Dopamine increase in the prefrontal cortex correlates with reversal of haloperidol-induced catalepsy...

- Brain Research Bulletin, Vol. 35, No. 2, pp. 125-133, 1994

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Dopamine Increase in the Prefrontal Cortex Correlates With Reversal of Haloperidol-induced

Catalepsy in Rats

SONIA TUCCI,’ RICHARD FERNANDEZ, TRINO BAPTISTA, EURO MURZI AND LUIS HERNANDEZ

Laboratory of Behavioral Physiology, Los Andes University, Me’rida, 5101-A, Venezuela

Received 12 February 1994; Accepted 16 March 1994

TUCCI, S., R. FERNANDEZ, T. BAPTISTA, E. MURZI AND L. HERNANDEZ. Dopamine increase in rhe prefrontal cortex correlates with reversal of haloperidol-induced catalepsy in rats. BRAIN RES BULL 35(2) 125- 133, 1994.-The mechanism by which forced swimming reverses haloperidol-induced catalepsy was examined by measuring dopamine (DA) turnover in the nucleus accumbens-ventromedial caudate (NAC-C) and the prefrontal cortex (PFC) in rats. DA and its metabolites 3,4-dihydroxi- phenylacetic acid (DOPAC) and homovanillic acid (HVA) were assessed by microdialysis and high pressure liquid chromatography with electrochemical detection (HPLC-ED) after systemic administration of a cataleptic dose of haloperidol (5 mg/kg) or saline. Haloperidol-induced catalepsy was temporarily suppressed by forced swimming. Haloperidol-treated rats showed an increase of DA, DOPAC, and HVA overflow in the PFC and the NAC-C. This increase was greater in the PFC of rats that were forced to swim. Rats that were not treated with haloperidol but were forced to swim (control group) showed an increase of DA, DOPAC, and HVA in the PFC but not in the NAC-C. Zero micrograms, 5 pg, 10 pg. and 20 pg of DA was bilaterally injected in the PFC of cataleptic rats to evaluate the hypothesis that DA in the PFC reverses catalepsy. Haloperidol-induced catalepsy was diminished by bilateral microinjections of IO pg and 20 pg but not by 5 pg of DA in the PFC. The higher the dose of DA, the longer the decrease of catalepsy. These results suggest that an increase of DA turnover in the PFC might mediate temporal suppression of haloperidol-induced catalepsy. The mechanism by which the mesocortical DA system reduces catalepsy is discussed.

Haloperidol Mesocortical dopamine Catalepsy reversal

Mesostriatal dopamine Mesolimbic dopamine Microdialysis

CATALEPSY is a condition of muscle rigidity in which vol- untary movements are impeded and the limbs and the trunk preserve awkward positions when passively moved (22). Cat- alepsy occurs in terminal Parkinson disease and neuroleptic intoxication (3,5,39). A similar clinical symptom has been de- scribed in catatonic schizophrenia and in other psychiatric and neurologic disorders (20,22). Catalepsy has been attributed to a lack of dopamine or to blockade of dopamine receptors in the corpus striatum or in the nucleus accumbens (4). Catalepsy reversal by I-DOPA, bromocriptine (a dopaminergic agonist), and anticholinergic drugs administration could be due to en- hancement of DA transmission in the striatum and the nucleus accumbens (10,28). This phenomenon has suggested an an- tagonistic relationship between dopamine and acetylcholine in the basal ganglia (7 1,75).

Stress suppresses catalepsy. The mechanism seems to be an enhancement of dopamine transmission because stress increases dopamine turnover (6,66,74). Interestingly, the increase in do- pamine turnover during stress seems to occur in specific dopa- minergic areas rather than been generalized. DOPAC has been reported to increase in prefrontal cortex homogenates but not in the striatum or the nucleus accumbens in rats (74).

Forced swimming in water transiently reverses catalepsy. This provides an animal model of catalepsy that is reversible by stress. Several experiments have shown that the prefrontal cortex might play a role in haloperidol-induced catalepsy reversal. Prefrontal cortex lesions attenuate haloperidol-induced catalepsy (66,78), suggesting that the output of the PFC might induce catalepsy. The main output of the PFC is a corticostriatal glutamatergic pathway. Activation or blockade of this pathway might contrib- ute or suppress catalepsy, respectively. In fact, the antagonists of NMDA receptors reverse haloperidol-induced catalepsy (25,54,67).

The development of in vivo monitoring techniques allows the evaluation of changes of DA turnover in freely moving rats (34,35). Haloperidol and stress effects have been evaluated in three dopamine terminal fields, i.e., prefrontal cortex, nucleus accumbens, and striatum. Haloperidol injections and stress in- creased dopamine turnover in these three brain areas (36,37). However, the stress-induced DA increase was greater in the pre- frontal cortex than in the striatum or the nucleus accumbens (1,18).

In the present experiments we assessed, by brain microdi- alysis, the dopamine turnover in the nucleus accumbens-ventro-

’ Requests for reprints should be addressed to Sonia Tucci, Apartado 109, MCrida. 5 101 -A. Venezuela.

125

medial caudate and the prefrontal cortex after a haloperidol sys- temic injection. A second experiment examined the effects of DA microinjections directly into the prefrontal cortex on haloperidol induced catalepsy.

METHOD

Subjects

Sixty-four male albino rats weighing between 250 and 350 g were individually housed with food and water ad lib. The room temperature was 23°C and the dark:Iight cycle was 12: 12 h.

Under ketamine (100 mg/kg) anesthesia the rats were placed on a stereotaxic instrument. For the microdialysis experiment, a guide shaft made of a 10 mm long, 21 gauge stainless steel tube was placed within the nucleus accumbens-ventromedial caudate (NAC-C) or the prefrontal cortex (PFC) of the rat. For the NAC- C and the prefrontal cortex the coordinates were: A: 10 mm, L: 1.2 mm and V: 3 mm; and A: Il.6 mm, L: 0.5 mm, V: 0.5 mm, respectively. The references were: A: anterior to the interaural axis, L: late& to the midsaggital suture, and V: ventral to the surface of the brain. For the ~croinje~tion experiment, guide shafts made of 20 mm long, 26 gauge stainless steel tube were bilaterally implanted within the medial prefrontal cortex of 26 rats. The coordinates were: A: 11.6 mm; L: 0.5 mm; and V: 3.0 mm. All coordinates were calculated according to the Paxinos and Watson Atlas (61).

~icrodialys~s Probes

The microdialysis probes were made of concentric fused silica capillary within a piece of 26 gauge stainless steel tubing (35). A piece of a cellulose hollow fiber was attached to the tip of the stainless steel tube. The effective length of the dialysis fiber was 4 mm. The fused silica capillary was 12 cm long, 75 pm id. and 150 pm o.d. It was strengthened with a polyimide cover. The microdialysis probe was needle shaped to facilitate insertion in the awake animal.

Microdialysis and Analysis Procedures

The day of the experiment the rat was placed in a cylindrical perfusion chamber. The inlet tube of a swivel joint was connected to a syringe filled with a modified Ringer solution containing 146 mM NaCl, 3.4 mM KC1 and 1.2 mM CaC12 at pH 6.0. The outlet tube of the swivel was connected to the inlet tube of a microdi- alysis probe. The syringe was placed in a syringe pump and the flow rate was set at 1 @min. Then the microdialysis probe was inserted into the guide shaft. The microdialysis probes protruded 5 mm from the tip of the guide shaft. Samples were collected every 20 min and analyzed by High Pressure Liquid Cluomatog- raphy and Electrochemical Detection (HLPC-ED). The HPLC system consisted of a model 222D, single piston, SSI pump con- nected to a model 1725 Rheodyne valve equipped with a 20 ~1 loop. The chemicals were separated in an ODS, 3 pm particle, 3.2 mm bore, 10 cm long Brownlee column. They were detected in a model 400, Princeton Applied research eI~~~hernic~ de- tector on a glassy carbon electrode set at 705 mV respect to a Ag-AgC1 reference electrode. DOPAC, DAm, and HVA were measured by comparison of the peak heights of the samples with the peak height of standards.

When the chemicals in four consecutive samples showed less than 10% variation, 28 animals received 5 m&g of haloperidol (H~dol~) in~a~~ton~ly. After the injection of h~o~~dol, ten

more samples were taken. Fifteen minutes alter the haloperidot injection, eight rats with microdialysis probes in the NAC-C and

five rats with probes in the PFC were placed 12 times every 5 mm into a water bath (forced swimming~ at room temperature (23°C). The rats swam to reach the border of the water container. Then, they were taken out and placed on a dry towel. Eight rats with probes in the NAC-C and seven with probes in the PFC were not forced to swim, and the catalepsy was assessed every 5 min. Catalepsy was evaluated by the following signs: immo- bility, back arching, limb extension, and preservation of an im- posed bizarre posture. This last sign was evaluated by putting the hind legs of the rat on a 7 cm high glass box. If all the signs were present and the hind legs of the rat remained on the box more than 60 s, it was considered cataleptic. Six rats with probes in the NAC-C and five rats with probes in the PFC were used to control the effect of forced swimming on DA activity in the me- solimbic and mesocorticai systems. The mi~rodialysis procedure and the forced swimming test in these animals were the same as in the previous ones except that they received intraperitoneal sa- line injections instead of haloperidol. For DA, DOPAC, and HVA the values were expressed as picograms in 20 ~1 without further correction, and then as a percent of the tirst sample. Sta- tistical analysis was done by one factor with repeated measure- ments ANOVA followed by Dunnet test, and time was the factor. For comparison of the haloperidol and forced swimming group vs. the haloperidol without forced swimming group, two-factor ANOVA tests followed by contrast analysis were used

Microirzjection Procedure

Twenty-five minutes after an intra~~toneal injection of halo- peridoi (5 m&g) two injectors made of 33 gauge stainless steel tubing protruding 2 mm from the tip of the guide shaft were bilaterally inserted into the PFC. Seven rats received bilateral vehicle (saline) injections; seven, 5 pg; six, IO /lg and six, 20 pg of dopamine dissolved in 0.5 ~1 of Ringer solution at the flow rate of 0.5 pl/min. These doses were selected for the following reasons. Microinjection expe~ments with narrow injectors com- monly are in the range of micrograms to assure a good concen- tration of neurotransmitter in the synaptic gap. For instance, to inhibit feeding 40 hg of dopamine or more has to be bilaterally injected into the lateral hypothalamus (62). Moreover, doses of 20 pg into the nucleus accumbens have been used to increase general locomotion in rats (41). Five minutes after the PFC in- jection, the forepaws of the rat were placed on a horizontal wood beam, 12 cm long, 1 cm wide, placed at 9 cm above the floor. The time that the rat remained in this position (descent latency) was measured to a maximum of 300 second (79). The test was repeated every half hour during a period of 90 min. The scores of the rats were compared by two-way ANOVA.

The rats were sacrificed with an overdose of chloroform 1 day after the microdialysis or the microinjection experiment and per- fused with a 10% formalin solution. They were decapitated and their heads included in formalin. After 5 days the brains were dissected out, immersed in formalin for 2 more days, frozen, and cut in 40 pm slices. The tracks of the probes or the tips of the injector cannulas were localized on the wet, unstained slices by the birefringency method.

RESULTS

Microdialysis Experiment

Fifteen minutes after the haloperidol injection all rats dis- played catalepsy, i.e., all the rats showed immobility, back arch-

MECHANISM OF CATALEPSY 127

PFC t NAC-C l

o- 0 100 200 300 Oh 0 100 300

MINUTES MINUTES

FIG. 1. After the administration of haloperidol (open circle curves) DA increased in the NAC-C (right side) and the PFC (left side). Forced swimming in the control group (open squares) increased DA only in the PFC. The group in which catalepsy was reversed by forced swimming (solid circles) showed levels of DA in the PFC significantly higher than the cataleptic group (open circles). These differences were not observed in the NAC-C. In addition, forced swimming prolonged DA activity en- hancement caused by systemic halopetidol. Asterisks indicate statisti- cally significant (p < 0.02) differences between catatonia suppressed (solid circles) and catatonic (open circles) groups.

ing, limb extension, and preservation of a bizarre posture for more than 60 s. These symptoms lasted for the rest of the exper- iment (200 min) in the rats that were not forced to swim. The rats receiving saline injection (control group) developed explor- atory activity for an average of 5 min after the injection. Those rats placed in water (both the ones receiving haloperidol and the control group) swam energetically to reach the border of the wa- ter container and often tried to jump out of the water. Once out of the water, they engaged in wet dog shakes and grooming be- havior and, after a few seconds, the rats that received haloperidol returned to their cataleptic state. The haloperidol-injected rats vocalized each time they were forced to swim. In contrast, the saline-injected rats never vocalized during the forced swimming test.

Dopamine

The basal levels of DA were higher in the NAC-C (10.3 2 2.2 pg/ZO ~1) than in the PFC (2.5 t 0.2 pg/20 pl), F(1, 27) = 18.59, p < 0.001. After the haloperidol injection, there was a significant increase of DA in the cataleptic groups in the PFC, F(13, 78) = 3.60, p -c 0.001, and in the NAC-C as well, F(13, 84) = 2.17, p < 0.02. Forced swimming increased DA in the PFC of the rats that received saline, F(6,Zl) = 8.69, p < 0.001, and in the rats that received haloperidol, F(13, 70) = 1.93, p < 0.05. However, there was a significant change of DA in the NAC- C of none of the two groups (saline injected control or haloper- idol injected and forced to swim group). The time course of the PFC DA increase was different in the three groups. DA increase for the hour of forced swimming in the forced to swim group that received saline. When forced swimming stopped, DA im- mediately returned to base line levels (see Fig. 1). DA increased and reached an asymptotic level in last five samples in the group that received haloperidol and was not forced to swim. In the group that received haloperidol and was forced to swim, DA increased in the first three samples and then continued increasing

in the last five samples. When the DA in the NAC-C of the haloperidol injected but not forced to swim group vs. the halo- peridol injected and forced to swim group were compared there was no significant difference, F(1, 112) = 3.01, NS. However, DA in the PFC of the haloperidol plus forced swimming group was significantly higher than in the haloperidol injected but not forced to swim group, F( 1, 96) = 28.78, p < 0.001 (see Fig. 1).

DOPAC

The basal levels of DOPAC were significantly higher in the NAC-C (1368.6 2 186.2 pg/20 ~1) than in the PFC (226.27 2 38.8 pg/ZO pl), F(1,27) = 31.3,~ < 0.001. There was an increase of DOPAC in the NAC-C in the rats receiving haloperidol but not forced to swim, F(l3, 98) = 6.93, p < 0.001, as well as in the rats receiving haloperidol and forced to swim, F(l3, 98) = 11.12, p < 0.001. By contrast, there was no increase of DOPAC in the NAC-C of the rats that received saline and were forced to swim. However, DOPAC increased in the PFC in the three groups: in the rats that received haloperidol and were not forced to swim, F(13, 98) = 19.23, p < 0.001; in the rats that received haloperidol and were forced to swim, F(13, 56) = 26.08, p < 0.001, and in the rats that received saline and were forced to swim, F(6, 28) = 10.31, p < 0.001. The time course of these DOPAC increases was different for the three groups and fol- lowed a similar pattern to the DA increase (see Fig. 2). When the DOPAC in the NAC-C of the haloperidol-injected but not forced to swim group vs. the haloperidol-injected and forced to swim group were compared, there was no significant difference. How- ever, DOPAC in the PFC of the haloperidol injected plus forced swimming group was significantly higher than in the haloperidol, but not forced to swim group, F( 1, 88) = 129.07, p < 0.001 (see Fig. 2).

HVA

Basal levels of HVA were also significantly higher in the NAC-C (705.49 k 102.1 pg/20 ~1) than in the PFC (223.4 2

PFC NAC-C *

INJECTION * INJECTION

400 -

0 100 200 300 Oi-x---- 200 300

MINUTES MINUTES

FIG. 2. After the administration of haloperidol there was a significant increase of DOPAC in all groups. Forced swimming alone (open squares) increased DOPAC only in the PFC. Again, DOPAC was significantly higher in the PFC dialysates of the group in which catalepsy was reversed by forced swimming (solid circles). In addition, DOPAC increased more in this group than in the group that received haloperidol but was not forced to swim (open circles). Asterisks indicate statistically significant (p < 0.02) differences between catatonia suppressed (solid circles) and catatonic (open circles) groups.

128

PFC * NAC-C

o- a 155 200 355

MINUTES

INJECTION

0- 0 lQ0 205 300

MINUTES

FIG. 3. After the administration of haloperidol there was a significant increase of HVA in all groups. Forced swimming alone (open squares) increased DOPAC only in the PFC. Again, HVA was significantly higher in the PFC dialysates of the group in which catalepsy was reversed by forced swimming (solid circles). Asterisks indicate statistically signifi- cant (p <: a.&?) differences between catatonia suppressed (solid circles) and catatonic (open circles) groups.

20.5 pg/20 ~0, F(1, 27) = 18.59, p < 0.001, HVA showed a trend similar to DOPAC. After the injection of haloperidol there was an increase of HVA in the NAC-C in the rats receiving haloperidd but not forced to swim, F(13,98) = 6.54, p < 0.001, as weli as in the rats receiving h~ope~do~ and forced to swim, F{ 13,98) = 4.77, p < 0.001. By contrast, there was no increase of WA in the NAC-C of the rats that received saline and were forced to swim. However, HVA increased in the PFC in the three groups: in the rats that received haloperidol and were not forced to swim, F( 13,98) = 19.23, p < 0.001; in the rats that received haloperidol and were forced to swim, F(l3, 56) = 26.08, p < 0.001, and in the rats that received saline and were forced to swim, F(7,32) = 2.35, p < 0.03. The time course of these HVA increases was different for the three groups and followed a sim- ilar pattern to the DA increase (see Fig. 3). There was no signif- icant difference between levels of HVA in the NAC-C of the haloperidol injected but not forced to swim group vs. the halo- peridol injected and forced to swim group. However, HVA in the PFC of the halo~~dol-injects plus forced swimming group was significantly higher than in the not forced to swim group, F( 1, 88) = 8 1.38, p < 0.001) (see Fig. 3).

The microdialysis probe tips in the FFC were focated between f 1.2- 11.7 mm A, 0.2-l mm L, and 5-7 mm V. The probes dialyzed the following structures: cingulate cortex 1 and 3, in- fralimbic, and dorsal peduncular cortex. In the NAC-C, the probes were located between 10.2-10.6 mm A, l-3 mm L, and 7.5-9 mm V and dialyzed the following structures: ventromedial caudate, nucleus accumbens core and shell, and olfactory tuber- cle (see Figs. 4 and 5).

Microinjection Experiment

Fifteen minutes after the injection of haloperidol the rats dis- played catalepsy as measured by the descent latency. This latency increased progressively in the group that received bilateral injec- tion of Ringer solution in the PFC and in the group that received 5 pg of dopamine. Descent latency was not statlsticdly different

when these two groups were compared. However, 30 min afkr the injection of haloperidot, the descent latency was significantly shorter in the group that received bilateral injections of IQ pg of dopamine in the PFC, F(1, 1 1) = 4.90, a /se 0.05, and in the group that received 20 pg, F( I, 1 1 j = 34.9, p .:: 0.00 I Sixty minutes after the bilateral injection of 20 p,g of dopamine, the descent latency was significantly shorter, F(l( 1 11 = 1 X.75, p s’ 0.01, than in the group that received intracerebral Ringer (see Fig. 6).

The microinjection cannula tips in the PFC were iocated bi- laterally between 11.2-12.2 mm A, 0.2- 1 mm L, and 3-6 mm V (the cannula tip). The cannuIas passed through the following structures: cingulate cortex 1 and 3, infralimbic and dorsal pe- duncular cortex (see Fig. 7).

DlSCUSSION

These results show that forced swimming increased DA, DOPAC, and HVA overflow in the PFC! but not in the NAC-C region. A single ~ntra~~toneal injection of 5 mg/kg of haloper- idol caused catalepsy and increased DA, DOPAC, and HVA overflow in the PFC and the NAC-C. These two effects were not additive but were synergistic in the PFC, i.e., the increase of DA, DOPAC, and HVA overflow in the PFC of the haloperidol in- jected and forced to swim rats was greater and longer than in the saline-injected and forced to swim rats as well as in the hduper- idol-injected but not forced to swim rats. In the NAC-C, forced

FIG. 4. Diaeram showine the tracks of the microdialysis cammias in the rat prefrontal cortex. The figures indicate the ank&r posterior coordi- nate of the brain slice according to the Paxinos and Watson atlas.

MECHANISM OF CATALEPSY 129

FIG. 5. Diagram showing the tracks of the microdialysis cannulas in the rat nucleus accumbens-ventromedial caudate. The figures indicate the anterior posterior coordinate of the brain slice according to the Paxinos and Watson atlas.

swimming prevented the DA but not the DOPAC and HVA over- flow increase due to haloperidol injection.

The microdialysis probes sampling from the area of the nu- cleus accumbens were located in a region that extended from the ventromedial portion of the caudate to the olfactory tubercle (OT), crossing the shell and the core of the nucleus accumbens septi (NAC). We have named this region NAC-C in the present article to emphasize that the probes were located in a functional and morphological unit. Although a distinction between the nu- cleus accumbens and the caudate was established in the past based on gross morphological analysis, currently the nucleus ac- cumbens and the olfactory tubercle are encased into the concept of ventral striatum to indicate continuity with the ventromedi~ part of the caudate (32). Grouping part of the caudate, the NAC, and the OT into a distinctive entity is based upon anatomical and pharmacological evidence. This entity receives DA projections from the Al0 cell group of the VTA (8) and from the PFC, amygdala, hipocampus, enthorhinal, perirhinal, and pyriform cortex (30,44,45). These structures are involved in cognition and emotion (13,23,53,65,68). By contrast, the putamen and the dor- sal caudate receive afferents from the A9 cell group of the sub- stantia nigra and from the neocortex (77).

Pharmacological features also distinguish the ventromedial caudate-ventral striatum region from the putamen-dorsal caudate. Microinjections of dopamine or dopamine agonists into the NAC-C area increase general locomotion but do not cause ste- reotypies (63). By contrast, micro~nj~tions of DA or DA ago- nists into the dorsal lateral caudate and the putamen cause steroptypies but do not increase general locomotion (42,46). Therefore, our probes, located in the NAC-C were monitoring DA overflow in an area where dopamine microinjections increase general locomotion and in which DA should play a role in cat- alepsy suppression. Surprisingly, forced swimming catalepsy suppression did not increase DA, DOPAC, or HVA overflow in

the NAC-C area. Interestingly, in the saline control group DA, DOPAC, and HVA overflow increased in the PFC but not in the NAC-C. Similar effects of stress have been reported. Electric shocks applied to the tail or to the foot, and tail pinching increase DA activity in the PFC but not in the NAC or the caudate (1,18,74).

How does forced swimming increase DA activity in the me- socortical system? The PFC is connected to the habenula via the stria medullaris (8,24). The stria medullaris contains afferents to the latera habenula stemming out of the nucleus accumbens, the ventral pallidum, the nucleus of the diagonal band, the entope- duncular nucleus, and the lateral preoptic area (56,57,60,72,76). The lateral habenula projects via the fasciculus retroflexus to the VTA and other midbrain nuclei such as the raphe nuclei (2,33). It has been proposed that the PFC-lateral habenula-VTA pathway is the anatomical substrate of a negative feedback that modulates the activity of the mesocortrcal system. Lesions of the habenula activate the mesocorticofrontal dopamine neurons in rats (50,58). It is possible that in our experiment, this feedback might be blocked by haloperidol enhancing DA overflow in the prefrontal cortex. However, the increase of PFC DA overflow induced by forced swimming or the prolonged DA overflow induced by forced swimming in halo~~dol-treated rats indicates that this negative feedback is overcome by stress, or that in freely moving conditions the PFC-lateral habenula-VTA inhibitory pathway does not control mesocortical DA turnover at all. This last alter- native is supported by recent microdialysis experiments in freely moving rats showing that electrical stimulation of the lateral ha- benula does not affect DA overflow either in the PFC or in the NAC (I 8).

However, this reasoning does not rule out a con~bution of the habenula-VTA connection to DA, DOPAC, and HVA over- flow increase in the PFC during forced swimming. The possi-

0 0 30 60 QO 120

MINUTES FIG. 6. Effect of intracortical injection of DA on haloperidol induced catalepsy. All the groups were compared with the group that received an in~~~toneal injection of halo~~dol and a bilateral equivolume~~ injection of Ringer solution into the medial prefrontal cortex (open cir- cles). The group that received 20 pg of dopamine (solid circles) showed shorter descent latency at the 30 min (one asterisk) and 60 min (two asterisks) after the haloperidol injection. The group that received 10 pg of dopamine (solid circles) had shorter descent latency 30 min after the haloperidol injection. The group that received 5 pg of DA (solid trian- gles) had no different descent latency than the control group. Asterisks indicate statistically signi~cant differences. For details see the text.

I-UC<‘I ET AI..

FIG. 7. Diagram showing the sites of microinjection of dopamine and Ringer in the PIT. The figures indicate the anterior Posterior coordinate of the brain slice according to the Paxinos and Watson atlas.

bility that the habenula-VTA connection might increase DA, DOPAC, and HVA overflow in the PFC during stress has been explored (49,50). It has been found that in addition to the lateral ha~nula-VTA i~ibito~ pathway, there is a medial habenula- VTA excitatory pathway. The neurons of this pathway release substance P on the mesocortical neurons (55). Foot shocks in- crease mesocortical DA activity by activation of this pathway (49). This mechanism has been confirmed by other authors (6). Therefore, forced swimming might increase DA, DOPAC, and HVA overflow in the PFC by activating the medial habenula- VTA excitatory pathway.

One corollary to this reasoning is that haloperidol and forced swimming increase DA, DOPAC, and HVA overflow by differ- ent mechanisms. Haloperidol injections at the dose of 5 mgkg increased DA, DOPAC, and HVA overflow in the PFC and the NAC-C. These results confirm previous observations showing that haloperidol is equip&em increasing DA activity in the PFC the NAC and the striatum (37). This effect is due to blockade of DA receptors and enhancement of the firing rate of dopaminergic neurons in general (14,16). By contrast, forced swimming in- creases the activity of mesocortical neurons probably by activa- tion of a pathway that directly and selectively excites the meso- cortical neurons. For this reason the effect of forced swimming did not add, but rather potentiated, the h~o~~dol-educe in- crease of DA, DOPAC, and WA overflow in the PFC. One

possible explanation is that the forced swimming test 1s more stressful in the cataleptic than in the normal rat. Another expla- nation is that the effects of DA receptor blockade are more pro- nounced on a mesocortical system challenged by stress than on the baseline DA levels of the mesocortical system. In any event, the results s&ongly suggest that haloperidol-induced DA increase and stress-induced DA increase occur by different mechanisms,

The present results do not concur with several experiments that have shown that dopamine in the nucleus accumbens and the ventromedial caudate facilitates locomotion (t 1,12,63). Ac- cording to this evidence, an increase of dopamine turnover in the NAC-C should be expected when stress activates a cataleptic animal or patient. Moreover, dopamine in the PFC has been in- volved in cognitive processes more than in locomotion. Dopa- mine depletion in the PFC causes a deficit in alternate delayed response tests, both in rats and primates (13,6_5,68). However, when the haloperidol injected rats were immersed in water, DA, DOPAC, and HVA overfiow increased furthermore in the PFC but not in the NAC-C. On the contrary, in the haloperidol injected and forced to swim group, the DA levels in the dialysates of the NAC-C had the tendency to be lower than the DA levels of the group that received haloperidol but was not forced to swim. This trend might be caused by the reciprocal relationship between me- socortical and mesolimbic dopamine already described. it has been found that when DA activity increases in the prefrontal cortex it decreases in the NAC and vice versa (69). In our ex- periments, the increase of DA activity in the prefrontal cortex caused by forced swimming might have attenuated the increase of DA, DOPAC, and HVA overflow caused by haloperidol in the NAC-C of the rats forced to swim.

The present results suggest that an increase of dopamine tumover in the PFC might mediate stress suppression of haloperidol-induced catalepsy. Putting the cataleptic animals into a water bath caused stress and increased dopamine turnover in the PFC but not in the NAC-C. In the second experiment, in the absence of stress, microin- jections of dopamine in the PFC reversed h~o~~dol-induct cat- alepsy, as shown by a diminish~ descent latency, The higher the dose. the shorter the descent latency and the longer the effect. The 20 pg dose significantly decreased the descent latency at 30 min and 60 min after the haloperidol injection. The 10 ,ug dose decreased the descent latency only 30 min after haloperidol injection, and the 5 pg dose did not decrease the descent latency. These results support the conclusion that dopamine in the PFC plays a critical role in stress supp~sion of catalepsy.

The question is: how could an increase of dopamine turnover in the PFC reverse haloperidol induced catalepsy’? Although still controversial, it seems that dopamine inhibits the neurons in the PFC. This effect has been shown in anesthetized rats (78). The main output of the PFC is a glutamatergic pathway that projects to the nucleus accumbens and the ventromedialcaudate (9,26,27,47,X&70). This pathway might play a prominent role in haloperidol-induced catalepsy. As we mentioned before, anticho- linergic drugs suppress haloperidol induced catalepsy (3 1,591. Therefore, it has been postulated that catalepsy is caused by an unbalance of dopamine, glutamic acid, and acetylcholine in the basal ganglia. Glutamate, dopamine. acetylcholine interaction in the basal ganglia play an important role in the control of general locomotion. The general conclusion is that glutamate and ace- tylcholine are antagonists of dopamine in certain situations. Sys- temic injections of D? receptor antagonists such as haloperidol or sulpiride increase acetylcholine release in the striatum, whereas systemic or intrastriatal injections of DZ receptor ago- nists decrease acetylcholine in striatal dialysates (7,21X Anticho- linergic agents suppress catalepsy in neuroleptic-heated animals or patients. Thus, it is possible that forced swimming decreases

MECHANISM OF CATALEPSY 131

acetylcholine in the basal ganglia. A direct assessment of ace- tylcholine is required to evaluate this hypothesis.

It has also been shown that glutamate acts on non-NMDA and NMDA receptors in the striatum and the nucleus accumbens to release dopamine and cause hipermotility (19,24,40,48,73). A massive release of glutamate might contribute to catalepsy sup- pression in the forced swimming situation. However, the lack of dopamine increase in the NAC-C during forced swimming in either normal or cataleptic rats indicates that the mechanism by which glutamate increase dopamine does not explain the sup- pression of catalepsy in the forced swimming situation. The glu- tamate-dopamine interaction might operate in the opposite di- rection, i.e., a reduction of glutamate in the basal ganglia might reduce catalepsy. Several experiments support this speculation. In animals that dopamine terminals have been destroyed by 6- hydroxydopamine, the catalepsy caused by dopamine depletion is suppressed by MK-801, an NMDA receptor blocker (15). It has been shown that either systemic or intrastriatal injections of NMDA receptor blockers suppress haloperidol-induced cata- lepsy (25,54,67,79). On this basis, it has been postulated that there exists an antagonistic balance between glutamate and do- pamine (17). The direct application of dopamine on the gluta- matergic terminals in striatum slices and synaptosomes inhibits glutamate release (51,64). This action seems to be mediated by D2 receptors which location (either pre- or postsinaptic) is still controversial (43,51). If this is the case, an increase of glutamate due to DA receptor blockade should occur in the basal ganglia

during haloperidoi-induced catalepsy. Then, forced swimming

catalepsy suppression might be due to a decrease of glutama-

tergic activity in the basal ganglia caused by the DA released in

I

2

3

4

5

6

I

8,

9.

10.

II.

the PFC. However, using capillary zone elctrophoresis and laser- induced fluorescence to measure glutamate in striatal dialysates in freely moving rats we found that basal glutamate decreases after injection of a cataleptic dose of haloperidol (38). In other words, a haloperidol cataleptic dose did not increase but rather decreased glutamate. Therefore, this finding does not support the glutamate release as a factor mediating haloperidol-induced cat- alepsy, but it does not discard the fact that glutamate release might be further diminished during forced swimming catalepsy suppression. In any event, haloperidol might cause catalepsy by biasing the glutamate-acetylcholine-dopamine balance toward glutamate and acetylcholine in detriment of dopamine. Then, it is possible that forced swimming-induced DA increase in the PFC could inhibit the glut~atergic co~icos~atal pathway, which could reestablish the balance toward dopamine in the basal ganglia suppressing catalepsy.

In conclusion, this work suggests that forced swimming sup- pression of haloperidol-induced catalepsy (an animal model of catalepsy suppression in Parkinson disease patients confronting a life-threatening situation) is not due to straight increase of do- pamine turnover in the basal ganglia. Catalepsy suppression in this situation seems to be triggered by the excitation of the me- socortical DA neurons and subsequent restablishment of a bal- ance between glutamate, acetylcholine, and dopamine in the basal ganglia. The main features of this balance have yet to be experimentally explored.

This experiments were supported by grant from BID-CONICIT BTS 37 and CDCHT- M-442, 1993.

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