The Effects of Haloperidol on Locomotor Activity and Dopamine Levels in the Brain

by Ashlee McElwain , Cambridge, OH

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Abstract

The purpose of the present study was to determine the effects of haloperidol on locomotor activity and dopamine levels in the brain. Male Sprague-Dawley rats were injected with haloperidol or placebo daily for thirteen days. During this period, the rats were behaviorally tested on the fourth and ninth days for locomotor activity and catalepsy in an open field. Haloperidol treated animals showed a decrease in activity and rears, and an increase in catalepsy. After behavioral testing, extracellular dopamine activity was measured using fast-scan cyclic voltammetry. The treated animals had a lower level of extracellular dopamine compared to the control and a reduced level of following an acute haloperidol challenge.



Antipsychotics drugs are used to treat mental illnesses including the most devastating, schizophrenia. Two kinds of drugs are used to treat this mental illness, which are neuroleptics and atypical antipsychotics. Neuroleptics include haloperidol (Haldol) and chlorpromazine (Thorazine). Atypical antipsychotics include clozapine (Clozaril), risperidone (Risperdal), quetiapine (Seroquel) and Olanzapine (Zyprexa).
Garris, P.A., Budygin, E.A., Phillips, P.E.M., Venton, B.J., Robinson, D.L., Bergstrom, B.P., Rebec, G.V., Wightman, R.M. (2003) were interested in the effects of haloperidol (Hal) on presynaptic mechanisms. Male Sprague-Dawley rats were anesthetized and prepared for surgery. Two types of surgery (survival and nonsurvival) were used in order to demonstrate which method is a better approach to experimentation—microdialysis (survival surgery) or fast-scan cyclic voltammetry (nonsurvival surgery). The two protocol for this experiment was a time course for Hal and the frequency dependence of evoked dopamine based on previous or latter to drug administration. Nomifensine was also used as a drug comparison to Hal. Behavioral analysis was conducted during the monitoring of dopamine. Hal caused an increase in catalepsy as the experiment progressed and there was a strong negative correlation between maximum dopamine levels and the score on the catalepsy test. They found that catalepsy was due to elevated dopamine levels in the synapses because neuroleptics bind to receptors and thus prevented the negative feedback control.
Typical neuroleptics demonstrate a high correlation between catalepsy and D2 receptor binding in the striatum (Torner, Sánchez-Hurtado, Aguilar-Roblero, 1998), which has been identified as the key brain structure for causing neuroleptics-related locomotor deficiencies (Vasconcelos, et al., 2003). This study was interested in characterizing haloperidol-induced catalepsy and determining if it was due to the efficacy or potency of haloperidol. They were also interested in the time course of haloperidol-induced catalepsy and D2 receptors. Male Wistar rats were used in this experiment in which they were decapitated in three-hour intervals in a 24-hour period. The brains were removed and the striatum was dissected out and stored. The striatum was disrupted and samples were centrifuged in buffer with the final pellet being resuspended. Binding assays performed on the striatal membrane were measured for radioactivity and the protein content was determined. Behavioral tests were also conducted in order to measure catalepsy. They used the four-cork method in which four corks are fixed onto a surface and the rats are placed on the corks, one paw on each cork. Catalepsy is measured by the movement of the head and/or paws on time intervals in which the animals were allowed to remain motionless for a maximum of 120 seconds. They found that there were significant diurnal rhythms in both the binding of receptors and haloperidol-induced catalepsy (Torner, et al., 1998).
Repeated daily administration can increase akinetic cataleptic response that is consistently context-dependent. The purpose was to study the effect of dizocilpine in increasing sensitivity of catalepsy. Three separate experiments were conducted using male Sprague-Dawley rats that were injected for seven or eight days and then tested for catalepsy. In experiment one, half of the rats were injected for eight days with haloperidol and MK-801 and the other half was injected with haloperidol and saline. The results indicated that haloperidol caused catalepsy and MK-801 had an anticataleptic effect when paired with haloperidol. Experiment two consisted of two groups of rats in which one was treated for seven days with haloperidol and saline and the other group was injected with haloperidol and MK-801. This differed from experiment one because the animals were tested daily after injections in the same room. The results indicated that there was an increase in cataleptic response in the rats injected with haloperidol, and also on the other group on the bar and grid. However, on day eight when the groups were treated with only haloperidol, the group previously treated with haloperidol and saline showed further increase in catalepsy on the bar, but there was no change on the grid. Experiment three was identical to experiment two with the exception of a reduced dose of haloperidol in an attempt to offset acute anticataleptic effects of MK-801 in the experimental group that received both haloperidol and saline. The results indicated that the cataleptic response was similar to that of experiment two, but on day eight, both groups were treated with half of the dose in experiment two. The group treated with haloperidol showed further increase in catalepsy, but the combined group’s cataleptic response decreased on the bar and one the grid (Schmidt, Tzschentke, Kretschmer, 1999).
The degree of locomotor activity can be tested using an open field and four cork test. Vasconcelos, S. M.M., Nascimento, V. S., Nogueira, Carlos R.A., Vieira, C. M.A.G., Sousa, F. C. F., Fonteles, M. M.F., Viana, G. S.B. (2003) reported that the effects of chronic haloperidol treatment on locomotor activity and catalepsy depend on time of drug withdrawal. High doses of haloperidol decreased locomotor activity, which could be due to inhibition of dopaminergic inhibition of the limbic forebrain. The results indicate that duration of treatment, dose of drug and withdrawal period all effect the relationship between catalepsy and decreased locomotor activity. In this study, male Wistar rats were injected with haloperidol for thirty days. After injections, the rats were behaviorally tested and binding essays were completed during periods of withdrawal- either one hour or one, three, seven or fifteen days. They measured catalepsy by positioning them on a bar and recording the length of time the rat stayed on the bar, in this case, sixty seconds. Locomotor activity was assessed by placing them in another open field with quadrants in which measurements were evaluated by the number of crossings between quadrants. In this study, all animals produced catalepsy after treatment with haloperidol until the third day of treatment where there was no effect.
Neurochemical analysis is useful for measuring neurochemical activity in the brain. After rats are anesthetized, they are mounted in a stereotaxic instrument in which they are immobilized. Skin and muscle layers are removed and holes are drilled according to landmarks on the skull (bregma and lambda) and electrodes (reference, stimulating and recording) are placed at certain coordinates. Fast-scan cyclic voltammetry is the method by which the potential is ramped by applying triangle wave in a forward sweep from –400mV to 1000mV and then a reverse sweep down to –400mV. Charging current, or background current, is the current that normally exists within the environment. Faradaic current is the current measured from the working electrode. Oxidation and reduction reactions are overlapped, and when subtracted from one another, produce the triangle wave in a background subtracted voltamagram. This is used to determine the species of what is being measured because each CV is different according to the species. This procedure allows scientists to experimentally observe how different drugs affect different systems and neurochemicals.
Phencyclidine, or PCP in high doses can cause schizophrenia-like symptoms to occur, allowing scientists to research its effects on the brain and locomotor activity. PCP induced psychosis is nearly indistinguishable from schizophrenia because it produces not only negative symptoms, but also positive symptoms. In this experiment, researchers used male Wistar rats to conduct the study using intracerebroventricularlly injection of drugs and then testing them on a hole board apparatus. The hole board is an open field in which it is divided into nine squares with sixteen evenly spaced holes (Takuma, et al., 2000). The rats were given intracerebroventricular injections of the drugs in which stereotaxic surgery was used to fix a cannula into the left lateral cerebral ventricle in the brain and then held by dental cement and silicon. The rats received injections in a pretreatment room three hours before testing and locomotor and dipping behaviors were observed and measured. The rats that were injected with PCP displayed different behaviors than the control. Locomotor activity increased while dipping behavior decreased, with slight ataxia, and sniffing and grooming were non-existent.
Neuroleptics are more effective in treating symptoms than older drugs; however, they can have severe side effects. Clozapine can be used to treat deficiencies in attention and memory and as of now is the only drug that can be used to treat non-responsive patients. Clozapine does not have the effects of other drugs such as raising prolactin levels, modest effects on body movements and can improve tardive dysinesia. Clozapine does cause other side effects, however, such as lowered blood pressure, weight gain, dizziness, drooling, and possibly seizures. Risperidone has few side effects including weight gain, hypotension and hyperprolactinaemia and extrapyramidal side effects (EPSE) (Gillam, 2002). This drug is used to treat both positive and negative symptoms and may have side effects of fatigue, dry mouth, dizziness, increased heartbeat, and lowered blood pressure (“World Fellowship for Schizophrenia and Allied Disorders [WFSAD], 2002). Quetiapine is used to treat positive and negative symptoms without causing hyperprolactinaemia, but can cause the development of cataracts (WFSAD, 2002). This drug seems to have little effect on weight gain or EPSE compared to other atypicals (Gillam, 2002). Olanzapine can be used to treat positive and negative symptoms and have low rates of side effects such as weight gain, drowsiness, constipation, but does not cause hyperprolactinaemia (WFSAD, 2002). Neuroleptics such as haloperidol (Haldol) and chlorpromazine (Thorazine) may be effective in treating psychosis of schizophrenia, but are not useful in treating the negative symptoms. Using a combination of these or other medications may improve the psychotic symptoms as well as lessen side effects. Tardive dyskinesia is a disorder in which the patient experiences involuntary movements of the body, somewhat resembling Parkinson’s disease, from long-term use of antipsychotics drugs.
The purpose of the present study was to determine the effects of haloperidol on locomotor activity with respect to catalepsy and dopamine levels within the brain. Male Sprague-Dawley rats were injected with haloperidol for fourteen days. They were then behaviorally tested for locomotor activity and catalepsy using an open field. After testing, they were mounted in a stereotaxic instrument and surgically prepared for fast-scan cyclic voltammetry. It was hypothesized that Hal treated animals would have decreased motor activity, but less dopamine in the synapses compared to control when stimulated after injection.

Materials and Methods

Subjects

Young adult male Sprague-Dawley rats (250-300g) were used in this experiment. They were individually housed under constant temperature and a 12:12, light:dark schedule (lights on at 8:00 A.M.). Water and food access was available ad lib. This experiment was approved by Muskingum College IACUC and procedures adhered to the NIH guide for the Care and Use of Laboratory Animals.
Methods

Drug Administration

Haloperidol was dissolved in DMSO. Rats were injected intraperitoneally daily with either .25 mg/kg haloperidol or vehicle control.


Specific Procedure and Results

Behavioral Testing and Analysis

Subjects were tested on the fourth and ninth days of injection. Twenty minutes after receiving their daily injection, animals were placed in a dimly lit room in an open field and observed for five minutes. This open field was a 3ft. x 2.0ft.x 1.0ft plastic tub divided into four 1.5ft x 1.0ft squares. During testing time, locomotor activity was measured by counting the number of squares the rat entered, the number of rears, number of cataleptic moments (freezes) and number of times grooming.
A 2x2 ANOVA (drug treatment x day) with a repeated measure was conducted for each of the variables. The results indicated that there was a drug effect on locomotor activity, F (1,12) = 9.076, p < .05. The haloperidol treated subjects moved into less squares than control (means of 5.875 v 17.855).
There was also a significant day effect on squares because as the days progressed, the subjects moved into less squares. The drug x day interaction was not statistically significant. Mean squares enter for Hal and vehicle treat are depicted in figure 1. There was also a significant difference in rearing between the control and the drug animals, F(1, 12) = 6.872, p < .05. The drugged subject reared less than the control subject, which is depicted in Figure 2 (means of 4.0 v 15.575).
A significant difference was also found between the drug and the control in the number of cataleptic moments F(1,12) = 47.624, p < .05. The results are depicted in Figure 3.



Neurochemical Methodology and Analysis

Following behavior testing, animals continued their daily injection regiment for five more days. On day fourteen, animals were surgically prepared for fast scan voltammetry. Surgical procedures followed a previously described method (Bergstrom et al., 2001). The carbon-fiber recording electrodes were locally constructed according to a similar method described by (Cahill et al., 1996). Fibers (r = 2.5 µm, Thornel T-650 Amoco, Greenville, SC) extended approximately 75-150 µm past the borosillicate capillary tube and were under computer control (Wiedemann et al., 1991). All electrochemistry was performed by an EI 400 potentiostat (Ensman Instruments, Bloomington, IN). Triangle waves were applied at 10 Hz. Voltage was ramped from -400 mV to 1000 mV back to -400 mV at a scan rate of 300 V/s. All recordings were obtained at a 60 Hz frequency (300µA) in the medial caudate putamen. The control and treated animals received the same dose of haloperidol (0.5mg/kg, I.P). Extracellular DA levels were recorded before and after the haloperidol injection. The control animal showed a 63% increase in DA levels after stimulation while the drug treated animals only displayed a 23% increase in DA levels after stimulation.
Each circle represents the concentration of extracellular DA measured by real-time voltammetry at 100 ms intervals. The results for the neurochemical analysis are summarized in figure 1.
Discussion
The results indicated that as time progressed with Hal treatment, locomotor activity decreased. There was a significant decrease in the number of squares the subjects moved into and also a day effect (locomotor decreased across time). The Hal treated subjects also reared less and had more cataleptic moment compared to control animals. The neurochemical analysis revealed that before an acute treatment with Hal, the drugged animals had higher levels of extracellular DA compared to control. However, after an acute injection, the control animals displayed a greater increase in extracellular DA levels than the drug treated animals.
The results indicated that as Haloperidol was administered to subjects, their locomotor activity progressively decreased over the thirteen-day period. This was also the case in the study conducted by Vasconcelos, et al. (2003). They observed that as the animals were injected with haloperidol across time, catalepsy increased and locomotor activity diminished. Garris, et al (2000) also supported these findings. Behavioral analysis was conducted during the monitoring of dopamine. Haloperidol demonstrated an increase in catalepsy as the experiment progressed and there was a strong negative correlation between maximum dopamine levels and the score on the catalepsy test. Schmidt, et al.(1999) also found that there was an increase in cataleptic response in the rats injected with haloperidol and also on the bar and grid. When treated with Hal, groups that were previously treated with Hal and saline, both displayed an increase in catalepsy.
Decreased locomotor activity was associated with the excess of dopamine in the synapses due to the blockade of the D2 dopamine autoreceptor, as also suggested by Garris, et. al. (2000). They found that catalepsy is due to elevated dopamine levels in the synapses because neuroleptics bind to receptors and thus preventing the negative feedback control. Torner, et al. (1998) also found that typical neuroleptics have a high correlation between catalepsy and D2 receptor binding in the striatum, which has been identified as the key brain structure for causing neuroleptics-related locomotor deficiencies. Vasconcelos, et. al. (2003) reported that high doses of Hal decreased locomotor activity, which could be due to inhibition of dopaminergic inhibition of the limbic forebrain. Because of the circuitry in the brain, the excess of extracellular dopamine leads to inhibitory responses to the basal ganglia, causing decreased motor activity.
The results for the neurochemical analysis indicate that chronic treatment with haloperidol causes only a slight increase in DA levels. DA levels did not increase as much as control because the neuron’s receptors down-regulated due to desensitization. Since the receptors were exposed to excessive amounts of dopamine previously for fourteen weeks, the receptors had become less sensitive to dopamine; therefore the Haloperidol injection during surgery did not have much of an affect compared to the control. The control subjects, however, did not receive previous treatment with Hal so they did not experience the desensitization or down-regulation of receptors. This is why the drug treated subject’s dopamine levels only increase 25 % while the control subject’s dopamine level increased 63 %.
Schizophrenia is the most debilitating mental illness that occurs in 1% of the population and affects everyone equally. Medications are necessary in the treatment of Schizophrenia and they can have serious side effects such as parkinsonian symptoms. It was concluded that Haloperidol, a D2 receptor antagonist, causes a decrease in motor activity due to the down-regulation of the D2 receptor and inactivation of the basal ganglia. It was also concluded that the drug treated subject was less sensitive to Haloperidol because of the down regulation of the D2 receptor caused by the prolonged exposure of excess dopamine levels in the synapses (desensitization). The control animals were not desensitized to the drug action, therefore dopamine levels were incredibly high.






References

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Figure Captions


Figure 1: Mean number of squares entered for Hal and vehicle treated animals on the two days of testing
Figure 2: Mean number of rears for Hal and vehicle treated animals on the two days of testing
Figure 3: Mean number of cataleptic moments for Hal and vehicle treated animals on the two days of testing
Figure 4: Synaptic effects of Haloperidol in Control and Chronically Treated Animals. Solid circles represent traces collected prior to administration of drug. Open circles represent traces collected after drug was administered. Each trace contained a background subtracted voltammogram that was determined to be DA