Tryptophan Adjunctive Therapy To Conventional Haloperidol Treatment In Schizophrenia: Effects On Serotonergic Mechanisms In Rat Brain
F Batool, D Haleem
eps functions, haloperidol, schizophrenia, serotonin, trplnaa ratio, tryptophan supplementations
F Batool, D Haleem. Tryptophan Adjunctive Therapy To Conventional Haloperidol Treatment In Schizophrenia: Effects On Serotonergic Mechanisms In Rat Brain. The Internet Journal of Pharmacology. 2007 Volume 6 Number 1.
We studied the effects of dietary amino acid L-tryptophan (TRP) as an adjunct to haloperidol administration on the modulation of extrapyramidal symptoms (EPS) and rat brain serotonin (5-hydroxytryptamine; 5-HT) functions in relation to schizophrenia. TRP added in the drinking water and haloperidol at doses of 5.0 mg/kg or saline were injected for two weeks twice daily with one week of withdrawal to 36 locally bred male albino Wistar rats. Motor/ Exploratory activities were scored in activity boxes and open field apparatuses. Catalepsy was monitored on an inclined surface. Results revealed significant increases (p<0.01) in locomotor activity and marked reduction in catalepsy in rats orally supplemented with TRP for 3 weeks plus haloperidol administration. Significant (p<0.01) increases were observed in the rat brain TRP and 5-HT metabolism. The findings suggest that amino acids, in particular, TRP can possibly attenuate EPS functions induced by haloperidol and enhanced brain 5-HT metabolism.
Modern medical science has made imposing progress in understanding the role of dietary amino acid supplementations in the maintenance of modern health and in the prevention of schizophrenia. Many of the neurotransmitter substances are present in foods, and therefore, can directly influence brain chemistry. Dietary factors 1 affecting the peripheral amino acid balance like carbohydrate and protein content influence the availability of the amino acid L-tryptophan (L-TRP) 1,2 for the central nervous system (CNS) and thus synthesis of 5-hydroxytryptamine (5-HT; serotonin). 2 5-HT is a chemical that produces from its precursor L-TRP in the CNS. The rate at which serotonergic neurons synthesize their 5-HT depends upon the availability of its precursor TRP. TRP is transported from the blood to the brain via an active carrier mechanism specific for this and other large neutral amino acids (LNAAs) like valine (VAL). 2 As a consequence, not the plasma concentration of TRP but the ratio of TRP to the sum of other five LNAAs (valine, leucine, isoleucine, tyrosine and phenylalanine) (TRP/ 5LNAAs ratio) reflects the best concentration of TRP in the CNS. 3,4 The administration of TRP or the consumption of carbohydrate rich diet/ meal all elevate brain TRP levels and, soon thereafter, the levels of serotonin and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA). 2 TRP occurs in low concentration (<1%) in most protein sources. In order to gain access to the brain, it must compete with other LNAAs via a common transport mechanism. Low protein diets sway the ratio of TRP to LNAA in favor of TRP, so that more TRP is transported into the brain (Fig1). Dietary supplementation of TRP can likewise increase the ratio of TRP to other LNAAs, and afford TRP an advantage when vying for entry into the brain. 3 It is, therefore, suggested that serotonin-containing neurons are under specific dietary control. L-VAL, a branched-chain amino acid (BCCA) competes with the TRP for transport into the brain and has previously been shown the decreased brain 5-HT synthesis. 5 However, it is reported that TRP load increases 5-HT synthesis in the brain and therefore may stimulate 5-HT release and functions. 4,6 In our study, effects of these amino acids manipulations are monitored on haloperidol-induced catalepsy and 5-HT metabolism in the medial prefrontal cortical (mPFC) region of rat brain. These findings will help to understand the role of serotonin in the precipitation of neuroleptic-induced catalepsy. The ability of antipsychotic drugs to modulate serotonergic as well as dopaminergic function has been suggested to be important for their efficacy and side-effect profile. 7 Motor-related side-effects are commonly encountered in the treatment of schizophreniform psychoses with so-called “classical” antipsychotic drugs such as haloperidol that are known to block central dopamine (DA) receptors 8,9 with their DA-D2 antagonistic potential. However, it is also known that there are interactions between dopaminergic and serotonergic neurons in the CNS which may be of relevance to the catalepsy syndrome.
There is also suggestive evidence from early laboratory studies for important DA/ 5-HT interaction the mediation of EPS functions, as displayed in the catalepsy model in rats. 10,11 Indeed, substantial evidence has long supported the role of 5-HT in the modulation of haloperidol-induced catalepsy. More recently, it has also been shown that stimulation of presynaptic 5-HT1A, as well as postsynaptic 5-HT2A/2C receptors may involve in the antagonism of catalepsy induced by DA receptor-blocking agents like haloperidol 11 , suggesting a novel principle for attaining clinically effective antipsychotic agents with fewer or no EPS. It seemed pertinent therefore to investigate central 5-HT mechanisms in animals receiving chronic neuroleptic therapy in combination with oral TRP and VAL supplementations. There has been extensive study on the effects of antipsychotics on the mPFC because of the possible importance of this region for cognitive, negative or positive symptoms of schizophrenia. 12 However; there has been relatively less study of the effects of antipsychotics with orally supplemented amino acid agents on the release of serotonin in the mPFC brain region.
While much of the interest with regard to the importance of 5-HT2A and D2 receptor affinities for antipsychotics has focused on the striatum and EPS, there has also been consideration of their importance for the ability of these drugs to modulate serotonergic function in the mesocortical system, and this area is believed to be relevant to cognition, negative symptoms and antipsychotic activity. 12,13 The mPFC is reported to have significant concentrations of 5-HT1A, 5-HT1B, 5-HT2A, 5-HT3 and 5-HT7 receptors. 14 Therefore the increase in extracellular 5-HT levels in the mPFC region can be expected to have significant effects on mesocortical 5-HT neurotransmission. Enhanced behavioral responses and 5-HT functions in mPFC were observed in neuroleptic 5-HT systems 15 but in our study oral supplementations of amino acids, primarily TRP augmented the release of 5-HT in the mPFC . An interaction between 5-HT and DA could partly account for the ability of brain TRP/ LNAAs ratio to increase extracellular 5-HT levels in mPFC in our study. It is also observed in our study that the treatment of animals with TRP, the precursor of 5-HT, produced marked increases in open field ambulation/ exploration and which were significantly more pronounced in rats treated with two weeks haloperidol administration following one week withdrawal. In relation to a connection between dietary BCCA amino acid VAL intake and brain functions, however, to date, only the production of the amine neurotransmitters appears clearly to have been linked to diet. As a consequence of relations between plasma TRP/ 5LNAAs ratio, the ingestion of BCCA VAL causes rapid elevation of their plasma concentrations, increases their uptake into the brain, and decreases the brain uptake and levels of the amino acid TRP. 15 Oral BCCA supplements have been examined as a treatment for neurologic disorder schizophrenia. BCAA VAL have also been administered to schizophrenic patients with tardive dyskinesia, a notable aberration of voluntary motor control develops in these patients taking antipsychotic drugs. 14,15 In our study all these observations are interpreted in the context of amino acids supplementation to antipsychotic therapy.
The present study was designed to test the effects of oral supplementations of amino acids (VAL and TRP) on behavioral responses, plasma and brain mPFC TRP with medial prefrontal cortical 5-HT metabolism in rats treated with two weeks administration of haloperidol following one week of drug withdrawal. The results will possibly suggest the contribution of serotonin and its precursor amino acid TRP as adjuncts for the treatment of EPS functions of conventional neuroleptic drug haloperidol and will also aid in the development of novel agents in relation to schizophrenia and its management.
Materials and Methods
Male Albino-Wistar rats with an average weight of 180±20 gms on arrival purchased from Agha Khan University (AKU), were group-housed (2 rats per cage) in an animal-keeping environmentally controlled room (ambient temperature 21±1 °C and relative humidity 55±5%) on a 12:12h light/ dark cycle (lights on at 7:00AM). A 5 day acclimatization period was allowed before animals were used in experiments. After this period, and 24h before the behavioral tests, the animals were individually housed in an environmentally controlled test room in transparent Perspex cages (dimensions 26×26×26 cm W×L×H). Food (standard rat diet) and tap water were continuously available to animals during experiment. The rats used for the treatment were all experimentally naive animals. All experimental protocols were approved by and performed in strict accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, US National Research Council, 1996) and the Local Ethical Committee guidelines for animal research.
Drugs And Injections
Haloperidol (Serenace; manufactured under license from G.D. Searle and Co. U.S.A, by Searle Pakistan Ltd. Laboratories) available in 5mg/kg ampoules was injected intraperitoneally (i.p.) twice daily between 9:00-10:00am and 3:00-4:00pm hrs in rats for two weeks continuously in the present study. The oral supplementation of drugs VAL and TRP freshly prepared in tap water (200ml measured volume) at dose of 2mg/ml (w/v) were administered orally added in drinking water for 3 weeks on alternate days. Control animals were administered orally with tap water for 3 weeks as the same schedule.
Monitoring Of Motor Activity In A Familiar Environment
The activity boxes used in the present investigation were specifically designed Perspex home cages (26 26 26 cm) with saw-dust covered floor and the experiment was conducted in a separate quiet room. The procedure used was essentially described before. 16 Water, VAL and TRP treated rats daily injected with haloperidol at a dose of 5mg/kg were observed in their home cages for 10 minutes to monitor the activity. During this time period numbers of cage crossings were counted after 3 weeks of treatment and after 2 weeks treatment and one week withdrawal of haloperidol in rats orally administered with water, VAL and TRP.
Monitoring Of Exploratory Activity In A Novel Environment
The procedure used was essentially as described earlier. 17 Experiment was conducted in a quiet room under white light. Rats treated with water, VAL and TRP were exposed to the open field area for 5 minutes. The activity was scored by counting squares crossed by each rat during 5 minutes. Latency to move in seconds and numbers of square crossed with all four paws were counted for 5 minutes. The open field behavior was observed in a balanced design, i.e. behaviors of water, VAL and TRP treated rats in combination with haloperidol injections were monitored alternately with time accuracy.
Rat Catalepsy Determinations
Catalepsy, defined as the acceptance and retention of abnormal posture, was measured by means of a bar test. Bar test determinations were carried out by gently removing rats (n=12) from their home cages and placing their forepaws over a horizontal bar, fixed at a height of 10cm with heads of animals towards upward on an inclined surface at an angle of 60 ° with the hind limbs abducted. 18 The length of time during which the animal retained this position was recorded by measuring the time from the placement of the rat until removal of one of its forepaws. Testing was performed 30 minutes postinjection of haloperidol after 2 weeks of treatment plus one week drug withdrawal, to monitor weekly changes on catalepsy and the time to withdrawal of legs by the rats was measured. A cut-off time of 180 seconds was employed to each rat in the treatment. Rats were removed from the bar if their latency on the bar test exceeded by 180sec.
To investigate the effects of oral supplementations of amino acids (VAL and TRP) on behavioral responses, plasma and brain mPFC TRP and mPFC 5-HT metabolism in rats treated with two weeks administration of haloperidol following one week withdrawal (taken as wash out period of drug). Animals of control (0.9% NaCl) and haloperidol (5mg/kg) treated groups were equally supplemented with VAL and TRP in their drinking water for three weeks continuously at a dose of 2mg/ml freshly prepared in tap water. Wash out period of the drug in the last week was taken as a measure to monitor withdrawal symptoms of haloperidol in rats. Activities were monitored weekly in the familiar (home cage; in terms of numbers of cage crossings/ 10min.) and novel (open field; in terms of latency to move in sec/ 5min. and numbers of squares crossed/ 5min.) environments. Cataleptic effects were also monitored after three weeks and two weeks plus one week withdrawal treatment. Effects on plasma and brain mPFC TRP, mPFC 5-HT and its metabolites were also determined in control and haloperidol treated rats with combine supplementation of VAL and TRP for three weeks. Animals were decapitated after one week withdrawal. Brain regions mPFCs were dissected out and immediately stored at –70 °C for the determination of TRP and 5-HT metabolism by high performance liquid chromatography with electrochemical detection (HPLC-EC). Plasma samples were also stored for TRP determinations. 18
Brain Dissection Technique
Animals were decapitated and the brains were removed immediately. Brain regions were dissected out as described earlier. 18 The cerebellum was pinched out by forceps. The brain dipped in ice cold saline was placed with dorsal side up in the molded cavity of a brain slicer. A fine fishing line wire was inserted into the slots of the slicer to make 1 mm thick slices of brains. Desired brain regions were identified with the aid of a stereotaxic atlas. Olfactory nucleus material was discarded. Medial prefrontal cortices were dissected out with the help of sharp scalpel bilaterally and stored at -70 o C in order to assay biogenic amines by HPLC-EC.
HPLC-EC Determinations Of Plasma TRP, Brain mPFC TRP And 5-HT Metabolism
Brain samples were homogenized as described previously. 18 mPFC 5-HT and its metabolites were determined by HPLC-EC as described before. 18 A 5µ Shim-Pack ODS separation column of 4.0 mm internal diameter and 150 mm length was used. Separation was achieved by mobile phase containing methanol (14%), octyl sodium sulfate (0.023%) and EDTA (0.0035%) in 0.1 M phosphate buffer of pH 2.9 at an operating pressure 2000–3000 psi on Shimadzu HPLC pump. Electrochemical detection was achieved on Shimadzu L-ECD-6A detector at an operating potential of 0.8 V (glassy carbon electrode vs an Ag/ AgCl reference electrode). TRP was determined in a separate run at an operating potential of 1.0V.
The results presented in this investigation are as means ± SD. The neurochemical and biochemical data were analyzed by two-way 2,3-ANOVA. Drug-induced behavioral responses i.e., numbers of cage crossings in home cages, open field activities (numbers of squares crossed) and cataleptogenic effects in Water, VAL and TRP treated rats were statistically analyzed by two-way 2,3- ANOVA (repeated measure design). Values p<0.05 were considered statistically significant.
Activity Reducing And Cataleptogenic Effects Of Orally Supplemented Amino Acids (VAL & TRP) In 3 Weeks HAL Treated Rats And 2 Weeks HAL Plus One Week Withdrawal:
Fig2 shows the effects orally supplemented amino acids (VAL & TRP) in rats treated with repeated administration (for 3 weeks) of haloperidol at a dose of 5mg/kg and 2 weeks haloperidol plus one week withdrawal on Home Cage Activity (in terms of numbers of cage crossings for 10min).
Data analyzed by 2way 2,3 ANOVA (repeated measure design) showed non-significant V+T effects (F=1.4453; df=2,30; p>0.01), significant single drug effect (F=241.41; df=1,30; p<0.01) and insignificant interaction (F=2.395; df=1,30; p>0.01) between V+T and drug effects. Data performed on home cage activity for one week withdrawal was analyzed by 2way 2,3 ANOVA (repeated measure design) showed significant V+T effect (F=28.011; df=2,30; p<0.01), non-significant single drug effect (F=2.9976; df=1,30; p>0.01) and significant interaction between V+T and drug effect (F=127.75; df=1,30; p<0.01).
Posthoc analysis by Newman-Keuls test performed on Home Cage Activity showed that repeated administration of haloperidol at a dose of 5mg/kg significantly (p<0.01) decreased motor activity in rats treated with water, VAL and TRP when compared with similarly treated saline injected rats. In contrast, following one week withdrawal effect of haloperidol significant (p<0.01) increases were observed in both saline injected and haloperidol injected groups orally administered with TRP from respective water treated rats. However, significant (p<0.01) decreases observed in repeatedly injected haloperidol rats orally administered with water and VAL from similarly treated saline injected rats.
Fig 3 shows the daily withdrawal effects of haloperidol following 6 th day on Open Field Activity in terms of latency to move and numbers of squares crossed in rats orally supplemented with water, VAL and TRP.
Data performed on Open Field Activity (latency to move) and (numbers of squares crossed) were analyzed by 2way 2,3 ANOVA (repeated measure design) showed insignificant V+T effect (F=1.474; df=2,30; p>0.01), insignificant single drug effect (F=0.2687; df=1,30; p>0.01) and significant interaction between V+T and drug effect (F=76.55; df=1,30; p<0.01) and insignificant V+T effect (F=2.743; df=2,30; p>0.01), significant single drug effect (F=65.108; df=1,30; p<0.01) and significant interaction between V+T and drug effect (F=37.43; df=1,30; p<0.01) on 6 th day of withdrawal from haloperidol respectively.
Posthoc analysis of data by Newman-Keuls test on open field exploratory activity showed significant (p<0.01) increases in exploratory activity following 6 th day withdrawal from haloperidol injection in rats orally supplemented with water, VAL and TRP for 3 weeks when compared with respective water treated rats.
Fig 4 shows the effects of repeated administration (for 3 weeks) of haloperidol at a dose of 5mg/kg on Catalepsy (immobile posture on an inclined surface) in rats orally supplemented with VAL and TRP added in their drinking water.
Data analyzed by 2way 2,3 ANOVA (repeated measure design) showed non-significant V+T effects (F=2.92; df=2,30; p>0.01), significant single drug effect (F=17120.1; df=1,30; p<0.01) and insignificant interaction (F=14.103; df=1,30; p>0.01) between V+T and drug effects.
Data performed on Catalepsy for one week haloperidol withdrawal were analyzed by 2way 2,3 ANOVA (repeated measure design) showed significant V+T effect (F=6.5379; df=2,30; p<0.01), significant single drug effect (F=247.4; df=1,30; p<0.01) and significant interaction between V+T and drug effect (F=28.56; df=1,30; p<0.01).
Posthoc analysis by Newman-Keuls test performed on Cataleptogenic effect showed that repeated administration of haloperidol at a dose of 5mg/kg significantly (p<0.01) produced 100% catalepsy in rats orally supplemented with water, VAL and TRP when compared with similarly treated saline injected rats. In contrast, following one week withdrawal effect of haloperidol no 100% catalepsy was observed in haloperidol injected groups orally administered with water, VAL and TRP when compared with similarly treated saline injected rats.
Fig 5 shows the effects of orally supplemented amino acids (VAL & TRP) plus one week haloperidol withdrawal on Plasma TRP and brain mPFC TRP (Fig 5) in rats.
Data analyzed by 2way 2,3 ANOVA (repeated measure design) showed significant V+T effects (F=18.797; df=2,30; p<0.01), significant single drug effect (F=13.04; df=1,30; p<0.01) and significant interaction (F=7.68; df=1,30; p<0.01) between V+T and drug effects for plasma TRP (Fig 5).
Data analyzed by 2way 2,3 ANOVA (repeated measure design) showed significant V+T effects (F=14.651; df=2,30; p<0.01), non-significant single drug effect (F=0.7973; df=1,30; p>0.01) and
significant interaction (F=25.162; df=1,30; p<0.01) between V+T and drug effects for mPFC TRP (Fig 5).
Posthoc analysis on data of plasma TRP showed significant (p<0.01) increases in repeatedly saline injected rats orally supplemented with VAL and TRP from respective water treated rats. However, significant (p<0.01) increases were also observed in repeatedly haloperidol injected group of rats orally supplemented with VAL and TRP when compared with respective water treated rats. Brain mPFC TRP showed insignificant (p>0.01) increases in repeatedly saline injected rats orally supplemented with VAL and TRP from respective water treated rats. However, significant (p<0.01) increases of mPFC TRP were observed in repeatedly haloperidol injected group of rats orally supplemented with VAL and TRP when compared with respective water treated rats.
Effects Of Orally Supplemented Amino Acids And Haloperidol Withdrawal For One Week On Brain mPFC 5-HT And Its Metabolites
Fig 6 shows the effects of orally supplemented amino acids (VAL & TRP) plus one week haloperidol withdrawal on brain mPFC 5-HT metabolism (Fig 6) in rats.
Data analyzed by 2way 2,3 ANOVA (repeated measure design) showed significant V+T effects (F=466.68; df=2,30; p<0.01), significant single drug effect (F=31.04; df=1,30; p<0.01) and significant interaction (F=126.13; df=1,30; p<0.01) between V+T and drug effects for mPFC 5-HT. Data analyzed for mPFC 5-HIAA showed significant V+T effects (F=138.66; df=2,30; p<0.01), significant single drug effect (F=23.28; df=1,30; p<0.01) and significant interaction (F=20.31; df=1,30; p<0.01) between V+T and drug effects. Data analyzed for mPFC 5-HIAA/ 5-HT ratio showed significant V+T effects (F=26.91; df=2,30; p<0.01), significant single drug effect (F=23.87; df=1,30; p<0.01) and significant interaction (F=26.23; df=1,30; p<0.01) between V+T and drug effects.
Posthoc analysis on data of brain mPFC 5-HT and its metabolites showed significant (p<0.01) increases in repeatedly haloperidol injected rats orally supplemented with VAL and TRP from respective saline and water treated rats. Significant (p<0.01) increases of mPFC 5-HIAA were observed in repeatedly haloperidol injected group of rats orally supplemented with VAL and TRP when compared with respective water treated rats. However significant (p<0.01) decreases were observed in repeatedly haloperidol injected group of rats orally supplemented with VAL and TRP when compared with respective saline and water treated rats.
The typical effect of administering a dopamine receptor antagonist is a suppression of spontaneous exploratory locomotor behavior and elicitation of a state known as catalepsy in animals. 4,19 Selective antagonists of D2 receptors such as haloperidol can elicit catalepsy. 8,9 Our study has demonstrated that administration of haloperidol at a dose of (5.0mg/kg) for 3 weeks produced maximal (100%) cataleptogenic effects (Fig. 4). However, oral supplementation of amino acids primarily TRP following one week withdrawal from repeated administration of haloperidol exhibited an increase in locomotion and exploratory activity and marked reduction in catalepsy (Fig 2 & 3). The present results on the effects of repeated administration of haloperidol are explainable in terms of either an increase in the responsiveness of postsynaptic 5-HT1A receptors or dopamine D2 receptors or both. We have reported earlier that acute anticataleptogenic effects of 8-OH-DPAT and smaller cataleptogenic effects of clozapine may result because of stimulation of somatodendritic 5-HT1A receptors. 6,20 In the present study augmented motor behaviors reflect an increase in the effectiveness of somatodendritic as well as postsynaptic 5-HT1A receptors following oral supplementation of amino acids with drug withdrawal effect (Fig 6). It is suggested that these changes of receptor responsiveness leading to a decrease in the normal inhibitory influence of 5-HT on motor activity. 21
Other authors have reported that the anticataleptogenic effect was also produced by the local application of 5-HT into the raphe region suggesting that the effects is produced by the stimulation of somatodendritic receptors. 22 Our results show that oral supplementations of amino acids and prior administration of haloperidol for 2 weeks plus one week withdrawal may be of help in the improvement of EPS induced by haloperidol (Fig 4). The most important finding of this study is that the magnitude of the anti-cataleptic effects of amino acid TRP correlates positively with its intrinsic activity towards serotonin receptors. This relationship suggests that amino acid TRP which inhibited haloperidol- induced catalepsy more extensively examined here, did so because of its higher intrinsic activity at 5-HT1A/2 receptors in mPFC. In our study special attention paid to the role of predominant 5-HT2 receptor blockade over D2 blockade. Whereas D2 receptor blockade seems to be essential for the treatment of positive symptoms of schizophrenia, it also underlies the induction of EPS. Predominant 5-HT2 receptor blockade may reduce the EPS liability and can ameliorate negative symptoms of schizophrenia. 22,23 Our results indicate that the cataleptic effects of haloperidol depend on the balance between the dopaminergic and serotonergic systems, and that the serotonergic system exerts an inhibitory influence on the dopaminergic system.
Studies have shown that brain serotonin levels were elevated by chronic haloperidol treatment and correlated very well with the behavioral responses 24 as observed in present investigation. These findings suggest a possible serotonergic involvement in neuroleptic induced short term EPS and late appearing tardive dyskinesia and in amelioration of schizophrenia through TRP supplementations. 25 Early studies have reported the use of a high dose (25mg/kg) of haloperidol twice over a 3-week interval in combination of a dietary TRP supplements. 25 The combination of TRP plus haloperidol was found to cause a long lasting increase in spontaneous chewing movements. These observations are interpreted in the context of TRP supplementation to antipsychotic therapy. Enhanced behavioral responses were observed in chronic neuroleptic-treated rats to drugs believed to act through central 5-HT systems. 26 In addition an increase in specific 5-HT receptor binding was measured, 27 indicating enhanced 5-HT mechanisms in these animals and this is plain in our findings that withdrawal from long term haloperidol therapy may thereby induce central 5-HT receptor supersensitivity in combination with TRP oral supplementation.
The prefrontal cortex plays a crucial role in higher brain functions. 14,28 It receives dense innervations from the brainstem aminergic nuclei, including the serotonergic dorsal and median raphe nuclei of the midbrain. The prefrontal cortex contains a very large density of 5-HT1A and 5-HT2 receptors located on pyramidal neurons. 14 The results of the present study are in line with the view that repeated administration of haloperidol exerts a stronger effect upon the cortical serotonergic system and that this effect is mediated via 5-HT2 (possibly 5-HT2A) receptors. 12,29 A decrease in the number of 5-HT2 receptors will reduce the excitatory effect of 5-HT released from serotonergic terminals on cortical neurons. 29 A hypothesis of cortical-subcortical imbalance with an increase in subcortical 5-HT function responsible for positive symptoms and a decrease in prefrontal 5-HT function responsible for negative symptoms in schizophrenia is proposed. 19,28,29 If enhanced serotonergic transmission in frontal cortex contributes to positive syndromes of schizophrenia, as suggested by studies on hallucinogens, 30 then the therapeutic effects of neuroleptic include not only a modification of the excitability through a direct blockade of 5-HT2A and other receptors located on cortical neurons, 12 but also through a sustained reduction in the responsiveness of these cells to 5-HT. The present study shows time-dependent increases in 5-HT synthesis following the supplementation of amino acids with drug withdrawal effects in mPFC brain region. The increases were more marked in rats repeatedly injected with haloperidol plus oral TRP supplementations (Fig5 & 6). An increase in the concentration of 5-hydroxyindoleacetic acid (5-HIAA), the metabolite of 5-HT, also occurs in the mPFC of rats injected with haloperidol at a dose of 5.0 mg/kg in the present study (Fig 6).
This interpretation can be further enlightened as TRP administration influences brain functions thought to be at least partly controlled by 5-HT neurons. 31 Such effects can be blocked by co-administration of 5-HT antagonists and enhanced by co-injection of 5-HT reuptake blockers. These and other findings thus, strongly support the notion that TRP-induced increments in 5-HT synthesis enhance transmitter release and interaction with postsynaptic receptors. The effects of diet-mediated changes in brain TRP and 5-HT on these brain functions are interesting to elucidate. It is reported that oral administration of TRP-free amino acid mixture significantly decreased basal 5-HT and 5-HIAA levels 100min. after ingestion (65 and 81% of basal value respectively) and remained at this level for another 140min. 32 These results thus show that removal of TRP from the balanced amino acid mixture decreased release of 5-HT in the rat brain. Our results strongly supported the evidence and are in general agreement with the conception that oral administration of amino acids in combination with haloperidol withdrawal from long term administration augmented the uptake of TRP in the brain (Fig5) and consequently increased 5-HT synthesis in mPFC which convincingly suggested that the rate of 5-HT formation varied directly with the availability of circulating precursor TRP. In future studies, it would be interesting to monitor the density of 5-HT1A/2 receptors in different brain regions of rats treated chronically with haloperidol and combination of new dietary ingredients.
Together the neurochemical and behavioral data suggest that increase in serotonergic neurotransmission in the mPFC plays an imperative role in improving EPS functions in rats. This increment in 5-HT metabolism in the mPFC emphasizes the involvement of this region in schizophrenia and its etiology. The results further suggest that dietary supplementation of TRP as a neutraceuticle act as an adjunct may be helpful for the management of schizophrenia. Blockade of mPFC 5-HT2A receptors may contribute to its improved therapeutic profile against negative symptoms and cognitive deficits. Studies involving local administration of serotonin agonists and/or antagonists in the pre- and postsynaptic regions (raphe and striatum respectively) may further strengthen the conclusion.
This study was supported, in part, by grants from the Pakistan Science Foundation (PSF) and University of Karachi, Pakistan.
Dr. Farhat Batool Assistant Professor Neurochemistry and Biochemical Neuropharmacology Research Laboratory, Department of Biochemistry, University of Karachi, Karachi-75270, Pakistan. E-mail: firstname.lastname@example.org Tel (Res): +92-021-4511794 (Mobile): +923333097217