l -Deprenyl stimulates the release of catecholamines in the rat medial basal hypothalamus in vivo

Neuroscience Letters 270 (1999) 79±82

L-Deprenyl

stimulates the release of catecholamines in the rat medial basal hypothalamus in vivo Srinivasan ThyagaRajan*, S. Kaleem Quadri

Neuroendocrine Research Laboratory, Kansas State University, Manhattan, KS 66506, USA Received 23 March 1999; received in revised form 25 May 1999; accepted 25 May 1999

Abstract In vivo release of catecholamines in the medial basal hypothalamus (MBH) by L-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, was measured in young male Sprague±Dawley rats with stereotaxically implanted push-pull cannulae in the MBH and perfused with 0 (control), 1.5, 2.5 or 10.0 mg deprenyl in 20 ml of saline. Perfusate samples were collected at 20-min intervals and analyzed for norepinephrine (NE) and dopamine (DA) by high performance liquid chromatography (HPLC)-EC. NE release in the MBH was enhanced following perfusion with 2.5 and 10.0 mg deprenyl while DA release was augmented after infusion of 10.0 mg of deprenyl. There were no signi®cant alterations in the release of NE and DA in the control and 1.5 mg deprenyl groups. These results suggest that deprenyl-induced in vivo release of catecholamines in the MBH may be involved in the reversal of some of the reproductive aging processes. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Norepinephrine; Dopamine; Monoamine oxidase; Arcuate nucleus; Ventromedial hypothalamus; Push-pull perfusion; Selegiline

l-Deprenyl, an irreversible monoamine oxidase-B inhibitor, has been shown to increase sexual activity and life span in old male rats [8,9,12]. It is possible that these effects are produced by changes in hypothalamic catecholamines that are known to affect reproductive functions [11,14,16]. We have found that deprenyl treatment alters the synthesis and metabolism of catecholamines and indoleamines in the hypothalamus and the striatum of old female rats, and in the rats with carcinogen-induced mammary tumors [15,17]. Although, deprenyl has been reported to inhibit the uptake of dopamine (DA) and increase DA turnover rate in the striatum [8], no information is available on the effects of deprenyl on the release of catecholamines in the hypothalamus. The present study was conducted to determine the effects of deprenyl on the actual release of norepinephrine (NE) and dopamine (DA) in the medial basal hypothalamus (MBH) because these neurotransmitters are known to regulate reproduction and reproductive aging processes. Young male Sprague±Dawley rats (4 months old; Amitec, Lincoln, NE) were implanted with push-pull cannu* Corresponding author. Center for Neuroimmunology, Loma Linda University School of Medicine, 11021 Campus Street, Room 320, Loma Linda, CA 92350, USA. Tel.: 11-909-558-8389; fax: 11-909-558-0432. E-mail address: [email protected] (S. ThyagaRajan)

lae in the ventromedial and arcuate nuclei, which form the MBH, as previously described [16]. The rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and later injected with atropine (2.2 mg/kg, i.p.). A 10-mm long (22 G) cannula with a removable stylet, which protrudes 0.5±0.7 mm beyond the outer cannula, was stereotaxically implanted in the MBH (coordinates DV: 10 mm, L: 0.1 mm, AP: 3.3 mm). One week after recovery, push-pull perfusion was performed in rats that had shown no signs of infection or abnormal physical movements. On the day of perfusion, the rats were brought to the perfusion chamber at least 1 h prior to the commencement of the procedure. An inner cannula assembly (push and pull cannulae constructed from 29 G stainless steel tubings) was attached to two polyethylene tubings (PE-20, Clay Adams, Parsippany, NJ) that were connected to two pump tubings running through two identically calibrated peristaltic pumps (Pharmacia, Sweden). The pumps pushed the arti®cial cerebrospinal ¯uid (ACSF) (CaCl2 0.087 g/l; NaCl 7.188 g/l; KCl 0.358 g/l; MgSO4 0.296 g/l and Na2HPO4 1.703 g/l mixed in pyrogen-free water and adjusted to pH 7.3) through the push tube and the perfusates were withdrawn through the pull tube at a ¯ow rate of 10 ml/min. After the collection of three 20-min pretreatment samples to determine the basal release rate of NE and DA, the rats in

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 48 1- 4

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S. ThyagaRajan, S.K. Quadri / Neuroscience Letters 270 (1999) 79±82

the experimental groups were perfused with ACSF containing 1.5 mg (n ˆ 5), 2.5 mg (n ˆ 9), or 10.0 mg (n ˆ 7) of ldeprenyl in 20 ml of 0.9% saline. The control rats (n ˆ 6) were perfused with ACSF containing the vehicle (saline) alone. After delivery of the vehicle or the vehicle containing deprenyl, the pumps were stopped for 15 min, and then 20min samples were collected for 300 min. The perfusates were collected on ice in polypropylene vials containing 0.5 M HClO4 added at a v/v ratio of 25:1. After collection, the perfusates were stored at 2708C until further analysis by high pressure liquid chromatography (HPLC)-EC. The perfusion period was between 10:00 and 17:00 h. Prior to analysis by HPLC-EC, the samples were thawed at 608C for 1 min and 25 ml from each sample was mixed with 10 ml of the internal standard (isoproterenol). At the time of HPLC-EC analysis, the samples were injected onto a C 18, 5 mm particle size, 250-mm long analytical column (Bioanalytical Systems, West Lafayette, IN) and the catecholamines were measured using HPLC-EC as described previously [13±17]. After the perfusion procedure, the animals were sacri®ced, their brains were removed, frozen on dry ice and stored at 2108C. Coronal sections of 60±70 mm thickness were cut using a cryostat and stained with cresyl violet to verify the position of cannula in the brain. Data were analyzed using analysis of variance (ANOVA) with repeated measures followed by Fisher's least signi®cant difference test. The locations of the cannulae in the control and the treatment groups were in the arcuate and ventromedial nuclei of the MBH (data not shown). The exact location of perfusion sites did not produce any within-group differences in the release of NE or DA. Therefore, the location of perfusion sites was ignored in data analysis.

Fig. 1. The effects of intrahypothalamic infusion of 0, 1.5, 2.5 or 10.0 mg deprenyl on norepinephrine release (pg/min) in the MBH. **Signi®cantly (P , 0:05) different from the control and 1.5 mg deprenyl groups.

Fig. 2. The effects of intrahypothalamic infusion of 0, 1.5, 2.5 or 10.0 mg deprenyl on the percent change in average release rate of norepinephrine (NE) in the MBH during the entire post-treatment period. **Signi®cantly (P , 0:05) different from the control and 1.5 mg of deprenyl groups.

Pretreatment NE release rates in the control group (2:5 ^ 0:3 pg/min), and 1.5 mg (3:6 ^ 0:81 pg/min), 2.5 mg (3:8 ^ 1:3 pg/min), and 10.0 mg (3:5 ^ 1:3 pg/min) deprenyl groups were not signi®cantly different from each other (Fig. 1). In the control group, there were no differences in the release of NE following the infusion of saline. The NE release rate in this group was 0:3 ^ 0:2 pg/min near the end of the perfusion period. Similar to the control group, the release of NE was not altered in the 1.5 mg deprenyl group but the decline in the release rate was prevented. In contrast to the control and 1.5 mg deprenyl groups, the release of NE increased (P , 0:05) in the 2.5 mg (23:5 ^ 6:8 pg/min) and 10.0 mg (28:1 ^ 5:4 pg/min) deprenyl groups. This increase in NE release that began at 35 min following infusion of 2.5 and 10.0 mg deprenyl was sustained for more than 290 min. The percent change in average NE release during the entire post-treatment period was signi®cantly (P , 0:0005) higher in the 10 mg (437:9 ^ 149:2%) and 2.5 mg (615:7 ^ 102:2%) deprenyl groups than those in the 1.5 mg (182:6 ^ 17:6%) deprenyl group and control (13:2 ^ 9:1%) group (Fig. 2). The pretreatment release rates for DA (pg/min) in the control group, and 1.5, 2.5 and 10.0 mg deprenyl groups (0:8 ^ 0:2, 0:9 ^ 0:1, 0:9 ^ 0:3 and 0:6 ^ 0:1, respectively) were not different from each other (Fig. 3). The DA release pro®le was not altered in the control group, and 1.5 mg, and 2.5 mg deprenyl groups during the entire post-treatment period. In contrast, 10.0 mg deprenyl produced a signi®cant (P , 0:05) increase in the release of DA at 35 min (2:0 ^ 0:4 pg/min). The percent change in average DA release during the entire post-treatment period was signi®cantly (P , 0:0005) higher in the 10 mg (88:5 ^ 17%) and 2.5 mg (53:4 ^ 10:4%) deprenyl groups than that in the 1.5

S. ThyagaRajan, S.K. Quadri / Neuroscience Letters 270 (1999) 79±82

mg (8:6 ^ 8:4%) deprenyl and the control (11:4 ^ 5:8%) groups (Fig. 4). The results of the present study demonstrate that deprenyl stimulates in vivo release of NE and DA in a dose-dependent manner from the MBH in freely moving male rats indicating that its actions are not restricted primarily to MAO inhibition. The rapid increase in the release of NE and DA after intrahypothalamic infusion of deprenyl is because l-deprenyl easily penetrates into tissues and rapidly distributed throughout the body; it readily enters the brain and the spinal cord and the l(2)-form is retained longer within the brain than the d(2)-isomer [6]. To our knowledge, there are no comparable studies on the effects of intrahypothalamic infusion of deprenyl on the in vivo release of catecholamines from the MBH. However, parenteral administration of a high dose of deprenyl was reported to decrease the in vivo release of DA metabolites by inhibiting the activity of MAO in the striatum [2] while in another study, deprenyl did not alter the in vivo release of DA metabolites in the striatum [7]. The observed increase in the release of hypothalamic NE and DA in the present study may be due to the ability of deprenyl to diffuse across neuronal membrane and act intraneuronally to alter Ca 21 release [19]. Another possibility is inhibition of catecholamineuptake into the synaptic cleft that may facilitate an increase in the extracellular pool of transmitters [10]. Similar increase in the release of NE and DA from the MBH was reported when MBH was challenged with various concentrations of deprenyl in vitro [13]. In the present study, the increase in the release of NE and DA was immediate suggesting that initially deprenyl may be directly stimulating the noradrenergic and dopaminergic activity in the hypothalamus but the subsequent sustained increase in the release of these neurotransmitters may result from the inhibition of MAO. The diverse physiological

Fig. 3. The effects of intrahypothalamic infusion of 0, 1.5, 2.5 or 10.0 mg deprenyl on dopamine release (pg/min) in the MBH. **Signi®cantly (P , 0:05) different from the control and 1.5 mg deprenyl groups.

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Fig. 4. The effects of intrahypothalamic infusion of 0, 1.5, 2.5 or 10.0 mg deprenyl on the percent change in average release rate of dopamine (DA) in the MBH during the entire post-treatment period. **Signi®cantly (P , 0:05) different from the control and 1.5 mg of deprenyl groups.

effects observed following chronic treatment of rodents are attributed to MAO inhibition both in the brain and periphery [8,15,17]. MAO is classi®ed into two types, A and B, based on the substrate speci®city. MAO-A degrades serotonin and norepinephrine, while MAO-B catabolizes phenylethylamine. Dopamine is a substrate for both MAO-A and MAO-B. The inhibitory effect of deprenyl on the activity of MAO depends on dose used and duration of treatment [4,20]. The half-life for the turnover of MAO in the brain following direct infusion of deprenyl is not known but it is approximately 10 days in rats and 30 days in primates after a single parenteral injection [1,5,18]. Deprenyl-induced improvement in sexual activity, life span, and behavioral de®cits in old male rats [3,8,9,12] may be due to prevention of age-associated decline in hypothalamic catecholamines that are known to regulate reproduction and reproductive aging processes [11,14,16]. NE is essential for the release of luteinizing hormone-releasing hormone from the hypothalamus during regular estrous cycles in young female rats and a decrease in its release results in cessation of estrous cycles in old rats [11,14,16]. Hypothalamic DA is inhibitory to prolactin secretion from the pituitary. Age-related decline in DA activity and hyperprolactinemia are some of the factors that promote mammary tumorigenesis [11]. We have demonstrated that deprenyl inhibits the metabolism of catecholamines and indoleamine resulting in an increase in the levels of these neurotransmitters in the hypothalamus and a decrease in serum prolactin in old female rats and rats with carcinogen-induced mammary tumors [15,17]. These changes in the concentrations of neurotransmitters by deprenyl were accompanied by reinstatement of estrous cycles and a reduction in the incidence of mammary tumors in old acyclic female rats, and suppression of tumor growth and tumor

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