Neuroscience Vol. 70, No. 4, pp. 849-859, 1996

~ ) Pergamon

0306-4522(95)00407-6

Elsevier Science Ltd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00

I N V O L V E M E N T OF V E N T R O L A T E R A L S T R I A T A L D O P A M I N E IN M O V E M E N T I N I T I A T I O N A N D EXECUTION: A MICRODIALYSIS AND BEHAVIORAL INVESTIGATION M. S. C O U S I N S a n d J. D. S A L A M O N E * Department of Psychology, University of Connecticut, Storrs, CT 06269-1020, U.S.A. Abstract--Previous studies have demonstrated that the ventrolateral region of the rat neostriatum is the site at which dopamine depletions produce profound motor deficits that interfere with food handling and lever pressing. In the present work, two experiments were undertaken to investigate the role of ventrolateral striatal dopamine in lever pressing. The first experiment was a detailed characterization of the motor impairments induced by injections of the neurotoxic agent 6-hydroxydopamine into the ventrolateral striatum. Behavioral output during lever pressing on a fixed ratio 5 schedule was recorded by a computerized system that measured the duration and response initiation time for each lever press. Response initiation time was defined as the time from offset of one lever press to the onset of the next one. Dopamine depletions resulting from 6-hydroxydopamine injections profoundly depressed lever pressing response rate. This deficit was largely due to a dramatic increase in the average response initiation time. Analysis of the distribution of response initiation times indicated that dopamine-depleted rats made relatively few responses with fast initiation times (e.g. 0-125 ms), and also that dopamine depletions led to a dramatic increase in the number of pauses in responding (i.e. response initiation times greater than 2.5 s). This slowing of the initiation of movement was very sensitive to the effects of dopamine depletions, and even animals with mild dopamine depletions (29.1% of control levels) showed increased initiation times. Analysis of response durations indicated that dopamine depletions resulted in a shift in the distribution of durations such that depleted rats had a modal response duration of 375-500 ms, in contrast to the control mode of 125-250 ms. There was an overall increase in average response duration among animals with more severe dopamine depletions, although rats with moderate depletions showed no change in average response duration. In the second experiment, in vivo dialysis methods were used to study the dynamic activity of ventrolatera| striatal dopamine during lever pressing. During the performance of a 30-min fixed ratio 5 lever pressing session, there was a small but significant increase (20.9% above baseline) in dopamine release. There was not a linear or curvilinear correlation between lever pressing rate and increases in dopamine release. The relatively modest increase in ventrolateral striatal dopamine release during lever pressing and the lack of relation between dopamine release and behavioral output may indicate that dopamine in the ventrolateral striatum plays mainly a permissive role in lever pressing. These results suggest that ventrolateral striatal dopamine depletions in rats produce deficits in skilled motor control that are similar to the motor deficits observed in patients with Parkinson's disease. Key words: caudate, putamen, nigrostriatal, Parkinson's disease, motor, operant.

studies. 3'12'44'51A l t h o u g h several papers have reported the effects o f widespread striatal D A depletions, considerable evidence indicates t h a t there is substantial heterogeneity o f function across distinct striatal subregions.2,9 11,14,23,24,39,40,52,50,51,57 In the rat, there

N e o s t r i a t a l d o p a m i n e ( D A ) is involved in various aspects o f m o t o r function. 25'45'~'49'54'55 In h u m a n s , d e g e n e r a t i o n o f nigrostriatal D A n e u r o n s is the major neuropathological sign o f P a r k i n s o n ' s d i s e a s e ) '~3,21 In rats, depletions o f striatal D A result in a p r o f o u n d s y n d r o m e o f m o t o r i m p a i r m e n t s t h a t is characterized by aphagia, adipsia a n d diminished responsiveness to sensory input. 3°'31'52-55 Striatal D A depletions have also been s h o w n to i m p a i r forelimb reaching a n d grasping in r o d e n t a n d p r i m a t e

are differences between the m o t o r or s e n s o r i m o t o r functions o f the medial a n d lateral neostriatum. ~1~'14'22'5°'51'59 Several studies have s h o w n t h a t the ventral p o r t i o n o f the lateral neostriatum, i.e. the ventrolateral s t r i a t u m (VLS), is i m p o r t a n t for m o t o r control functions t h a t involve the head, orofacial a n d forepaw regions. T h e VLS is the m o s t effective region for inducing oral stereotypy with local injections o f stimulants. 23 The VLS is the striatal locus within which c h o l i n o m i m e t i c drugs are m o s t effective for inducing t r e m u l o u s jaw m o v e m e n t . ~4'27"4s T r e m u l o u s

*To whom correspondence should be addressed. Abbreviations: DA, dopamine; EDTA, ethylenediaminete-

tra-acetate; FR5, fixed ratio of five lever presses to receive one food pellet; 6-OHDA, 6-hydroxydopamine; VLS, ventrolateral striatum. 849

850

M.S. Cousins and J. D. Salamone

jaw m o v e m e n t s are also induced by VLS D A depletions, the depletions in other striatal sites were s h o w n to be lneffectwe.- Ibotenate lesions o f the VLS p r o d u c e d deficits in food biting. 39'4° The VLS was shown to be the most effective site for neurolepticinduced suppression of food intake? Also, the VLS was d e m o n s t r a t e d to be the m o s t effective site at which D A depletions impaired forepaw reaching, 44 food intake, 22'51 feeding rate and food handling. 51 One o f the behaviors that is very sensitive to disruption o f VLS D A is lever pressing. 95° In a recent article 5° it was s h o w n that the VLS is the most effective site at which D A depletions could disrupt lever pressing in rats. Depletions o f VLS D A p r o d u c e d substantial and persistent decreases in the n u m b e r o f fixed ratio 5 (FR5) lever pressing responses (i.e. five presses to receive one food pellet) relative to control rats and also c o m p a r e d to rats with D A depletions in the nucleus accumbens or the medial striatum. 5° VLS D A depletions also produced a p r o f o u n d alteration o f the pattern o f interresponse times shown. 5° The interresponse time is the time between the onset o f each response, and this d a t u m represents the reciprocal o f the local rate o f responding. VLS D A depletions decreased the relative proportion o f high rate (short-duration) interresponse time, and significantly increased the p r o p o r t i o n o f low rate (long-duration) interresponse time? ° A l t h o u g h an analysis o f interresponse times provides a detailed description o f the pattern o f lever pressing responses, it is i m p o r t a n t to recognize that there are two separate c o m p o n e n t s o f the IRT. Each deflection o f the lever lasts for a particular period o f time, which represents the response duration. Once a lever press is terminated, there is a time period before another response is initiated (i.e. response initiation time). Previous work has indicated that administration of D A antagonists increases average response duration. ~6~7'4~ However, little is k n o w n a b o u t the role o f VLS D A in parameters o f response output such as response initiation or duration. Therefore, the first experiment o f the present work was designed to provide a detailed characterization of the effects o f VLS D A depletions on the temporal parameters o f FR5 lever pressing. Behavioral output during lever pressing on an FR5 schedule was recorded by a computerized system that measured the duration and response initiation time for each lever press, and determined the distributions o f these two parameters for each animal. VLS D A depletion was induced by local injection o f the neurotoxic agent 6-hydroxyd o p a m i n e (6-OHDA). A l t h o u g h it has been well d o c u m e n t e d that VLS D A depletions produce p r o f o u n d m o t o r impairments, little is k n o w n a b o u t the dynamic activity o f VLS D A during the p e r f o r m a n c e o f behaviors such as FR5 lever pressing. Thus, the second study involved the use o f in vivo dialysis m e t h o d s to investigate D A release during FR5 performance. Previous studies on the neurochemical c o n c o m i t a n t s of lever •

"

~2

'

pressing have largely focused on D A in the nucleus accumbens. It has been d e m o n s t r a t e d that large increases (i.e. 50-90% above baseline) in accumbens D A release accompany lever pressing on a continuous schedule, 2°'34 a lever press avoidance schedule 36 and an FR5 schedule. 47 A l t h o u g h several studies have employed various e x vivo m e t h o d s for studying the relation between lever pressing and D A turnover in striatal tissue, 7'~8 the present study represents the first microdialysis investigation specifically focusing on VLS D A release during lever pressing.

EXPERIMENTAL PROCEDURES

Subjects

A total of 41 male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were used for these experiments. They were singly housed with a 12-h light-dark (lights on 07.00) in a colony room maintained at 23'C. Rats were initially food deprived to 85% of their free feeding body weight but were then allowed to grow up to 95% of their original free feeding body weight during the pre-surgical testing (initial free feeding body weights were between 270 and 325 g). Water was available ad libitum. These experiments were approved by the University Animal Care Committee, which supervises the care and use of animals. Consistent with University policy, all efforts were made to minimize animal suffering and the number of animals used. Beharioral procedures

For both experiments testing was performed in operant chambers (28 x 23 x 23cm3). Rats were trained for 30 min/day, five days/week on an FR5 lever pressing schedule for 45 mg food pellets (Bioserve Inc.). For the first experiment, food-deprived rats were placed in an operant chamber for 30 min/day and trained on a continuous schedule for one week before being switched to an FR5 lever pressing schedule. A microcomputer was used to control the schedule, gather the data during the 30-min test session and to measure the temporal pattern of responding. The computer recorded the number of lever press responses across the 30-min behavioral session. For each response, including those that followed reinforcement, the response initiation time (i.e. time from offset of one lever press to onset of the next lever press) and the response duration were recorded. These three time measures for each response were counted as being within one of three sets of 10 time bins, with the first eight bins each representing an interval of 125 ms (0 125 ms, 126 250ms, 251-375 ms, etc. up to 1.0s). Bin 9 included all response initiation times or durations from 1.0 to 2.5 s, and bin 10 included all times that were greater than 2.5 s. For the second experiment, rats in the FR5 group were initially trained on a continuous schedule for five days before being switched to the FR5 schedule• After two weeks of training on the FR5 schedule, rats were trained in preparation for the dialysis test sessions. For this training, all rats were placed in the operant chamber in the morning, and received their behavioral treatment in a 30 min session during mid-day, after which the rats remained in the operant chamber for 4-5 h before being returned to their home cage. During the 30-rain mid-day test session the room lights were dimmed but small house lights in the operant chamber were turned on. In addition, a lever was placed in the chamber and those rats trained to lever press received food pellets upon completion of the FR5 schedule. A food-deprived control group was used in the dialysis experiment, and these

Striatal dopamine and movement initiation rats were placed into the chamber during the entire training period but never received food in the chamber. During the behavioral test period, a lever was placed in the chamber for food-deprived controls, but lever pressing was not reinforced in this group.

Ventrolateral striatal dopamine depletion by injection of 6-hydroxydopamine For the first experiment, rats received i.p. injections of 10.0 mg/kg pargyline 30 min prior to surgery and 50 mg/kg sodium pentobarbital anesthesia. VLS D A depletions were obtained by bilateral injection of 6 - O H D A (Research Biochemicals) through 30-gauge stainless steel injectors into the VLS (AP + l . 4 m m , M L _+4.0mm, DV - 7 . 2 m m with respect to bregma; incisor bar 5.0 m m above the interaural line). A total of 12.5/~g of the free base 6 - O H D A was dissolved in 2.5#1 of 0.1% ascorbate (2.5/~1 of 5.0/~g//~l 6-OHDA) and injected in each side. Control rats received injections of 2.5#1 of the 0.1% ascorbate solution at the same site as the 6-OHDA-injected rats. The injection was driven at a flow rate of 0.5/~l/min by a Harvard Apparatus syringe pump. The injectors were left in place for 2 min after infusion to allow for diffusion into the tissue.

Dialysis probe construction and implantation Rats were anesthetized with sodium pentobarbital and implanted, on either the right or left side of the skull, with a 16-gauge stainless steel guide cannula aimed 3.0 m m above the VLS (A + l . 4 m m from bregma, L + 4 . 0 m m from bregma, V - 4 . 2 m m below skull; incisor bar 5.0 m m above interaural line). The guide cannula was secured to the skull with machine screws and acrylic cement, and a 19-gauge stainless steel stylet maintained the patency of the guide cannula. Two weeks after guide cannula implantation, each rat was again anesthetized for dialysis probe insertion. Loop style dialysis probes were constructed in the laboratory from dialysis fiber (200 # m , 15,000 mol. wt cut-off). The probe tip was placed to extend 3.0 m m beyond the tip of the guide cannula and was then cemented in place. A fluid swivel was used to allow free movement of the animal. The probe was perfused with artificial cerebrospinal fluid (147.2 m M NaCI, 1.4 m M CaC12, and 4.0 m M KCI) by a syringe p u m p at 1.5 #l/min. The collection tubes contained 1.0 #1 of frozen 11 N perchloric acid.

851

Neurochemical analysis of dopamine Tissue and microdialysis samples were analysed for D A content using a high-performance liquid chromatography system with electrochemical detection that has been described previously. 3~36'47 The phosphate-buffered mobile phase (pH 4.5) also contained 7.0% methanol, E D T A and 2.8 ml 0.4 M sodium octyl sulfate. Standards of D A (Sigma Chemical Co.) were assayed daily before and after the dialysis samples. For the dialysis experiment, baseline levels of D A [mean ( + S.E.M.) pg DA/40/~1 sample] were: control 21.4 (+4.5) and FR5 17.5 ( + 1.6).

Procedure : effects depletions

of ventrolateral

striatal

dopamine

Rats were trained for three weeks (30-rain sessions, five days per week) on the FR5 task prior to surgery. These rats received either ascorbate vehicle (n = 11) or 6 - O H D A (n = 14) injected into the VLS as described above. After two days of post-surgical recovery, rats received an additional three weeks of behavioral testing (30-min sessions, five days per week on days 3-7, 10-14 and 17 21 after surgery). To maintain body weights some rats received supplemental feedings of lab chow in the home cage following the daily behavioral test session. All rats that received intra-VLS 6 - O H D A had to receive wet m a s h in order to maintain their body weight. Some rats that received 6 - O H D A also required tube feeding (Bioserve liquid diet) for a few days after surgery. All supplementary feeding was done after the daily operant session had been conducted.

Procedure: microdialysis study of ventrolateral striatal dopamine release Previously trained rats were implanted with a guide cannula aimed 3 . 0 m m above the VLS, and these rats were then implanted with a dialysis probe into the VLS and tested the next day. Dialysis samples were collected in 30-min periods throughout the day. The two dialysis samples prior to the mid-day test session served as a neurochemical baseline. The rats were then exposed either to the FR5 schedule (n = 11) or the control procedure (n = 6), as described above, for a single 30-min session. After termination of the behavioral test period, levers were removed and five additional dialysis samples were collected.

Data analysis Histology After the dialysis experiment, the rats were overdosed on sodium pentobarbital anesthesia and perfused with 0.9% saline and 10% formalin. The dialysis probes were removed after the perfusion and the brains were dissected from the skull and stored in 10% formalin. Coronal sections (50/~m) through the brain were made of the area surrounding the dialysis probes and mounted on microscope slides. The neurochemical data from dialysis probes that were verified to penetrate the VLS were analysed further.

Dissection for tissue assays After the D A depletion experiment, rats were placed in ether for approximately 1 min before being decapitated. Brains were quickly removed and immediately frozen. Coronal sections (0.7 m m thick) were cut through the nucleus accumbens, medial striatum and VLS. Tissue samples from each section were placed in 200/11 of chilled 0.1 N perchloric acid and homogenized. The samples were then centrifuged for 4 m i n at 16,000r.p.m. and frozen at - 2 0 ° C . Samples were analysed within 48 h of the decapitation, as described below.

For the D A depletion experiment, weekly means were calculated for total n u m b e r of responses, average response initiation time and average duration for each animal. As described below, the rats that received 6 - O H D A were further divided into two groups based upon the extent of the D A depletion (low depletion, n = 7; high depletion, n = 7). These data were analysed by a 3 × 3 (week × group) factorial A N O V A with repeated measures on the week factor. Planned comparisons that used the error term from the overall analysis (see Refi 26, pp. 109 118, 209-212) were used to test for differences between each D A depletion group and the control group. The number of comparisons was restricted to two (number of groups minus 1; see Ref. 26). Analysis of simple main effects was used to identify the sources of any significant interactions. Data obtained for total number of responses and average response duration did not violate the assumption of homogeneity of variance. However, there was substantial heterogeneity of variance in the response initiation measure. Therefore, average response initiation times were log transformed prior to performance of the A N O V A to reduce variability. For analysis of time bins for initiation times and response durations, data in each bin were calculated as weekly totals in that time bin expressed as a percentage of the total number of responses

852

M.S. Cousins and J. D. Salamone

for the week. This measure was used in order to correct for the group differences in total number of responses so that the relative frequency distributions of the initiation times and durations could be examined. Each time bin was analysed non-parametrically by using the Kruskal-Wallis non-parametric ANOVA, and specific comparisons between DA-depleted and control groups were made using the Mann-Whitney U-test. The modal time bin for each distribution was analysed using the Mann-Whitney U-test. For the dialysis experiment, DA levels in the last two baseline samples were calculated as picogram values, and for additional statistical analyses the DA content in each sample was also calculated as a percentage of the baseline.

RESULTS

Table 2. Mean response initiation time (in seconds) for weeks I-3 of post-surgical testing; data shown are for rats in the control group, and with low or high dopamine depletion Week 1

Week 2

Week 3

Control Mean (±S.E.M.)

1.16 (0.10)

0.88 (0.05)

0.93 (0.11)

Low VLS DA depletion Mean (±S.E.M.)

6.53* (1.97)

2.86* (0.80)

1.65 (0.26)

High VLS DA depletion Mean (±S.E.M.)

16.40" (6.69)

7.61" (1.88)

2.91" (0.57)

*P < 0.05, compared to control group in that week.

Neurochemical and behavioral effects of ventrolateral striatal dopamine depletions Neurochemical analysis of dopamine depletions. Based u p o n the h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y analyses, the m e a n ( ± S.E.M.) tissue levels o f D A (calculated as ng D A per m g wet weight o f tissue) in nucleus accumbens, medial n e o s t r i a t u m and VLS o f control a n d 6 - O H D A - t r e a t e d rats were as follows. Nucleus accumbens: control, 2.93 ( ± 0.51); 6 - O H D A , 2.88 ( + 0 . 2 2 ) ; medial neostriatum: control, 8.37 ( ± 1.97); 6 - O H D A , 5.18 ( ± 0 . 5 5 ) ; VLS: control, 5.09 ( ± 1 . 1 0 ) ; 6 - O H D A , 0.94 ( ± 0 . 7 4 ) . There were no significant effects of 6 - O H D A injection o n D A levels in nucleus a c c u m b e n s a n d medial striatum, a n d only VLS D A levels were significantly reduced by injections of 6 - O H D A into the VLS (t = 4.2, d.f. = 23, P < 0.01). F o r analyses o f the behavioral data in terms o f the extent of VLS D A depletion, the g r o u p t h a t received injections of 6 - O H D A into the VLS were further divided into a low depletion g r o u p In = 7, VLS D A levels = 1.48 n g / m g (_+0.26), 29.1% of m e a n control values] a n d a high depletion g r o u p [n = 7, VLS D A levels = 0.41 n g / m g ( ± 0 . 0 7 ) , 8.1% o f m e a n control values]. Behavioral effects of dopamine depletions. Depletion of D A in the VLS substantially impaired F R 5 lever

Table 1. Mean number of lever presses per day for weeks 1-3 of post-surgical testing; data shown are for rats in the control group, and with low or high dopamine depletion Control Mean (±S.E.M.) Low VLS DA depletion Mean ( ± S.E.M.) High VLS DA depletion Mean (±S.E.M.)

Week 1

Week 2

Week 3

1139.6 (48.6)

1431.9 (32.0)

1543.6 (48.7)

381.2* (76.1)

765.1 * (147.3)

1045.8" (115.9)

244.5* (98.3)

387.8* (126.5)

675.3* (124.6)

*P < 0.05, compared to control group in that week.

pressing. Table 1 displays the effects of VLS D A depletion on total n u m b e r o f responses over the three weeks o f post-surgical testing. A N O V A indicated t h a t there was a n overall significant effect of D A depletion o n total n u m b e r o f responses [F(2,22) = 38.7, P < 0.001], a significant effect of test week [ F ( 2 , 4 4 ) = 118.4, P < 0.001] a n d a significant D A depletion x week interaction [F(4,44) = 4.8, P < 0.05]. P l a n n e d c o m p a r i s o n s showed t h a t there was a significant reduction in responding in b o t h the low depletion a n d high depletion 6 - O H D A groups relative to controls d u r i n g all three weeks of post-surgical testing. The data o n average response initiation time are s h o w n in Table 2. A N O V A indicated there was an overall significant effect o f D A depletion o n average response initiation time [ F ( 2 , 2 2 ) = 26.5, P < 0.001], a significant effect of test week [F(2,44) = 77.0, P < 0.001] a n d a significant D A depletion x week interaction [F(4,44) = 13.7, P < 0.001]. P l a n n e d c o m p a r i s o n s showed that there was a significant increase in response initiation time in the low depletion 6 - O H D A g r o u p relative to controls during weeks 1 a n d 2 after surgery. The high depletion 6 - O H D A group h a d significantly increased average response initiation times c o m p a r e d to the control g r o u p d u r i n g all three weeks of post-surgical testing. T h e effects of VLS D A depletions o n the distributions o f response initiation times are s h o w n in Fig. I A - C . The average response d u r a t i o n s for each g r o u p are s h o w n in Table 3. A N O V A indicated there was an overall significant effect of D A depletion on response d u r a t i o n [ F ( 2 , 2 2 ) = 3 . 7 , P 2.5

Response initiation bins (s)

(c)

50

~ 3o "Jr

51

20

0'

0.125

0.250

0.375

0.500

0.625

0 . 7 5 0 0.875

1.000 1.01-2.5 >2.5

Response initiation bins (s)

• Control 17]Low VLS depletion [ ] High VLS depletion Fig. I. Relative distribution of response initiation times, showing the mean percentage of total initiation times within each of the I0 time bins recorded. Data shown are for rats in the control group, and with low or high D A depletion. *P