hypothalamic functions whose anatomical substrate is located in the vicinity of the perifornical lateral hypothalamus. However, nothing is known on the physiological role of PACAP in the pfLH. This area contains dopamine D 2 receptors negatively linked to adenylate cyclase and inhibiting the production of cAMP [20]. Blockade of pfLH D 2 receptors with sulpiride, a selective D 2 receptor blocker [4], induces feeding [14], drinking [14], locomotion [15], hyperthermia [18], DA release in the nucleus accumbens and reward [17,19]. Were the PACAP receptors located in one set of LH neurons bearing D 2 receptors, then PACAP administration in the pfLH should reproduce all or some of the sulpiride effects. The present report shows that PACAP38 induces preferentially drinking when administered in the pfLH. Twenty-two male Wistar rats were used for this experiment. The animals were individually housed with water and purina chow pellets ad libitum, 12-12 h light-dark cycles and 22°C ( + 2°C) room temperature. Under ketamine anesthesia chronic bilateral intracerebral guide cannulas [16] were stereotaxically implanted in each rat. Those cannulas were 18 mm long pieces of 27-gauge stainless
M.P.d. Parada et al. / Brain Research 696 (1995) 254-257
steel tubing aimed 2 mm above the intended target. With the incisors bar 3.5 mm below the interaural line the lateral (1.6 mm from the midsagittal sinus) and ventral (6.5 mm from the surface of the skull) coordinates were the same in all rats. The AP coordinate was 1.3 mm (anterior group; n = 12) and 2.6 mm (posterior group; n = 10) posterior to the bregma. This last group was used as a control for anatomical specificity. After at least one week of post-surgical recovery each animal received bilateral pfLH microinjections of PACAP38 (1 nmol), sulpiride (45 nmol) or Ringer solution. Those injections were applied in three different sessions, four days apart, and in counterbalanced order. The injections were performed following a remote insertion procedure [16] using 20 cm long injectors made out of fused silica tubing. Fifteen minutes before one injection each non-deprived animal was taken from its home cage and connected with a tether to a swivel joint. Long PE-20 guide tubes were connected to the stainless steel intracerebral guide cannulas [16] and the animal was then placed in a plexiglas box. At the injection time the long capillary injectors were slid through the PE-20 guide and intracerebral guide cannula down to the LH. The injection volume was always 0.5 /zl delivered in 30 s. The injectors remained in place for 20 more seconds to allow diffusion of the drugs. PACAP38 (Sigma Chemical Co.) was dissolved in a Ringer's solution with the same ionic composition as the cerebrospinal fluid. DL-Sulpiride (Sigma Chemical Co.) was dissolved in Ringer's solution containing 1 N acetic acid (9:1). Immediately after the injection the animal was returned to its home cage where it had access to water from and inverted graduated cylinder. Water intake was measured 1 h after the injections. Both PACAP and sulpiride induced water intake in non-deprived rats when administered in an area previously shown effective for sulpiride [14] (anterior group). As shown in Fig. 1 the dipsogenic effect of PACAP38 (19.7 + 4.1 m l / h ) was more than two-fold larger than that of sulpiride (7.8 ___1.4 ml/h), and a paired t-test showed this difference statistically significant (t = 3.61; df = 11; P < 0.005). By contrast, both substances were less effective when administered 1.3 mm behind the effective place (posterior group). At this location animals drank 2.4 + 0.83 m l / h after PACAP38 and 1.5 __. 0.58 m l / h after sulpiride. Comparisons, by means of unpaired t-test, of the effects induced by each substance on both locations showed significant differences (t = 3.71; df = 20; P < 0.001 for PACAP38; t = 3.81; df = 20; P < 001 for sulpiride), but there must be more subtle anatomical differences in sensitivity to PACAP38 since the anterior group could be subdivided into two subgroups. One subgroup of 7 rats with a mean water intake of 29.9 _ 3.5 m l / h (including one rat that drank 47 m l / h ) and the other subgroup (5 rats) with a low water intake of 5.4 + 1.2 m l / h after PACAP38. The tip of the injector cannulas ended in the vicinity of the perifornical LH, but with this preliminary and relatively
255
[ 30-
p
]
~'¢01001$ it.
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I.g ~<0"001 t
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.r, 10
PACAP38
1 nmol/0.5 BI
SULRFaDE 45 nrnol/0.5 ~1
RFIGER 0.5 ~1
Fig. 1. Mean water intake ( + S.E.M.) induced by bilateral intrahypothalamic microinjections of PACAP38 or sulpiride in non-deprived rats. Each substance was applied in two different places located in the same sagittal and ventral planes but in different antero-posterior planes 1.3 mm apart. The anterior sites (dark columns) were 1.3 mm posterior to bregma in an area close to the perifornical LH where previous studies had shown a dipsogenic effect for sulpiride. The posterior sites (open columns) were 2.6 mm behind bregma in an area where sulpiride induced hyperlocomotion. Sulpiride was effective again and induced drinking from the anterior area, but PACAP38 was more potent. Neither drug induced an appreciable drinking when injected into the posterior location. Ringer injections in the anterior location were also ineffective.
small group of animals it was difficult to establish the exact location of the most effective PACAP-sensitive area. More systematic explorations and mapping studies are needed. A linear regression analysis (Fig. 2) carried out on water intake data after sulpiride and after PACAP38, showed a positive correlation ( r = 0.821; F1.20 = 41.5; P < 0.0001). The obvious interpretation of this analysis is that the dipsogenic potencies of both substances are anatomically correlated, suggesting the existence of a common mechanism triggered by both drugs, with PACAP38 being more potent than sulpiride. We don't have the dose-response relationships in order to make an appropriate comparison of the potencies of both drugs, but taking into account the slope (2.3) of the linear regression equation (Fig. 2) and the dose difference (1 vs. 45 nmol), it can be suggested that PACAP is around 90 times more potent than sulpiride to induce drinking. More experimental work has to be conducted in order to clearly establish the molecular or neurochemical actions responsible for the drinking effects of both drugs. However, it is tempting to speculate that some neural elements involved in drinking and located in the pfLH carry both D 2 and PACAP I receptors, and that D 2 blockade or PACAP I activation have the same molecular consequences differing just in the
256
M.P.d. Parada et al. / Brain Research 696 (1995) 254-257 50-
previously reported [15] and there was no hyperactivity after PACAP. The potent dipsogenic effect displayed after PACAP administration in the LH and reported in this paper, strongly suggests that this peptide could play an important role in the rat lateral hypothalamus as a regulatory peptide for drinking behavior. Since the relatively high amounts of water consumed by some rats must have repercussions on the organic fluid volume and osmolarity and on the arterial blood pressure, it follows that PACAP38 might intervene in the regulation of those physiological variables as well.
y = 0.50539+ 2.3045x R = 0.821 / 40
30
I- 0.
•
20-
lo
Acknowledgements 0-0
-=.
-=, 5
1'0
15
2'0
WATER INTAKE AFTER SULPIRIDE (ml/hr)
Fig. 2. Scattergram showing a positive correlation between the magnitudes of the drinking responses induced by PACAP38 and sulpiride. The whole set of rats (n = 22) used for the present experiments were included in this analysis. The slope of the curve was greater than 2 indicating that at the doses employed PACAP38 was twice as potent as sulpiride.
magnitude of the response. Activation of the adenylate cyclase followed by an increase in the intracellular cAMP levels could be the common effect that triggers drinking behavior. However, the PACAP38 secretagogue activity, previously studied assessing the adrenaline release from chromaffin cells and apparently cAMP-independent [3,22], should also be considered as an alternative explanation. An equivalent of this PACAP secretagogue activity has been demonstrated in the CNS where PACAP administration has been shown to release acetylcholine (ACh) from hippocampal neurons [9]. The hypothetical existence of the same relationship between PACAP38 and cholinergic elements in the lateral hypothalamus would explain the similar effects of PACAP38 and sulpiride. ACh release could mediate the drinking responses induced by both drugs. This hypothesis is supported by the facts that sulpiride administration in the LH releases ACh [19] and ACh in the LH seems to be involved in drinking [2]. Finally, we cannot discard that the dipsogenic effect of PACAP38 might be the result of an interaction of several actions of the peptide, including the two mentioned before. One apparent difference between sulpiride and PACAP38 effects on behavior is that the peptide seems to be more specific for drinking, whereas sulpiride triggers several other behaviors including feeding and certain kind of psychomotor stimulation. For instance, PACAP38 injections performed during pilot experiments and directed to the LH area from where a feeding response is usually obtained with sulpiride, failed to increase food intake. During the present experiments we did not test the animals with food, but they became hyperactive with sulpiride as
These experiments were supported by Grants CDCHTULA M-498-95-03-A and BID-CONICIT BTS-37.
References [1] Arimura, A., Somogyv~iri-Vigh, A., Miyata, A., Mizuno, K., Coy, D.H. and Kitada, C., Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes, Endocrinology, 129 (1991) 2787-2789. [2] Grossman, S.P., Direct adrenergic and cholinergic stimulation of hypothalamic mechanisms, Am. J. Physiol., 202 (1962) 872-882. [3] Isobe, K., Nakai, T. and Takuwa, Y., CaZ+-dependent stimulatory effect of pituitary adenylate cyclase activating polypeptide on catecholamine secretion from cultured porcine adrenal medullary chromaffin cells, Endocrinology, 132 (1993) 1757-1765. [4] Jenner, P., Elliot, P.N.C., Clow, A., Reavill, C. and Marsden, C.D., A comparison of in vitro and in vivo dopamine receptor antagonism produced by substituted benzamide drugs, J. Pharm. PharmacoL, 30 (1978) 46-48. [5] K6ves, K., Arimura, A., G6rcs ,T.G. and Somogyv~iri-Vigh, A., Comparative distribution of immunoreactive pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in rat forebrain, Neuroendocrinology, 54 (1990) 159-169. [6] Lam, H.C., Takahashi, K., Ghatei, M.A., Kanse, S.M., Polak, J.M. and Bloom, S.R., Binding sites of a novel neuropeptide pituitaryadenylate-cyclase-activating-polypeptide in the rat brain and lung, Eur. J. Biochem., 193 (1990) 725-729. [7] Massuo, Y., Ohtaki, T., Masuda, Y., Nagai, Y., Suno, M., Tsuda, M. and Fujino, M., Autoradiographic distribution of pituitary adenylate cyclase activating polypeptide (PACAP) binding sites in the rat brain, Neurosci. Lett., 126 (1991) 103-106. [8] Masuo, Y., Ohtaki, T., Masuda, Y., Tsuda, M. and Fujino, M., Binding sites for pituitary adenylate cyclase activating polypeptide (PACAP): comparison with vasoactive intestinal polypeptide (VIP) binding site localization in rat brain sections, Brain Res., 575 (1992) 113-123. [9] Masuo, Y., Matsumoto, Y., Tokito, F., Tsuda, M. and Fujino, M., Effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) on the spontaneous release of acetylcholine from the rat hippocampus by brain microdialysis, Brain Res., 611 (1993) 207-215. [10] Masuo, Y., Suzuki, N., Matsumoto, H., Tokito, F., Matsumoto, Y., Tsuda, M. and Fujino M., Regional distribution of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat central nervous system as determined by sandwich-enzyme immunoassay, Brain Res., 602 (1993) 57-63
M.P.d. Parada et al. / Brain Research 696 (1995) 254-257
[11] Miyata, A., Arimura, A., Dahl, R.R., Minamino, N., Uehara, A., Jiang, L., Culler, M.D. and Coy, D.H, Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells, Biochem. Biophys. Res. Commun., 164 (1989) 567-574. [12] Miyata, A., Jiang, L., Dahl, R.D., Kitada, C., Kubo, K., Fujino, M., Minamino, N. and Arimura, A., Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38), Biochem. Biophys. Res. Commun., 170 (1990) 643-648. [13] Ohtaki, T., Watanabe, T., Ishibashi, Y., Kitada, C., Tsuda, M., Gottschall, P.E., Arimura, A. and Fujino, M., Molecular identification of receptor for pituitary adenylate cyclase activating polypeptide, Biochem. Biophys. Res. Commun., 171 (1990) 838-844. [14] Parada, M.A., Hernandez, L. and Hoebel, B.G., Sulpiride injections in the lateral hypothalamus induce feeding and drinking in rats, Pharmacol. Biochem. BehaL,., 30 (1988) 917-923. [15] Parada, M.A., Hernandez, L. and Santiago, C., An improved circular tilt-cage shows that intrahypothalamic injections of sulpiride increase locomotion, Brain Res. Bull., 21 (1988) 873-880. [16] Parada, M.A., Puig de Parada, M. and Hoebel, B.G., A remote insertion technique for intracerebral microinjections in freely moving animals, J. Neurosci. Methods, 50 (1993) 237-241. [17] Parada, M.A., Puig de Parada, M. and Hoebel, B.G., Rats self-inject a dopamine antagonist in the lateral hypothalamus where it acts to
[18]
[19]
[20]
[21]
[22]
257
increase extracellular dopamine in the nucleus accumbens, Pharmacol. Biochem. Behat,., 51 (1995) in press. Parada, M.A., Puig de Parada, M., Rada, P. and Hernandez, L., Sulpiride increases and dopamine decreases intracranial temperature in rats when injected in the lateral hypothalamus: an animal model for the neurole!btic malignant syndrome? Brain Res., 674 (1995) 117-121. Parada, M.A., Puig de Parada, M., Rada, P., Hernandez, L. and Hoebel, B.G., Sulpiride is self-injected in the lateral hypothalamus where it releases acetylcholine and induces also dopamine release in the nucleus accumbens, Soc. Neurosci. Abstr., (1994) 103.6. Pizzi, M., Coen, E., Memo, M., Missale, C., Carruba, M.O. and Spano, P.F., Evidence for the presence of D 2 but not D I dopaminc receptors in rat hypothalamic perifornical area, Soc. Neurosci. Lett., 67 (1986) 159-162. Shivers, B.D., Gorcs, T.J., Gottschall, P.E. and Arimura, A., Two high affinity bindings sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions, Endocrinolog)', 128 (1991) 3055-3065. Watanabe, T., Masuo, Y., Matsunoto. H., Suzuki, N., Ohtaki, T.. Masuda Y., Kitada, C., Tsuda, M. and Fujino, M., Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline, Biochem. Biophvs. Res. Commun., 182 (1992) 403-411.
ELSEVIER
Short communication
Dipsogenic effect of pituitary adenylate cyclase activating polypeptide (PACAP38) injected into the lateral hypothalamus M. Puig de Parada *, M.A. Parada, L. Hernfindez Los Andes University, Department of Physiology, School of Medicine, Apartado postal 109, M~rida 5101-A, Venezuela
Accepted 13 June 1995
Abstract
PACAP38 bilaterally injected in the vicinity of the perifornical lateral hypothalamus (pfLH) induced drinking behavior in rats. The animals (n = 12) drank 19.7 + 4.1 ml of water during the hour following PACAP38 microinjections (1 nmol/0.5 txl). In the same rat sulpiride microinjections (45 nmol/0.5 /.d) had relatively mild effects (7.8 + 1.4 ml/h). The dipsogenic effects of sulpiride and PACAP38 were well correlated suggesting that both substances trigger drinking behavior activating the same hypothalamic mechanisms. Neither sulpride nor PACAP38 promoted drinking when injected just 1.3 mm behind the effective zone. This negative result is an evidence of the neuroanatomical specificity of the dipsogenic effects of both substances. These preliminary results suggest that PACAP38 in the pfLH could be a neuropeptide regulating drinking behavior and perhaps body fluid volume and osmolarity and arterial blood pressure. Keywords: PACAP; Microinjection;DA; Lateral hypothalamus;Sulpride; D2 receptor;Drinking; Osmolarity;Blood pressure
Pituitary adenylate cyclase activating polypeptide 38 (PACAP38) is a bioactive neuropeptide recently isolated from ovine hypothalamic tissues [11]. PACAP38 stimulates adenylate cyclase and increases cAMP levels in rat anterior pituitary cell cultures [11]. The 27 N-terminal amino acids of PACAP38 termed PACAP27 [12] has similar potency to activate pituitary adenylate cyclase. PACAP is widely distributed in peripheral tissues [1] as well as in the central nervous system (CNS) [1,5,10] in rats. It has been suggested that PACAP38 is a neurotransmitter/neuromodulator [7] that acts through two types of receptors [6,13]. Type I PACAP-binding site binds PACAP27 and PACAP38 with high affinity, is not displaceable by vasoactive intestinal polypeptide (VIP) and it is said to be PACAP specific. Type II PACAP-binding site is not specific for PACAP and binds also VIP [21]. The highest concentration of PACAP has been detected in the hypothalamus [1,10]. Immunohistochemistry and sandwich-enzyme immunoassay have shown high concentrations of PACAP in the lateral hypothalamus (LH) [10] and the perifornical nuclei [5]. Moreover, the LH is the
* Corresponding author. Fax: (58) (74) 63-4587; E-maih [email protected] 0006-8993/95/$09.50 © 1995 ElsevierScienceB.V. All rights reserved SSDI 0006-8993(95)00824-1
hypothalamic area with the highest density of type I PACAP-binding sites [8]. These facts suggest that PACAP could be involved in the regulation of one or more of the hypothalamic functions whose anatomical substrate is located in the vicinity of the perifornical lateral hypothalamus. However, nothing is known on the physiological role of PACAP in the pfLH. This area contains dopamine D 2 receptors negatively linked to adenylate cyclase and inhibiting the production of cAMP [20]. Blockade of pfLH D 2 receptors with sulpiride, a selective D 2 receptor blocker [4], induces feeding [14], drinking [14], locomotion [15], hyperthermia [18], DA release in the nucleus accumbens and reward [17,19]. Were the PACAP receptors located in one set of LH neurons bearing D 2 receptors, then PACAP administration in the pfLH should reproduce all or some of the sulpiride effects. The present report shows that PACAP38 induces preferentially drinking when administered in the pfLH. Twenty-two male Wistar rats were used for this experiment. The animals were individually housed with water and purina chow pellets ad libitum, 12-12 h light-dark cycles and 22°C ( + 2°C) room temperature. Under ketamine anesthesia chronic bilateral intracerebral guide cannulas [16] were stereotaxically implanted in each rat. Those cannulas were 18 mm long pieces of 27-gauge stainless
M.P.d. Parada et al. / Brain Research 696 (1995) 254-257
steel tubing aimed 2 mm above the intended target. With the incisors bar 3.5 mm below the interaural line the lateral (1.6 mm from the midsagittal sinus) and ventral (6.5 mm from the surface of the skull) coordinates were the same in all rats. The AP coordinate was 1.3 mm (anterior group; n = 12) and 2.6 mm (posterior group; n = 10) posterior to the bregma. This last group was used as a control for anatomical specificity. After at least one week of post-surgical recovery each animal received bilateral pfLH microinjections of PACAP38 (1 nmol), sulpiride (45 nmol) or Ringer solution. Those injections were applied in three different sessions, four days apart, and in counterbalanced order. The injections were performed following a remote insertion procedure [16] using 20 cm long injectors made out of fused silica tubing. Fifteen minutes before one injection each non-deprived animal was taken from its home cage and connected with a tether to a swivel joint. Long PE-20 guide tubes were connected to the stainless steel intracerebral guide cannulas [16] and the animal was then placed in a plexiglas box. At the injection time the long capillary injectors were slid through the PE-20 guide and intracerebral guide cannula down to the LH. The injection volume was always 0.5 /zl delivered in 30 s. The injectors remained in place for 20 more seconds to allow diffusion of the drugs. PACAP38 (Sigma Chemical Co.) was dissolved in a Ringer's solution with the same ionic composition as the cerebrospinal fluid. DL-Sulpiride (Sigma Chemical Co.) was dissolved in Ringer's solution containing 1 N acetic acid (9:1). Immediately after the injection the animal was returned to its home cage where it had access to water from and inverted graduated cylinder. Water intake was measured 1 h after the injections. Both PACAP and sulpiride induced water intake in non-deprived rats when administered in an area previously shown effective for sulpiride [14] (anterior group). As shown in Fig. 1 the dipsogenic effect of PACAP38 (19.7 + 4.1 m l / h ) was more than two-fold larger than that of sulpiride (7.8 ___1.4 ml/h), and a paired t-test showed this difference statistically significant (t = 3.61; df = 11; P < 0.005). By contrast, both substances were less effective when administered 1.3 mm behind the effective place (posterior group). At this location animals drank 2.4 + 0.83 m l / h after PACAP38 and 1.5 __. 0.58 m l / h after sulpiride. Comparisons, by means of unpaired t-test, of the effects induced by each substance on both locations showed significant differences (t = 3.71; df = 20; P < 0.001 for PACAP38; t = 3.81; df = 20; P < 001 for sulpiride), but there must be more subtle anatomical differences in sensitivity to PACAP38 since the anterior group could be subdivided into two subgroups. One subgroup of 7 rats with a mean water intake of 29.9 _ 3.5 m l / h (including one rat that drank 47 m l / h ) and the other subgroup (5 rats) with a low water intake of 5.4 + 1.2 m l / h after PACAP38. The tip of the injector cannulas ended in the vicinity of the perifornical LH, but with this preliminary and relatively
255
[ 30-
p
]
~'¢01001$ it.
20
I.g ~<0"001 t
I-z ILl I-' "~
.r, 10
PACAP38
1 nmol/0.5 BI
SULRFaDE 45 nrnol/0.5 ~1
RFIGER 0.5 ~1
Fig. 1. Mean water intake ( + S.E.M.) induced by bilateral intrahypothalamic microinjections of PACAP38 or sulpiride in non-deprived rats. Each substance was applied in two different places located in the same sagittal and ventral planes but in different antero-posterior planes 1.3 mm apart. The anterior sites (dark columns) were 1.3 mm posterior to bregma in an area close to the perifornical LH where previous studies had shown a dipsogenic effect for sulpiride. The posterior sites (open columns) were 2.6 mm behind bregma in an area where sulpiride induced hyperlocomotion. Sulpiride was effective again and induced drinking from the anterior area, but PACAP38 was more potent. Neither drug induced an appreciable drinking when injected into the posterior location. Ringer injections in the anterior location were also ineffective.
small group of animals it was difficult to establish the exact location of the most effective PACAP-sensitive area. More systematic explorations and mapping studies are needed. A linear regression analysis (Fig. 2) carried out on water intake data after sulpiride and after PACAP38, showed a positive correlation ( r = 0.821; F1.20 = 41.5; P < 0.0001). The obvious interpretation of this analysis is that the dipsogenic potencies of both substances are anatomically correlated, suggesting the existence of a common mechanism triggered by both drugs, with PACAP38 being more potent than sulpiride. We don't have the dose-response relationships in order to make an appropriate comparison of the potencies of both drugs, but taking into account the slope (2.3) of the linear regression equation (Fig. 2) and the dose difference (1 vs. 45 nmol), it can be suggested that PACAP is around 90 times more potent than sulpiride to induce drinking. More experimental work has to be conducted in order to clearly establish the molecular or neurochemical actions responsible for the drinking effects of both drugs. However, it is tempting to speculate that some neural elements involved in drinking and located in the pfLH carry both D 2 and PACAP I receptors, and that D 2 blockade or PACAP I activation have the same molecular consequences differing just in the
256
M.P.d. Parada et al. / Brain Research 696 (1995) 254-257 50-
previously reported [15] and there was no hyperactivity after PACAP. The potent dipsogenic effect displayed after PACAP administration in the LH and reported in this paper, strongly suggests that this peptide could play an important role in the rat lateral hypothalamus as a regulatory peptide for drinking behavior. Since the relatively high amounts of water consumed by some rats must have repercussions on the organic fluid volume and osmolarity and on the arterial blood pressure, it follows that PACAP38 might intervene in the regulation of those physiological variables as well.
y = 0.50539+ 2.3045x R = 0.821 / 40
30
I- 0.
•
20-
lo
Acknowledgements 0-0
-=.
-=, 5
1'0
15
2'0
WATER INTAKE AFTER SULPIRIDE (ml/hr)
Fig. 2. Scattergram showing a positive correlation between the magnitudes of the drinking responses induced by PACAP38 and sulpiride. The whole set of rats (n = 22) used for the present experiments were included in this analysis. The slope of the curve was greater than 2 indicating that at the doses employed PACAP38 was twice as potent as sulpiride.
magnitude of the response. Activation of the adenylate cyclase followed by an increase in the intracellular cAMP levels could be the common effect that triggers drinking behavior. However, the PACAP38 secretagogue activity, previously studied assessing the adrenaline release from chromaffin cells and apparently cAMP-independent [3,22], should also be considered as an alternative explanation. An equivalent of this PACAP secretagogue activity has been demonstrated in the CNS where PACAP administration has been shown to release acetylcholine (ACh) from hippocampal neurons [9]. The hypothetical existence of the same relationship between PACAP38 and cholinergic elements in the lateral hypothalamus would explain the similar effects of PACAP38 and sulpiride. ACh release could mediate the drinking responses induced by both drugs. This hypothesis is supported by the facts that sulpiride administration in the LH releases ACh [19] and ACh in the LH seems to be involved in drinking [2]. Finally, we cannot discard that the dipsogenic effect of PACAP38 might be the result of an interaction of several actions of the peptide, including the two mentioned before. One apparent difference between sulpiride and PACAP38 effects on behavior is that the peptide seems to be more specific for drinking, whereas sulpiride triggers several other behaviors including feeding and certain kind of psychomotor stimulation. For instance, PACAP38 injections performed during pilot experiments and directed to the LH area from where a feeding response is usually obtained with sulpiride, failed to increase food intake. During the present experiments we did not test the animals with food, but they became hyperactive with sulpiride as
These experiments were supported by Grants CDCHTULA M-498-95-03-A and BID-CONICIT BTS-37.
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