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Reduction of rat striatal thyrotropin-releasing hormone receptors produced by repeated methamphetamine administration
BKlL PSYCKIATRY 1989:25:191-199
191
Reduction of Rat Striatal Thyrotropin-Releasing Hormone Receptors Produced by Repeated Meth~phet~in~ Ad~inis~ation Makoto Nakashima, Saburo Kajita, and Saburo Otsuki
It has been reported previously that repeated, but not continuous, administration of methamphetamine (MAP) to animals produces progressive and sustained enhancement of MAP-induced behavior (behavioral sensitization), which may be related to functional changes in central dopamine (DA) systems. To investigate the possible involvement of thyrotropin-rele~ing hormone (TRH), a neuro~dulator of DA, both i~unore~tive TRH (IR-TRH) levels and specij?c TRH binding were examined in rat brain regions aj?er MAP administration either repeatedly (4 mglkg intraperitoneally once a day for 14 consecutive days) or continuously (about 4 mglkglday for 13 consecutive days). Although no significant changes were observed in IR-TRH levels in any regions of the brainfollowing repeated MAP injections, speciJc TM binding in the striatum sigm~antly decreased. Scatchard a~iysis revealed that the decrease was due to a redu~~on in the ~imum number of binding sites (B-1. Pretreatment with haloperidol prior to each MAP injection prevented this decrease. Continuous MAP administration had no efSecton regional specific TRH binding. These results suggest that repeated MAP administration caused lasting dysfunction in the brain TRH system, which may be implicated in the behavioral sensitization.
Introduction It has been well documented that both chronic amphetamine (AMP) and methamphetamine (MAP) abuse cause gradual increases in psychotic symptoms (AMP or MAP psychosis) similar to those seen in paranoid schizop~nia (Cannel 1958; Snyder 1973; Janowski and Risch 1979). Once the psychotic state has developed in a patient exposed to AMP abuse, symptoms may recur abruptly, even after years of abstinence, following reexposure to AMP or to psychological stress (Tatetsu 1963; Gold and Bowers 1978; Sato et al. 1983). These observations suggest that chronic AMP abuse produces long-lasting changes in neural systems critically affecting susceptibility to the psychotic state. Similarly, in animals, stereotyped behavior has been shown to be produced and enhanced by repeated (Segal and Mandell 1974; Klawans and Margolin 1975; Klawans et
From the Department of Neumpsycbiitry, Okeyama University Medical School, Okayema, Japan. Supported in pert by a research greet for the biological study of schizopbwnia from the Ministry of Health and Welfare of Japan. Address reprint requeststo Dr. Makoto Nakashima, De+tmem of Ne~yc~~, Okayeme University Me&cal School, 25- 1, Skta-cbo, Okayama, 700, Japan. Received June 8, 1987; revised November 14, 1987. a 1989 Society of Biological Psychiatry
ooo6-3223/89/$03.50
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et al.
al. 1975b), but not by continuous (Nelson and Ellison 1978; Odo et al. 1985), AMP or MAP adminis~ation. Moreover, subsequent AMP adminis~tion following abstinence has resulted in augmentation of stereotyped behavior (Kilbey and Ellinwood 1979; Sato 19’79;Weiner et al. 1979). This behavioral sensitization has been proposed as an animal model of AMP psychosis (Segal and Schuckit 1983; Robinson and Becker 1986). It is well known that AMP and MAP affect the central nervous system (CNS) by enhancing the release of dopamine (DA) into the synaptic clefts, while inhibiting DA “uptake at DAergic nerve terminals (Carlsson 1970). Although some studies have suggested that behavioral sensitization is related to functional changes in central DA systems, there is no convincing evidence for this in the steady state (for review see Robinson and Becker 1986). Recently, several peptides have been found to act as neuro~~smi~ers or neuromoduiators in CNS, and among these, thyrotropin-pleasing hormone (TRH) has been shown to influence the functions of other putative neurotransmitters, especially DA. For example, it enhances the release of DA in the striatum (Fukuda et al. 1979) and nucleus accumbens (Kerwin and Pycock 1979; Sharp et al. 1982). Such a relationship between this neuropeptide and DA may he important in behavioral sensitization. In the present study, in order to investigate the possible involvement of brain TRH systems in MAP-induced behavioral sensitization, we examined (I) changes in regional immunoreactive TRH (IR-TRH) levels and specific TRH binding after daily administration of MAP for 14 days, (2) modification of specific TRH binding after pretreatment with haloperidol (HPD) prior to each MAP injection, and (3) regional specific TRH binding after continuous MAP administration by an osmotic pump for 13 days.
Methods Animals and Drug Administration Male Sprague-Dawley rats weighing 200-250 g were maintained on a 12&r light-dark cycle (6:OOAM to 6:oO PM, light), under conditions of constant temperature (22 2 2°C) and humidity (65% + 5%). Food and water were accessible ad libitum. Drug administration was always at about noon. Experiment I. Twenty rats were divided into two groups: MAP-treated and controls. They were injected int~~~tone~ly with either MAP (4 mg/kg) or saline once a day for 14 days. All animals were left untreated for 7 days, and were then killed by decapitation. Each group was subdivided into two and subjected to either TRH-radioimmunoassays (TRH-RIA) (Ogawa et al. 1984) or specific TRH-radioreceptor assays (TRH-RRA) (Ogawa et al. 1985). Experiment 2. Twenty-one rats were divided into three groups: h~o~~dol (HPD) and MAP-treated (H-M group), HPD and saline-treated (H-S group), and one group treated by saline only (S-S group). They were injected intraperitoneally with either HPD (2 mg/kg) or saline, and subsequently with either MAP (4 mg/kg) or saline once a day for 14 days. All animals were then maintained for 7 days, killed by decapitation, and the brain tissues subjected to TRH-RRA. In both this and in the next experiment, IRTRH was not measured because si~ific~t change was observed in specific TRH bin~ng, not IR-TRH, in Experiment 1.
Behavioral Sensitization and TRH
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Experimenf 3. Twelve rats were divided into two groups. Under anesthesia (pentobarbital 50 mg/kg, ip), cutaneous incisions were made in the backs of all rats, and in one group, osmotic pumps containing 12.4 mg MAP (model 2ML2, ALZA; Dainippon Pharmaceutical Co., Ltd.) were subcutaneously implanted (continuous MAP group). The other rats received no osmotic pumps and served as controls (control group). Thirteen days later, all animals were once again cutaneously incised while under pentobarbital anesthesia, and the pumps were removed. The amount of MAP administered to each rat was estimated to be 3.9-4.1 mg/kg/day. Five days later, all animals were killed by decapitation. In each experiment, the brain was removed following decapitation, placed on ice, and rapidly dissected according to a modification of the method of Glowinski and Iversen (1966) into tissue moities from seven regions; the frontal cortex, nucleus accumbens, striatum, hypothalamus, thalamus and midbrain, amygdala and piriform cortex, and hippocampus. TRH-RIA The brain tissues were immediately homogenized with 10 vol of acidified ethanol (ethanol: 0.1 N HCl = 1:1 v/v) and then centrifuged at 12,000 x g for 20 min at 4°C. The resulting supematant was dried at 40°C under a continuous N2 gas stream (Ogawa et al. 1985). The dried extract was dissolved in RIA buffer [0.14 M phosphate buffer, 25 mu ethylene diamine tetraacetic acid (EDTA), and 0.5% bovine serum albumin, pH 7.41 immediately before the determination of IR-TRH by double-antibody TRH-RIA (Ogawa et al. 1985). To each assay tube was added 0.1 ml RIA buffer, 0.1 ml anti-TRH rabbit antiserum (1: 10,000 dilution) (Utsumi et al. 1975), 0.1 ml TRH solution in concentrations form 36 fg/ml to 36 ng/ml or each sample, and 0.1 ml of ‘251-labeled TRH (15,00018,000 cpm) (New England Nuclear, Boston, MA). After incubation at 4°C for 48 hr, 0.1 ml 5% sheep anti-rabbit y-globulin antiserum was added and incubation continued at 4°C for a further 24 hr. The samples were then centrifuged at 3000 rpm for 30 min, and the radioactivity in each pellet was counted by automatic gamma counter. The antiTRH antibody used in the present study was highly specific for TRH, as previously described (Utsumi et al. 1975). TRH-RRA The procedures for receptor preparation and the TRH receptor binding assay were carried out using methods described previously (Ogawa et al. 1984). Briefly, brain tissues were homogenized in 50 mu Tris-HCI buffer, pH 7.6, and precipitates prepared by centrifugation at 11,500 X g. Samples of each receptor preparation were incubated on ice for 3 hr with 12 nrvr[3H]TRH, either in the presence or absence of unlabeled TRH (100 PM). The reaction was terminated by rapid filtration through Whatman GFK glass fiber filters. Specific binding was determined as the difference in radioactivity bound to receptors in the presence and in the absence of unlabeled TRH. In this TRH receptor assay, the ratio of specific to total binding was usually about 60%. The buffer employed for all dilutions and additions was 50 mu Tris-HCI, pH 7.6, containing 50 pg/rnl bacitracin. The brain region in which specific 13H]TRH binding of MAP group differed significantly from that of control group was reexamined by Scatchard analysis of saturation isotherms (Ogawa et al. 1984).
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Table 1. Regional IR-TRH after the Drug-Free Period for 7 Days following mg/kg) Treatment for 14 Days
Repeated MAP (4
Region
FC
Nucleus accumbens
Control
0.9 ? 0.3
17.8 2 2.9
group MAP
0.7 k 0.2
20.9 t
2.7
Hypothalamus
Thalamus and midbrain
1.5
101.1 ? 4.2
7.2 * 0.9
3.0 t
8.9 + 1.3
89.4 2 8.2
5.2 k 1.0
3.0 + 0.3
Striatum 10.6 ?
AM
andPC 0.4
Hippocampus 2.2 ” 0.4 2.1 t
0.7
group All values are expressed m pg/mg protein as means t SEM(n = 5) FC, frontal cortex; AM, amygdda; PC, piriform cortex.
Protein Assay Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard.
Statistical Analysis All data were subjected to Analysis of Variance (ANOVA), followed by comparisons of individual means using a two-tailed f-test.
Results
Experiment 1 No significant changes were observed in IR-TRH levels in any of the brain regions (the frontal cortex, nucleus accumbens, striatum, hypothakirnus, thalamus and midbrain, amygdala and piriform cortex, and hippocampus) after 7 days of drug-free period following repeated MAP for 14 days (Table 1). Specific TRH receptor binding was also unchanged in all brain regions except the striatum, where a significant decrease was observed (Figure 1). Scatchard analysis of specific TRH binding saturation isotherms in the striatum revealed that this decrease was due to a reduction in the maximum number of binding sites (B,,,,), with no reduction in binding affinity (Table 2).
Experiment 2 Pretreatment with HPD prior to each MAP injection for 14 days prevented the MAPinduced reduction in striatal TRH binding (Figure 1). Treatment with HPD alone for 14 days had no effect on specific TRH binding in any brain region.
Experiment 3 No significant changes in TRH receptor binding activity were observed in any brain region after the drug-free period for 5 days following continuous MAP treatment by osmotic pumps for 13 days (Table 3).
BIOL PSYC?MTRY 1989;25:191-199
Behavioral Sensitization and TRH
195
l-1
*
2
z 8
I
M
4
I
I
Figure 1. Regional specific binding of TRH (% of control) The data from the MAP group are described in Table 2. The aoimals in the H-M and H-S groups were treated for 14 days with either MAP (4 mg/kg) or saline, respectively, following pretreatment with haloperidol (2 mg/kg, prior to each MAP or saline injection). Statistical analysis was performed using the original data.
196
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Table 2. Scatchard Analysis of Specific TRH Binding in the Striatum Group
K,I (nM)
Control
5.0 ‘T 0.4
MAP
4.4 f 0.3
B,,,(fmol/mg protein) 24.9 t 1.1 20.9 + 0.4”
-
n 4 5
Values are means i SEM. “p i 0.02
Discussion In the present study, we found that TRH binding in the striatum significantly decreased after repeated MAP injections for 14 days followed by a 7-day drug-free period (Figure 1) and that this decrease was due to a reduction in the number of TRH receptors (Table 2). Moreover, under the same conditions striatal TRH binding was also significantly reduced after rechallenge with MAP (our unpublished data). Spindel et al. (1981) previously reported a reduction in striatal TRH levels, possibly reflecting an increased release of TRH, following acute D-amphetamine (AMP) administration. Thus, in the present study, long-term repeated MAP administration may have facilitated TRH. TRH receptor has been reported to be specifically down-regulated as a result of chronic administration of TRH (Ogawa et al. 1983). Taken together, these findings suggest that the reduced TRH binding observed in this study might reflect a down-regulation of TRH receptors. Although no significant changes were observed in IR-TRH levels, it is still possible that TRH biosynthesis increased during long-term MAP treatment. It might be difficult to detect such a change using this method, as IR-TRH might reflect the released and storaged TRH. In vivo dialysis, by which many transmitters, such as catecholamines, could be measured in vivo, may detect an increased release of TRH after repeated MAP injections. The reduction of striatal TRH binding after repeated MAP was completely prevented by pretreatment with HPD (Figure 1). This finding is consonant with the report of Spindel et al. (1981). They found that an AMP-induced decrease in striatal TRH content, reflecting an increased release of TRH, could be blocked by pretreatment with either HPD or CYmethyltyrosine. Long-term repeated MAP treatment thus induces and enhances behavioral sensitization (see Introduction), following which, a down-regulation of TRH receptors may be induced by this facilitated TRH release, secondarily caused by changes in DAergic
neurotransmission Unlike repeated MAP injection, continuous AMP or MAP administration has been reported to induce no behavioral sensitization (Nelson and Ellison 1978; Odo et al. 1985). The lack of significant changes in specific TRH binding in any brain region following continuous MAP administration in this study (Table 3) supports the view that the downregulation of TRH receptors may be closely linked to behavioral sensitization by repeated MAP administration. Although many of the behaviors sensitized by AMP and MAP are thought to be caused by release of DA from nigrostriatal neurons, there is no consistent evidence for this in the steady state. As TRH facilitates the release of DA in the striatum (Cohn et al. 1975; Fukuda et al. 1979), it is possible that a relatively long-lasting dysfunction in the striatal TRH system, presumably hyperfunction, underlies the susceptibility to behavioral sensitization. It has been proposed that behavioral sensitization in animals is a suitable model for both MAP psychosis and schizophrenia (Segal and Schuckit 1983; Robinson and Becker 1986). Our results suggest, then, that the TRH system is involved in these diseases.
Behavioral Sensitization and TRH
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and in a ~stmo~em brain study of schi~op~nic patients, changes in TRH-like immunoreactivity were observed in some regions (Manberg et al. 19SS), lending further support to this suggestion. Additional study, such as an examination of the time course of changes in specific TRH binding after cessation of MAP treatment, with measurement of IR-TRH using in vivo dialysis, will be needed to confirm this hypothesis.
We are indebted to Professor M. Sato (Department of Psychiatry, Tohoku University, School of Medicine) and Dr. N. Ogawa (Department of Neurochemistry, Institute for Neurobiology, Okayama University Medical School) for helpful comments and critical reading of the manuscript.
References Carlson A (1970): Amphetamine and brain catecholamines. In Costa E, Garattini S (eds), Amphetamines and Related Compounds. New York: Raven Press, pp 289-300.
Cohn ML, Cohn M, Taylor FH (1975): Thyrotropin releasing factor (TRF) regulation of rotation in the non-lesioned rats. Brain Res %:134-137. Connel PH (1958): Amp~tamine Psychosis. London: Mausdley Monographs, no. 5. Fukuda N, Miyamoto M, Narumi S, Nagai Y, Shima T, Nagawa Y (1979): Thyrotropin-releasing hormone (TRH): Enhancement of dopamine dependent circling behavior and its own circlinginducing effect in unilateral striatal lesioned animals. Folk Pharmacol Jpn 75:251-270. Glowinski J, Iversen LL (1966): Regional studies of catecholamines in the rat brain--J. J Biochem 13:6X5-669. Gold MS, Bowers MB (1978): Neurobiological vulnerability to low-dose amphetamine psychosis. Am J Psychiatry 135:1546-1548.
Janowski DS, Risch C (1979): Amphetamine psychosis and psychotic symptoms. Psychopharmacology 65173-77.
Kerwin RW, Pycock CJ (1979): Thyrotropin releasing hormone stimulates release of [3H]-dopamine from slices of rat nucleus accumbens in vitro. Br J F~r~co~ 67:323-325. Kilbey MM, Ellinwood EH (1979): Reverse tolerance to stimulant-induced abnormal behavior. Life Sci 20:1063-1076. Klawans HL, Margolin DI (1975): Amphetamine-induced dopaminergic hypersensitivity in guinea pig. Arch Gen Psychiatry 32~725-732. Klawans HL, Margohn DI, Dava H, Crosset P (1975b): Sensitivity to D-~phe~ne and apomo~hine-induced stereotyped behavior induced by chronic D-~phe~ine ad~is~ation, J Neural Sci 25283-289. Lowry OH, Rosenbrough NJ, Fan AL, Randall RJ (1951): Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275.
Manberg PJ, Nemeroff CB, Bissette G, Kizer JS, Prange AJ Jr (1985): Neuropeptides in CSF and post-mortem brain tissue of normal controis, schizophrenics and Huntington’s choreics. Frog Neura-P~c~p~r~co~
Biol Fiches
9~97-108.
Nelson LR, Ellison G (1978): Enhanced stereotypes after repeated injections but not continuous amphetamines. Neuropharmacology 17: 1081-1084. Odo S, Nakazawa T, Toru M (1985): Effects of prolonged methamphetamine treatment and its withdrawal on behavior and brain monoamine metabolism in mice. Psychiatr NeurotJpn 87: 15~5 175 (in Japanese). Ogawa N, Mizuno S, Nukina I, Tsukamoto S, Mori A (1983): Chronic th~otropin releasing hormone (TRH) administration on TRH receptors and muscarinic cholinergic receptors in CNS . Brain Res 263:348-350.
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Ggawa N, Mizuno S, Mori A, Kuroda H (1984): Chronic ~y~rgoto~ne ~s~tion sets on receptors for enkephalin and ~~o~pin releasing hormone in aged-rat brain. Pep~~s 5:5356. Ogawa N, Hirose Y, Mot-i A, Kajita S, Sato M (1985): Involvement of thyrotropin-releasing hormone (TRH) neural system of the brain in pentylenetetrazol-induced seizures. Reg Peptides 12:249-256.
Robinson TE, Becker JB (1986): Enduring changes in brain and behavior produced by chronic ~phe~ne a~is~ation: A review and evaluation of animal models of ~phe~ne psychosis. Bruin Res Rev 11: 157-198. Sato M (1979): An experimental study of onset and relapse mechanisms of the chronic methamphetamine psychosis. Psychiatr Neurol Jpn 81:21-32 (in Japanese). Sato M, Chen CC, Akiyama K, Gtsuki S (1983): Acute exacerbation of paranoid psychotic state after long-term abstinence in patients with previous rne~~phe~ne psychosis. Bid Psychiatq 18:429d40. Segd DS, Mandell AJ (1974): Long-term administration of amphetamine: Progressive augmentation of motor activity and stereotypy. Pharmacol Biochem Behuv 2:249-255. Segal DS, Schuckit MA (1983): Animal models of stimulant-induced psychosis. In Creese I (ed), Stimulants: Neurochemical, Behavioral and Clinical Perspectives. New York: Raven Press, pp
131-137. Sharp I’, Bennett GW, Marsden CA (1982): ~~~opin-~ie~ing hormone analogues increase dopamine release from slices of rat brain. J Neurochem 39:1763-1746. Snyder SH (1973): Amphetamine psychosis: A ‘model’ schizophrenia mediated by catecholamines. Am J Psychiahy 130:61-67.
Spindel ER, Pettibone DI, Wurtman RT (1981): Thyrotropin-releasing hormone (TRH) content of rat striatum: Modification by drugs and lesions. Brain Res 216~323-331. Tatetsu S (1%3): Me~~phe~ne psychosis. F&u P~chia~ Neural Jpn suppi 7:377-380. Utsumi M, Tateiwa M, Makimura H, Yamada A, Mori H, Kusaka T, Sakoda M, Baba S (1975): Diurnal variation of the human urinary TRH excretion measured by radioimmunoassay. Enabcrinol Jpn 22509-516.
Weiner WJ, Goetz CG, Nausieda PA, Klawans HL (1979): Amphetamine-induced hypersensitivity in guinea pig. Neurology 29:1054-1057.
191
Reduction of Rat Striatal Thyrotropin-Releasing Hormone Receptors Produced by Repeated Meth~phet~in~ Ad~inis~ation Makoto Nakashima, Saburo Kajita, and Saburo Otsuki
It has been reported previously that repeated, but not continuous, administration of methamphetamine (MAP) to animals produces progressive and sustained enhancement of MAP-induced behavior (behavioral sensitization), which may be related to functional changes in central dopamine (DA) systems. To investigate the possible involvement of thyrotropin-rele~ing hormone (TRH), a neuro~dulator of DA, both i~unore~tive TRH (IR-TRH) levels and specij?c TRH binding were examined in rat brain regions aj?er MAP administration either repeatedly (4 mglkg intraperitoneally once a day for 14 consecutive days) or continuously (about 4 mglkglday for 13 consecutive days). Although no significant changes were observed in IR-TRH levels in any regions of the brainfollowing repeated MAP injections, speciJc TM binding in the striatum sigm~antly decreased. Scatchard a~iysis revealed that the decrease was due to a redu~~on in the ~imum number of binding sites (B-1. Pretreatment with haloperidol prior to each MAP injection prevented this decrease. Continuous MAP administration had no efSecton regional specific TRH binding. These results suggest that repeated MAP administration caused lasting dysfunction in the brain TRH system, which may be implicated in the behavioral sensitization.
Introduction It has been well documented that both chronic amphetamine (AMP) and methamphetamine (MAP) abuse cause gradual increases in psychotic symptoms (AMP or MAP psychosis) similar to those seen in paranoid schizop~nia (Cannel 1958; Snyder 1973; Janowski and Risch 1979). Once the psychotic state has developed in a patient exposed to AMP abuse, symptoms may recur abruptly, even after years of abstinence, following reexposure to AMP or to psychological stress (Tatetsu 1963; Gold and Bowers 1978; Sato et al. 1983). These observations suggest that chronic AMP abuse produces long-lasting changes in neural systems critically affecting susceptibility to the psychotic state. Similarly, in animals, stereotyped behavior has been shown to be produced and enhanced by repeated (Segal and Mandell 1974; Klawans and Margolin 1975; Klawans et
From the Department of Neumpsycbiitry, Okeyama University Medical School, Okayema, Japan. Supported in pert by a research greet for the biological study of schizopbwnia from the Ministry of Health and Welfare of Japan. Address reprint requeststo Dr. Makoto Nakashima, De+tmem of Ne~yc~~, Okayeme University Me&cal School, 25- 1, Skta-cbo, Okayama, 700, Japan. Received June 8, 1987; revised November 14, 1987. a 1989 Society of Biological Psychiatry
ooo6-3223/89/$03.50
192
BIOL PSYCHIATRY 1989:2S:l91-199
M. Nakashima
et al.
al. 1975b), but not by continuous (Nelson and Ellison 1978; Odo et al. 1985), AMP or MAP adminis~ation. Moreover, subsequent AMP adminis~tion following abstinence has resulted in augmentation of stereotyped behavior (Kilbey and Ellinwood 1979; Sato 19’79;Weiner et al. 1979). This behavioral sensitization has been proposed as an animal model of AMP psychosis (Segal and Schuckit 1983; Robinson and Becker 1986). It is well known that AMP and MAP affect the central nervous system (CNS) by enhancing the release of dopamine (DA) into the synaptic clefts, while inhibiting DA “uptake at DAergic nerve terminals (Carlsson 1970). Although some studies have suggested that behavioral sensitization is related to functional changes in central DA systems, there is no convincing evidence for this in the steady state (for review see Robinson and Becker 1986). Recently, several peptides have been found to act as neuro~~smi~ers or neuromoduiators in CNS, and among these, thyrotropin-pleasing hormone (TRH) has been shown to influence the functions of other putative neurotransmitters, especially DA. For example, it enhances the release of DA in the striatum (Fukuda et al. 1979) and nucleus accumbens (Kerwin and Pycock 1979; Sharp et al. 1982). Such a relationship between this neuropeptide and DA may he important in behavioral sensitization. In the present study, in order to investigate the possible involvement of brain TRH systems in MAP-induced behavioral sensitization, we examined (I) changes in regional immunoreactive TRH (IR-TRH) levels and specific TRH binding after daily administration of MAP for 14 days, (2) modification of specific TRH binding after pretreatment with haloperidol (HPD) prior to each MAP injection, and (3) regional specific TRH binding after continuous MAP administration by an osmotic pump for 13 days.
Methods Animals and Drug Administration Male Sprague-Dawley rats weighing 200-250 g were maintained on a 12&r light-dark cycle (6:OOAM to 6:oO PM, light), under conditions of constant temperature (22 2 2°C) and humidity (65% + 5%). Food and water were accessible ad libitum. Drug administration was always at about noon. Experiment I. Twenty rats were divided into two groups: MAP-treated and controls. They were injected int~~~tone~ly with either MAP (4 mg/kg) or saline once a day for 14 days. All animals were left untreated for 7 days, and were then killed by decapitation. Each group was subdivided into two and subjected to either TRH-radioimmunoassays (TRH-RIA) (Ogawa et al. 1984) or specific TRH-radioreceptor assays (TRH-RRA) (Ogawa et al. 1985). Experiment 2. Twenty-one rats were divided into three groups: h~o~~dol (HPD) and MAP-treated (H-M group), HPD and saline-treated (H-S group), and one group treated by saline only (S-S group). They were injected intraperitoneally with either HPD (2 mg/kg) or saline, and subsequently with either MAP (4 mg/kg) or saline once a day for 14 days. All animals were then maintained for 7 days, killed by decapitation, and the brain tissues subjected to TRH-RRA. In both this and in the next experiment, IRTRH was not measured because si~ific~t change was observed in specific TRH bin~ng, not IR-TRH, in Experiment 1.
Behavioral Sensitization and TRH
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Experimenf 3. Twelve rats were divided into two groups. Under anesthesia (pentobarbital 50 mg/kg, ip), cutaneous incisions were made in the backs of all rats, and in one group, osmotic pumps containing 12.4 mg MAP (model 2ML2, ALZA; Dainippon Pharmaceutical Co., Ltd.) were subcutaneously implanted (continuous MAP group). The other rats received no osmotic pumps and served as controls (control group). Thirteen days later, all animals were once again cutaneously incised while under pentobarbital anesthesia, and the pumps were removed. The amount of MAP administered to each rat was estimated to be 3.9-4.1 mg/kg/day. Five days later, all animals were killed by decapitation. In each experiment, the brain was removed following decapitation, placed on ice, and rapidly dissected according to a modification of the method of Glowinski and Iversen (1966) into tissue moities from seven regions; the frontal cortex, nucleus accumbens, striatum, hypothalamus, thalamus and midbrain, amygdala and piriform cortex, and hippocampus. TRH-RIA The brain tissues were immediately homogenized with 10 vol of acidified ethanol (ethanol: 0.1 N HCl = 1:1 v/v) and then centrifuged at 12,000 x g for 20 min at 4°C. The resulting supematant was dried at 40°C under a continuous N2 gas stream (Ogawa et al. 1985). The dried extract was dissolved in RIA buffer [0.14 M phosphate buffer, 25 mu ethylene diamine tetraacetic acid (EDTA), and 0.5% bovine serum albumin, pH 7.41 immediately before the determination of IR-TRH by double-antibody TRH-RIA (Ogawa et al. 1985). To each assay tube was added 0.1 ml RIA buffer, 0.1 ml anti-TRH rabbit antiserum (1: 10,000 dilution) (Utsumi et al. 1975), 0.1 ml TRH solution in concentrations form 36 fg/ml to 36 ng/ml or each sample, and 0.1 ml of ‘251-labeled TRH (15,00018,000 cpm) (New England Nuclear, Boston, MA). After incubation at 4°C for 48 hr, 0.1 ml 5% sheep anti-rabbit y-globulin antiserum was added and incubation continued at 4°C for a further 24 hr. The samples were then centrifuged at 3000 rpm for 30 min, and the radioactivity in each pellet was counted by automatic gamma counter. The antiTRH antibody used in the present study was highly specific for TRH, as previously described (Utsumi et al. 1975). TRH-RRA The procedures for receptor preparation and the TRH receptor binding assay were carried out using methods described previously (Ogawa et al. 1984). Briefly, brain tissues were homogenized in 50 mu Tris-HCI buffer, pH 7.6, and precipitates prepared by centrifugation at 11,500 X g. Samples of each receptor preparation were incubated on ice for 3 hr with 12 nrvr[3H]TRH, either in the presence or absence of unlabeled TRH (100 PM). The reaction was terminated by rapid filtration through Whatman GFK glass fiber filters. Specific binding was determined as the difference in radioactivity bound to receptors in the presence and in the absence of unlabeled TRH. In this TRH receptor assay, the ratio of specific to total binding was usually about 60%. The buffer employed for all dilutions and additions was 50 mu Tris-HCI, pH 7.6, containing 50 pg/rnl bacitracin. The brain region in which specific 13H]TRH binding of MAP group differed significantly from that of control group was reexamined by Scatchard analysis of saturation isotherms (Ogawa et al. 1984).
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Table 1. Regional IR-TRH after the Drug-Free Period for 7 Days following mg/kg) Treatment for 14 Days
Repeated MAP (4
Region
FC
Nucleus accumbens
Control
0.9 ? 0.3
17.8 2 2.9
group MAP
0.7 k 0.2
20.9 t
2.7
Hypothalamus
Thalamus and midbrain
1.5
101.1 ? 4.2
7.2 * 0.9
3.0 t
8.9 + 1.3
89.4 2 8.2
5.2 k 1.0
3.0 + 0.3
Striatum 10.6 ?
AM
andPC 0.4
Hippocampus 2.2 ” 0.4 2.1 t
0.7
group All values are expressed m pg/mg protein as means t SEM(n = 5) FC, frontal cortex; AM, amygdda; PC, piriform cortex.
Protein Assay Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard.
Statistical Analysis All data were subjected to Analysis of Variance (ANOVA), followed by comparisons of individual means using a two-tailed f-test.
Results
Experiment 1 No significant changes were observed in IR-TRH levels in any of the brain regions (the frontal cortex, nucleus accumbens, striatum, hypothakirnus, thalamus and midbrain, amygdala and piriform cortex, and hippocampus) after 7 days of drug-free period following repeated MAP for 14 days (Table 1). Specific TRH receptor binding was also unchanged in all brain regions except the striatum, where a significant decrease was observed (Figure 1). Scatchard analysis of specific TRH binding saturation isotherms in the striatum revealed that this decrease was due to a reduction in the maximum number of binding sites (B,,,,), with no reduction in binding affinity (Table 2).
Experiment 2 Pretreatment with HPD prior to each MAP injection for 14 days prevented the MAPinduced reduction in striatal TRH binding (Figure 1). Treatment with HPD alone for 14 days had no effect on specific TRH binding in any brain region.
Experiment 3 No significant changes in TRH receptor binding activity were observed in any brain region after the drug-free period for 5 days following continuous MAP treatment by osmotic pumps for 13 days (Table 3).
BIOL PSYC?MTRY 1989;25:191-199
Behavioral Sensitization and TRH
195
l-1
*
2
z 8
I
M
4
I
I
Figure 1. Regional specific binding of TRH (% of control) The data from the MAP group are described in Table 2. The aoimals in the H-M and H-S groups were treated for 14 days with either MAP (4 mg/kg) or saline, respectively, following pretreatment with haloperidol (2 mg/kg, prior to each MAP or saline injection). Statistical analysis was performed using the original data.
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Table 2. Scatchard Analysis of Specific TRH Binding in the Striatum Group
K,I (nM)
Control
5.0 ‘T 0.4
MAP
4.4 f 0.3
B,,,(fmol/mg protein) 24.9 t 1.1 20.9 + 0.4”
-
n 4 5
Values are means i SEM. “p i 0.02
Discussion In the present study, we found that TRH binding in the striatum significantly decreased after repeated MAP injections for 14 days followed by a 7-day drug-free period (Figure 1) and that this decrease was due to a reduction in the number of TRH receptors (Table 2). Moreover, under the same conditions striatal TRH binding was also significantly reduced after rechallenge with MAP (our unpublished data). Spindel et al. (1981) previously reported a reduction in striatal TRH levels, possibly reflecting an increased release of TRH, following acute D-amphetamine (AMP) administration. Thus, in the present study, long-term repeated MAP administration may have facilitated TRH. TRH receptor has been reported to be specifically down-regulated as a result of chronic administration of TRH (Ogawa et al. 1983). Taken together, these findings suggest that the reduced TRH binding observed in this study might reflect a down-regulation of TRH receptors. Although no significant changes were observed in IR-TRH levels, it is still possible that TRH biosynthesis increased during long-term MAP treatment. It might be difficult to detect such a change using this method, as IR-TRH might reflect the released and storaged TRH. In vivo dialysis, by which many transmitters, such as catecholamines, could be measured in vivo, may detect an increased release of TRH after repeated MAP injections. The reduction of striatal TRH binding after repeated MAP was completely prevented by pretreatment with HPD (Figure 1). This finding is consonant with the report of Spindel et al. (1981). They found that an AMP-induced decrease in striatal TRH content, reflecting an increased release of TRH, could be blocked by pretreatment with either HPD or CYmethyltyrosine. Long-term repeated MAP treatment thus induces and enhances behavioral sensitization (see Introduction), following which, a down-regulation of TRH receptors may be induced by this facilitated TRH release, secondarily caused by changes in DAergic
neurotransmission Unlike repeated MAP injection, continuous AMP or MAP administration has been reported to induce no behavioral sensitization (Nelson and Ellison 1978; Odo et al. 1985). The lack of significant changes in specific TRH binding in any brain region following continuous MAP administration in this study (Table 3) supports the view that the downregulation of TRH receptors may be closely linked to behavioral sensitization by repeated MAP administration. Although many of the behaviors sensitized by AMP and MAP are thought to be caused by release of DA from nigrostriatal neurons, there is no consistent evidence for this in the steady state. As TRH facilitates the release of DA in the striatum (Cohn et al. 1975; Fukuda et al. 1979), it is possible that a relatively long-lasting dysfunction in the striatal TRH system, presumably hyperfunction, underlies the susceptibility to behavioral sensitization. It has been proposed that behavioral sensitization in animals is a suitable model for both MAP psychosis and schizophrenia (Segal and Schuckit 1983; Robinson and Becker 1986). Our results suggest, then, that the TRH system is involved in these diseases.
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and in a ~stmo~em brain study of schi~op~nic patients, changes in TRH-like immunoreactivity were observed in some regions (Manberg et al. 19SS), lending further support to this suggestion. Additional study, such as an examination of the time course of changes in specific TRH binding after cessation of MAP treatment, with measurement of IR-TRH using in vivo dialysis, will be needed to confirm this hypothesis.
We are indebted to Professor M. Sato (Department of Psychiatry, Tohoku University, School of Medicine) and Dr. N. Ogawa (Department of Neurochemistry, Institute for Neurobiology, Okayama University Medical School) for helpful comments and critical reading of the manuscript.
References Carlson A (1970): Amphetamine and brain catecholamines. In Costa E, Garattini S (eds), Amphetamines and Related Compounds. New York: Raven Press, pp 289-300.
Cohn ML, Cohn M, Taylor FH (1975): Thyrotropin releasing factor (TRF) regulation of rotation in the non-lesioned rats. Brain Res %:134-137. Connel PH (1958): Amp~tamine Psychosis. London: Mausdley Monographs, no. 5. Fukuda N, Miyamoto M, Narumi S, Nagai Y, Shima T, Nagawa Y (1979): Thyrotropin-releasing hormone (TRH): Enhancement of dopamine dependent circling behavior and its own circlinginducing effect in unilateral striatal lesioned animals. Folk Pharmacol Jpn 75:251-270. Glowinski J, Iversen LL (1966): Regional studies of catecholamines in the rat brain--J. J Biochem 13:6X5-669. Gold MS, Bowers MB (1978): Neurobiological vulnerability to low-dose amphetamine psychosis. Am J Psychiatry 135:1546-1548.
Janowski DS, Risch C (1979): Amphetamine psychosis and psychotic symptoms. Psychopharmacology 65173-77.
Kerwin RW, Pycock CJ (1979): Thyrotropin releasing hormone stimulates release of [3H]-dopamine from slices of rat nucleus accumbens in vitro. Br J F~r~co~ 67:323-325. Kilbey MM, Ellinwood EH (1979): Reverse tolerance to stimulant-induced abnormal behavior. Life Sci 20:1063-1076. Klawans HL, Margolin DI (1975): Amphetamine-induced dopaminergic hypersensitivity in guinea pig. Arch Gen Psychiatry 32~725-732. Klawans HL, Margohn DI, Dava H, Crosset P (1975b): Sensitivity to D-~phe~ne and apomo~hine-induced stereotyped behavior induced by chronic D-~phe~ine ad~is~ation, J Neural Sci 25283-289. Lowry OH, Rosenbrough NJ, Fan AL, Randall RJ (1951): Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275.
Manberg PJ, Nemeroff CB, Bissette G, Kizer JS, Prange AJ Jr (1985): Neuropeptides in CSF and post-mortem brain tissue of normal controis, schizophrenics and Huntington’s choreics. Frog Neura-P~c~p~r~co~
Biol Fiches
9~97-108.
Nelson LR, Ellison G (1978): Enhanced stereotypes after repeated injections but not continuous amphetamines. Neuropharmacology 17: 1081-1084. Odo S, Nakazawa T, Toru M (1985): Effects of prolonged methamphetamine treatment and its withdrawal on behavior and brain monoamine metabolism in mice. Psychiatr NeurotJpn 87: 15~5 175 (in Japanese). Ogawa N, Mizuno S, Nukina I, Tsukamoto S, Mori A (1983): Chronic th~otropin releasing hormone (TRH) administration on TRH receptors and muscarinic cholinergic receptors in CNS . Brain Res 263:348-350.
Behavioral Sensitization and TRH
BIOL PSYCHIATRY 1989;25:191-199
199
Ggawa N, Mizuno S, Mori A, Kuroda H (1984): Chronic ~y~rgoto~ne ~s~tion sets on receptors for enkephalin and ~~o~pin releasing hormone in aged-rat brain. Pep~~s 5:5356. Ogawa N, Hirose Y, Mot-i A, Kajita S, Sato M (1985): Involvement of thyrotropin-releasing hormone (TRH) neural system of the brain in pentylenetetrazol-induced seizures. Reg Peptides 12:249-256.
Robinson TE, Becker JB (1986): Enduring changes in brain and behavior produced by chronic ~phe~ne a~is~ation: A review and evaluation of animal models of ~phe~ne psychosis. Bruin Res Rev 11: 157-198. Sato M (1979): An experimental study of onset and relapse mechanisms of the chronic methamphetamine psychosis. Psychiatr Neurol Jpn 81:21-32 (in Japanese). Sato M, Chen CC, Akiyama K, Gtsuki S (1983): Acute exacerbation of paranoid psychotic state after long-term abstinence in patients with previous rne~~phe~ne psychosis. Bid Psychiatq 18:429d40. Segd DS, Mandell AJ (1974): Long-term administration of amphetamine: Progressive augmentation of motor activity and stereotypy. Pharmacol Biochem Behuv 2:249-255. Segal DS, Schuckit MA (1983): Animal models of stimulant-induced psychosis. In Creese I (ed), Stimulants: Neurochemical, Behavioral and Clinical Perspectives. New York: Raven Press, pp
131-137. Sharp I’, Bennett GW, Marsden CA (1982): ~~~opin-~ie~ing hormone analogues increase dopamine release from slices of rat brain. J Neurochem 39:1763-1746. Snyder SH (1973): Amphetamine psychosis: A ‘model’ schizophrenia mediated by catecholamines. Am J Psychiahy 130:61-67.
Spindel ER, Pettibone DI, Wurtman RT (1981): Thyrotropin-releasing hormone (TRH) content of rat striatum: Modification by drugs and lesions. Brain Res 216~323-331. Tatetsu S (1%3): Me~~phe~ne psychosis. F&u P~chia~ Neural Jpn suppi 7:377-380. Utsumi M, Tateiwa M, Makimura H, Yamada A, Mori H, Kusaka T, Sakoda M, Baba S (1975): Diurnal variation of the human urinary TRH excretion measured by radioimmunoassay. Enabcrinol Jpn 22509-516.
Weiner WJ, Goetz CG, Nausieda PA, Klawans HL (1979): Amphetamine-induced hypersensitivity in guinea pig. Neurology 29:1054-1057.