Regionally specific effects of diazepam on brain serotonin metabolism in rats: Sustained effects following repeated administration

Life Sciences,Vol. 59, No. 15, pp. PL 239~246,19% Copyriat o 1996 ElsevimScienceInc. Printedin the USA_ Au rightsreserved 0024-3zos/% s15.00 t .oa

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ELSEVIER

PhvRiu4coLoGYLETERs Accelerated Communicadon

REGIONALLY SPECIFIC EFFECTS OF DIAZEPAM ON BRAIN SEROTONIN METABOLISM IN RATS: SUSTAINED EFFECTS FOLLOWING REPEATED ADMINISTRATION Darakhshan Department

J. Haleem* and Farhat

Batool

of Biochemistry, Neurochemistry and Biochemical Neuropharmacology Research Unit, University of Karachi, Karachi 75270, Pakistan (Submitted April 30,19%, accepted May 20,19%, received in final form August 8, 1996)

Abstract: The effects of single ( lmgkg) and repeated (lmgkg 2* daily for 4 days) diazepam adminktration are investigated on brain regional 5-hydroxytryptamine (5-HT; serotonin) and 5-hydroxy indoleacetic acid (5-HlAA) concentration in rats. Daily treatment decreased food intakes but body weights did not decrease. Adminktration of diazepam (lmgkg) to 4 day saline injected rats on the 5th day decreased 5-HT levels in the hippocampus and increased it in the hypothalamus. 5-HlAA levels 4 day diazepam injected rats were increased in the striatum and decreased in the hypothalamus. injected with saline on the 5th day also exhibited sihnihu changes of 5-HT and 5-HlAA. Cortical levels of 5-HlAA were also smaller in these rats. Adminktration of diazepam to 4 day diazepam injected rats again decreased 5-HT in the hippocampus and 5-HlAA in the hypothalamus. 5-HT and 5-HlAA were both decreased in the striatum. Regionally specific effects of diazepam on brain serotonin metabolism are discussed in relation to their possiile functions. K~YWords: dimpam, aerototi,

HIT, anxiety, tryptophan, brain regions, hippocampus Introduction

Evidence supporting the involvement of central 5-hydroxytryptamine (5-HT; serotonin) in anxiety related behaviour and in the mechanism of action of benzodiazepine (BZ) anxiolytics is well documented ( 1). Although a number of BZs exist, they share a common property of binding with high aBinity to specific recgnition sites in the brain (2). Pharmacological actions of BZs are mediated via binding of BZ recognition sites and subsequent facilitation of gamma amino butyric acid (GABA) ergic neurotransmission (3). Inhibitory effects of GABA on 5-HT neurons decrease 5-HT cell firing (4). Despite a tkily well documented role of serotonin in the antianxiety effects of BZs, neurochemical studies on the effects of BZs on brain 5-HT metabolism /turnover are not very consistent. Pratt et al (5) observed an increase in the concentration of tryptophan, the precursor of 5HT, following the admit&ration of BZs. Although raised tryptophan levels indicated enhanced 5-HT * To whom correspondence

and reprint request may be addressed

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formation, the synthesis rate of 5-HT monitored as the accumubtion of S-hydroxytrytophan (SHTF’) following the admmistration of NSD 1015 did not always increase (6). It was suggested by Collinge et al (7) that BZs increase brain 5-HT concentration probably by decreasing 5-HT turnover. On the other hand Pratt et al (5) found an increase in 5-HT, 5-hydroxyindoleacetic acid (5-HIAA; a major metabolite of 5-HT) and tryptophan concentration in mice injected with clonazepam Variations in the density of BZ binding sites occur in 5-HT rich regions of the brain (89). Inhibitory effects of BZs (4) exerted at the level of cell bodies and/or nerve terminals may produce regionally dil%rent effects on the metabolism and activity of serotonergic neurons. The present study was, therefore, designed to investigate the effects of diazepam on brain regional 5-HT metabolism in rats. Since these drugs are taken repeatedly before their therapeutic efficacy becomes apparent, effects of repeated admimstration are also monitored. Materials

and Methods

Locally bred male Albino Wistar rats weighing 180-200 g, purchased from Pakistan Council of ScientiRc and Industrial Research (PCSIR) laboratories Pakistan were housed individually under 12 h light dark cycle (lights on at 6:OOh) in a quiet room with bee access to cubes of standard rodent food as described earlier ( 10) and water atleast 4 days before experimentation. Diazepam (F. Holhmum-La Roche Ltd., Base1 Switzerland) available in Smg/ml ampoules, diluted to required concentration in saline was injected i.p. Control animals were injected with saline. Effectv of diazepam on brain regional tryptophan

conceniration:

Rats injected with 1,3 and Smg/kg diazep am or saline were decapitated 1 h later and brain regions dissected out (10) were stored for the fhrorimeteric determination of tryptophan ( 11,12). Effects of single and repeated diazepam

administration

on brain regional S-HTmetabolism:

Daily diazepam treatment was performed by injecting the drug at doses of lmgkg 2*daily for 4 days between 9:00-10:00 h and 17:00-18:00 h. Control animals were injected with saline at the same times. Food intakes and body weights were measured daily each morning before injecting the drug. Daily food intakes are shown as g/lOOg body weight and growth rates as percentage of weights on the preceding day. Aller 4 day drug administration, effects of diazepam challenge on brain regional 5-HI metabolism were determined on the 5th day. Diazepam at doses of lmg&g was injected to a group of 4 day saline injected and another group of 4 day diazepam injected rats. A group of 4 day saline and another group of 4 day diazepam injected rats received saline injection. Animals were decapitated 1 h later. Brain regions dissected out (10) on ice were stored at -70°C for the determination of 5-HI and 5HIAA concentration by high performance liquid chromatography with electrochemical detection (HPLC-EC;lO). Statistical Anafysis:

Data on the effects of various doses of diazepam on brain regional tryptophan concentration were analyzed by one way analysis of variance (ANOVA). Effects of single and repeated diazepam admimstration on brain regional 5-HT and 5-HIAA concentration were analyzed by two way ANOVA. Repeated measure design was adopted for the data on daily changes of food intakes and growth rates. Posthoc comparisons were done by Newman- keuls test. P values > 0.05 were considered insignilicant.

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Results Fg 1 shows the effects of diazepam injected at doses of 1, 3 and SmgIkg on brain regional levels of tryptophan. ANOVA (df 3,20 ) revealed a sign&ant effect on hypothalamic (F=66.2 pO.O1). Posthoc analysis showed that the levels of tryptophan in the hypothalamus and striatum increased at all the doses of drug used. Increases in the cortex were significant at doses of 3 and %&kg whereas, in the hippocampus sign&ant increases occurred only at doses of 5 mg/kg.

Control

Imglkg

DZ

3mglkg

BRAIN REGIONAL

I

Hypothalamus

Hippocampus

DZ

MmBl

Smglkg

DZ

TRYPTOPHAN

Striatum

COlteX

Brain Stem

Fig 1 Effects of diazepam (1,3 and 5 mg/kg ) on brain regional levels of tryptophan Values are means + SD (n=6) 1 h after saline or drug injection. Significant differences by Newman-keuls test, *p
Fig 2 shows the effects of repeated diazepam administration on daily changes of food intakes and growth rates. Analysis by two way ANOVA (repeated measure design) showed a significant effect of drug (F=14 df 1,22 pO.O5), daily administration (F=l.l df 4,88 p>O.O5) and insignificant interaction (Fz1.6 df4,88 p>O.O5) between two factors for daily changes of growth rate. Posthoc analysis showed that diazepam administration at doses of lmg/kg 2*day decreased daily food intake values doring 4 day treatment. Fig 3 shows the effects of ImgIkg diazpam challenge on brain regional 5-HT, 5-HIAA concentration in 4 day saline and 4 day diazepam injected rats. Two way ANOVA on data of the hypothalamus showed s&Scant single (F=lOS dfl,lO pcO.01) and repeated (F=143 df 1,20 pCO.01) administration effect and an insignificant interaction (F=O.X df 1,20 p>O.O5) between two tictors for 5HIAA Significant single (F= 6.6 df 1,20 pcO.05) and insign%cant repeated (F=3.0 df 1,20 p>O.O5) administration effect and insignificant interaction (F=4.0 df 1,20 p>O.O5) was observed for 5-HT. Posthoc analysis showed that admit&ration of diazepam to 4 day saline injected rats decreased 5HIAA and increased 5-HT concentration in the hypothahunus. 4 day diazqam injected rats injected

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with saline on the 5th day also exhibited enhanced 5-HT and reduced 5-HIAA levels. Diazepam injected to 4 day diazepam injected rats resulted in further decrease of 5-JBAA but 5-HT levels did not increase. i-+Saline DAILY CHANGES OF FOOD INTAKE

z .p

-

+ - Diazepam]

DAILY CHANGES OF GROWTH RATE ‘15:

Treat

DAYS

Fig 2 Effects of diazepam (1 mg/kg2*daily ) on daily changes of food intakes and growth rates. Values are means + SD (n=12). Significant differences by Newman-keuls test: *p
Two way ANOVA performed on data of the hippocampus revealed signilicant effect of single (F=53 dfl,20 pcO.01) and insignificant effect of repeated (F=2.8 df 1,20 p>O.O5) administration and significant interaction (F=8 df 1,20 pcO.05) for 5-HT. Signiticant single (F=5.5 df I,20 pcO.05) insignificant repeated (F=4 df 1,20 p>O.O5) admimstration effect and insignificant interaction (F= 0.1 df 1,20 p>O.O5) was observed for 5-HJAA concentration. Posthoc analysis showed that administration of diazepam at doses of 1 mgkg decreased 5-HT concentration in both 4 day saline and 4 day diazepam injected rats. 4 day diazepam injected rats injected with saline on the 5th day also exhibited smaller levels of 5-HT. Differences of 5-HIAA concentration by Newman-keuls test were, however, insignificant.

ANOVA performed on 5-HT and 5-HUA concentrations of the s&turn revealed significant single (F=5.3 df 1,20 pcO.05) and insignificant repeated (F=2.2 df 1,20 p>O.O5) admimstration effect and a significant (F=6 df 1,20 pcO.05) interaction for 5-HT. Insignificant single (F=0.4 df 1,20 p>O.O5), repeated (F=O.S df 1,20 p>O.O5) admimstration effect but a significant interaction (F=33 df 1,20 p
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ANOVA performed on the cortical data revealed sign&ant single (F=8.6 df 1,20 pcO.01) and ins&nificant repeated (F=1.6 df 1,20 p>O.OS) adminktration effect and an ins&&ant interaction single (F=5.3 df 1,20 p
I

4 Day Saline

4 Day Saline

4 Day Diazepan

4 Day Diazepam

+ Saline

+ Diazepam

+ Saline

+ Diazepan

Hypdhalamus

Hippocampus

Sttiatum

B. BRAIN REGIONAL

800

Hypothalamus

Hippocampus

Striatum

Brain Stem

5-HI/U

Cotex

Brain Stem

Fig 3 Effects of lmg/kg diazepam challenge on brain regional 5-HT and 5-HIAA concentrations in 4 day saline and 4 day diazepam injected rats. Values are means f SD 1h after the last diazepam or s+ne injection. Significant differences by Newman-keuls test: *p
ANOVA performed p>o.O5), repeated (F=O.5 df 1,20 p>O.O5) for 5-m adminktration F=O.9 df 1,20

on data of the brain stem revealed insignificant single (F=O.O7 dfl,20 1,20 pBO.05) adminktration effect and insignificant interaction (F=O.S df and 5-m (single admit&ration F=O.4 dfl,20 p>O.O5; repeated p>O.O5; interaction F=1.9 dfl,20 p>O.O5) concentration.

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Discussion Tryptophan and diazepam (13) are both specific ligands for indole site on aIbumin. AdmiGtration of diazepam increases free ttyptophan concentration in plasma because the amino acid is freed horn its binding to plasma albumin. Studies on plasma albumin levels (14) would help to explain the extent of binding. Elevated levels of ttyptophan in many brain regions as observed in the present study (fig 1) may underlie enhanced availabii of free tryptophan to the brain ( 15). Increase in brain tryptophan concentration following the administration of diazepam has been also reported previously (5). The present study shows that these increases occur particularly in the nerve terminal regions. Regional variations were not observed in the effects of diazepam on brain tryptophan concentration (fig 1). Administration of diazepam at doses of 1,3 and 5 mg/kg significantly elevated tryptophan levels in many brain regions. On the other hand, regional variations were exhibited in the effects of diazepam on brain 5-HT and 5-HIAA concentrations.Thus administration of diazepam at doses of 1 mg/kg decreased 5-HT concentration in the hippocampus, and increased it in the hypothalamus. The levels of 5-HIAA decreased in the hypothalamus and increased in the striatum. (Fig 3). The observed decreases of hippocampal 5-HT concentration following the administration of diazepam may not be the resultant of decreased local availability of tryptophan because ttyptophan levels did not decrease in this brain region (fig 1). Enhanced 5-HT release may also not explain these decreases because 5-HIAA levels were not altered. The present data, therefore, show that the conversion of tqptophan to 5-HT is decreased in the hippocampi of diazpam injected rats. Similarly, accumulation of 5-HTP following the admimstration of NSD 10 15 decreased in the hippocampi of rats injected with 3 mgIkg diazepam (16). In addition, the present study shows that a sustained effect on hippocampal 5-HT decreases is produced by the repeated admiration of diazepaa Thus 4 day diazepam injected rats, injected with saline on the 5th day also exhibited decreased hippocampal levels of 5-HT and these were decreased more when 4 day diazepam injected rats were m-injected with diazepam on the 5th day. Evidences suggest that hippocampal changes of serotonin may be involved in the anxiolytic effects of BZs. Serotonergic drugs have been shown to produce anxiolytic effects by stimulating presynaptic somatodemhitic 5-HT receptors in the raphe nucleus resulting in an attenuation of serotonin neuronal 6ring particularly in the hippocampus (17). Lack of diazepam’s effect on the inhibition of 5-HT metabolism in the brain stem as observed in the present study (fig 3) suggests that the inhibitory effects on hippocampal 5-HT levels are possibly manifested by local BZs receptors. Indeed, local infusion of diazepam into the hippocampus also decreased 5-HT synthesis in this brain area whereas, infusion of the drug into the median raphe (origin of serotonergic neurons to the hippocampus) thiled to at&t serotonin synthesis in the hippocampus (15). This is fiuther supported by the fact that only very low density of BZ binding sites occur in the raphe region and brain stem whereas, hippocampus is rich in BZ binding sites (8,9). On the other hand, 5-HT levels in the hypothalamus were increased by the administration of diazepam The increases following single administration are explainable in terms of increased precursor availabii (fig 1) for the neurotransmitter synthesis and decreased release as indicated by decreased 5HIAA concentration (fig 3). Decreases of S-HIAA concentrations but not the increases of 5-H’f were more pronounced in the hypothalami of 4 day diazepam injected rats m-injected with diazepam on the

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5th day. A sustained effect on both 5-HT increases and 5-HJAA decreases was observed in 4 day diazepam injected rats injected with saline on the 5th day. A role of hypothalamus in the anti-anxiety effects of BZs has not been reported. This region of the brain is known to have a role in the regulation of appetite (18). Pharmacological manipulations which tend to increase 5-HT finmtions in the hypothahrmus are anorexiogenic (19). Deccreases of daily food intake in diazepam injected rats as observed in the present study (fig 2) may therefore occur due to enhanced 5-HT fin&on at local hypophagic sites (20). Jnsigniiicant effects of repeated diazepam administration on body weight changes are, however, not explainable by these decreases of food intake. Hypothalamus is also known to have a role in neuroendocrine regulations. Various stress stimuli and serotonergic drugs stimulate hypothahuno-pituitary adrenocortical (H-PA) axis to increase plasma levels of ghrcocotticoids (21). Inhibitory effects of diazepam on the stimuahion of H-PA axis (22) may well explain the lack of diazepam’s effect on body weight changes. Indeed, administration of glucocotticoids has been shown to decrease body weights in rats (23). The increases of striatal 5-HT turnover, as indicated by enhanced 5-HJAA concentration following single administration of diazepam (fig 3) together with increased local levels of tryptophan (fig I ) suggest that BZGABA receptor mediated inhibitory effects (4) on 5-HT neurons are minimal in this brain region. Although sttiatal levels of 5-HJAA were also higher in 4 day diazepam injected rats injected with saline on the 5th day. Admimstration of diazepam to 4 day diazepam injected rats decreased both 5-HT and 5-HJAA concentration in the striatum (fig 3). On the other hand, cortical levels of 5-HLAA were smaller in 4 day diazepam injected rats injected with saline on the 5th day and administration of diazepam to 4 day diazepam injected rats normahzed 5-HIAA levels in this brain region. Striatum is rich in dopamine (DA) nerve terminals. DA and 5-HT are know to interact antagonistically in this brain region to control motor behaviour (24). Diazepaminduced changes of striatal5HT metabolii may well be involved in the sedative effects ofthe drug. Ln conclusion, the present study shows that systemically administered diazepam increases brain regional tryptophan concentration. However, 5-HT and 5-HJAA levels are changed in a region speciftc mamrer. Diazepaminduced changes of brain regional 5-HT metabolism are explainable in terms of 1. an increase in the availability of tryptophan to the brain possibly due to an increase of plasma free tryptophan concentration, 2. inhiiion of serotonin release manifested by the facilitation of GABAergic neurotransmission and 3. interaction of BZs and/or 5-HT with other neurotransmitters. Evidences suggest that inhibitory effects of diazepam on particularly hippocampal 5-HT are anxiolytic (1,16). Changes of 5-HT metabolism in other brain regions may well be involved in the neuroendocrine, anorectic and psychomotor effects of diazpam. The present study also shows that repeated administration of diazepam produces sustained effects on serotonin metabolism in many brain regions. References 1.

2. 3. 4. 5.

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