Skilled motor deficits in rats induced by ventrolateral striatal dopamine depletions: behavioral and pharmacological characterization

BRAIN RESEARCH Brain Research 732 (1996) 186-194

Research

report

Skilled motor deficits in rats induced by ventrolateral striatal dopamine depletions: behavioral and pharmacological characterization Michael

S. Cousins,

John D. Salamone

*

Department of Psychology, University of Connecticut, Storrs, CT O6269-102O,USA Accepted 23 April 1996

Abstract Rats were tested in an instrumental lever pressing procedure, in which a computer program recorded detailed parameters of responding such as response initiation and duration. Initially, rats with ventrolateral striatal dopamine depletions and control rats were tested on days 3–5 after surgery. Dopamine depletions produced by local injections of 6-hydroxydopamine substantially reduced the number of lever presses emitted. Dopamine depleted animals showed significant increases in average response initiation times, average length of fast initiation times, average length of pauses and total pause time. The distribution of initiation times was altered so that DA depleted rats showed significant reductions in the relative number of very high rate responses and also showed increases in the relative number of pauses. On day 7 after surgery, dopamine-depleted rats received one of three drug treatments: injections of ascorbate vehicle, injections of 20.0 mg/kg L-DOPA, and injections of 40.0 mg\kg L-DOPA. Injections of 40.0 mg/kg L-DOPA led to some improvement in several parameters of instrumental responding. Compared to the previous baseline day, the group that received 40.0 mg\kg L-DOPA showed a significant increase in number of responses on the drug treatment day, and also showed significant decreases in average response initiation time and total pause time. The group that received 40.0 mg/kg L-DOPA also showed significant increases in number of responses (expressed as a percent of the previous day) when compared to the control group that received injections of ascorbate vehicle. These results indicate that L-DOPA can partially reverse the skilled motor deficits produced by ventrolateral striatal dopamine depletions, and suggest that this test may be useful for the assessment of antiparkinsonian drugs. Keywords: Dopamine; Parkinson; Striatum; L-DOPA; Motor; Putarnen

1. Introduction In humans, progressive degeneration of nigrostriatal dopamine (DA) neurons is the neuropathological hallmark of Parkinson’s disease [5,18,25]. Animal models involving DA depletions in primates [55] and rodents [63] have been used to study the neurochemical mechanisms underlying parkinsonian motor dysfunctions and to investigate possible treatments. Considerable research has focused on the use of the neurotoxic agent 6-hydroxydopamine (6-OHDA) to deplete neostriatal DA in rats. Extensive depletions of striatal DA in rats have been shown to produce a severe motor deficit that is characterized by akinesia, aphagia, adipsia and reduced sensorimotor responsiveness [37,38,51-53,56]. It has been suggested that such studies represent a rat model of some of the neurochernical and motoric features of Parkinson’s disease [63]. However, an

* Corresponding author. Fax: + 1 (203) 486-2760.

important test for the validity of an animal model is that the model should be sensitive to the effects of drugs that are effective in the treatment of the human disorder [48]. Evidence indicates that antiparkinsonian drugs, such as L-DOPA or anticholinergics, can increase locomotor activity in rats made akinetic by DA-depleting brain lesions [20,35,57]. This suggests that the motor deficits produced by striatal DA depletions in rats could be useful for the assessment of antiparkinsonian drug treatments. Despite the progress that has been made in this area, several important questions remain about the use of rat DA depletion models for Parkinson’s disease. Although locomotion is a useful measure of motor activity in DA-depleted rats, it also is important to investigate other aspects of motor function. Parkinson’s disease results in a number of motor symptoms, including rigidity, tremor, and deficits in skilled motor usage [18]. Skilled motor usage is not often emphasized as a major deficit in Parkinson’s disease, but according to patient reports it is one of the most common and disturbing symptoms [34]. Laboratory studies

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have demonstrated that parkinsonian patients have difficulties with skilled hand or arm movements [24,29,32]. MPTP-treated monkeys have been shown to have deficits in skilled forelimb control [8,59]. Striatal DA depletions in rats produced deficits in reaching and grasping [17,61] as well as forelimb usage during feeding [50] and lever pressing [49]. Another important consideration is that the functions of the rodent striatum, like those of human and non-human primates, show considerable regional heterogeneity. Several studies have shown that the ventrolateral striatum (VLS) is the region at which DA depletions are most likely to produce skilled motor impairments. The VLS is the most effective site at which DA depletions impair forelimb reaching [46] forepaw usage during feeding [50] and lever pressing [49]. Detailed analyses of the motor pattern of lever pressing in rats with VLS DA depletions has indicated that these rats have substantially increased interresponse times [50]. The lever pressing deficit in rats with VLS DA depletions is characterized by a pronounced slowing of response initiation times [11]. Anatomical evidence suggests that the lateral striatum of the rat is the homologue of the primate putamen, and that the VLS therefore represents the portion of the somatotopically organized putamen that is involved in head, orofacial and forelimb motor control [9,12,27,39,43,49,50,60,62]. The notion that the VLS is the rat homologue of the ventral putamen is particularly important in view of the fact that the putamen is the striatal region that is most severely depleted of DA in parkinsonian patients [45]. Therefore, VLS DA depletions in rats look particularly promising as an animal model of aspects of Parkinson’s disease because these depletions involve darnage to the same terminal region that is most severely impaired in human patients, and also because such depletions produce profound motor deficits, The present work was designed to study the effects of VLS DA depletions, and to determine if L-DOPA could improve lever pressing performance in DA-depleted rats. Computerized analysis of lever pressing allowed for the assessment of detailed temporal parameters of responding, including several measures related to response initiation.

2. Materials and methods 2.1. Subjects A total of 33 male Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN) were used in the present study. These rats were individually housed, with a 12-h light-dark cycle (lights on 07.00 h) in a colony room maintained at 23°C. Testing was conducted between 08.00–12.00 h. Rats were food deprived to 85% of their free feeding body weight during initial training, but then were maintained at 9590 of their original free feeding body weight during the presurgical testing period (initial body

187

weights were between 275 and 325 g). Water was available ad libitum. 2.2. Behavioral procedures Tests of instrumental responding were performed in standard operant chambers (Meal Systems; 28 X 23 X 23 cm). Initially, food-deprived rats were trained to press a lever for 45 mg food pellets (Bioserve, Frenchtown, NJ) on a continuous reinforcement schedule before being shifted to a fixed ratio 5 (FR5) reinforcement schedule. Rats continued training on the FR5 schedule for 2 weeks (30 rein/day, 5 days/week). A microcomputer was used to control the schedule, count the total number of lever presses, and measure the temporal pattern of responding during the 30-min test session. The response duration times (i.e., the time period during which the lever was deflected) and response initiation times (i.e., the time between the offset of a lever press and the onset of the next lever press) were recorded for each lever press response. Duration and response initiation times for each lever press were sorted as being in one of 21 time bins. The first 20 bins each represented an interval of 125 ms (e.g., 0-125 ms, 126-250 ms, 251-375 ms, etc., Up to 2.5 s). Bin 21 included all responses that had duration or initiation times greater than 2.5 s. The average initiation and duration times, the relative number of very high rate responses (i.e., the percentage of lever press responses that had initiation times less than 125 ins), and the average length of fast initiation times (i.e., the average length of initiation times that were less than 2.5 s) were recorded. A pause was defined as an initiation time greater than 2.5 s, and the average length of pauses (i.e., the average length of all initiation times that exceeded 2.5 s), total pause time and the relative number of pauses (i.e., the relative number of responses with initiation times greater than 2.5 s) were recorded. 2.3. Ventrolateral striatal dopamine depletion by injection of 6-OHDA Rats received 1P injections of 20.0 mg/kg pargyline 30 rnin prior to surgery and 50.0 mg/kg sodium pentobarbital anesthesia, Depletions of DA in the VLS were obtained by bilateral injections of the neurotoxic agent 6-OHDA (Research Biochemical Int). Solutions of 6-OHDA were injected through 30 ga stainless steel injectors into the VLS (AP + 1.4 mm, ML +4.0 mm, DV –7.2 mm with respect to bregma; incisor bar +5.0 mm relative to the interaural line). Each injection consisted of 12.5 p,g of free base 6-OHDA dissolved in a total of 2.5 p,l of 0.1% ascorbic acid (2.5 pl of 5.0 pg/@ 6-OHDA solution). Control rats received injections of 2.5 pJ of the O.l?ioascorbate solution at the same site as the 6-OHDA-treated rats. The injection was delivered at a flow rate of 0.5 p,l/min by a Harvard Apparatus syringe pump. The bilateral injectors were left

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in the VLS for 2 min after the infusion to allow for diffusion into the tissue. 2.4. Neurochemical analyses of tissue dopamine Rats were placed in a COZ chamber on day 3 following drug treatment (day 10 postsurgery) for 30 s before being decapitated. Brains were quickly removed and immediately frozen. Coronal sections 0.7 mm thick were cut through the brain and a 16 gauge stainless steel tube was used to dissect cylindrical samples from the nucleus accumbens, medial striatum, and adjacent segments of VLS (dorsal or dVLS, and ventral or vVLS). Tissue samples were placed in 200 pl of chilled 0.1 N perchloric acid and homogenized. The samples were centrifuged for 4 rnin at 16,000 rpm and then frozen at –20”C. The supernatant obtained from each sample was analyzed using a high performance liquid chromatography system with electrochemical detection that has been previously described [12,49]. The mobile phase was a phosphate buffer (pH 4.5) that contained 7.0% methanol, EDTA, and 0.4 M sodium octyl sulfate. Standards of DA (Sigma Chemical) were assayed before and after the tissue samples.

after surgery, and was designed to assess the effects of L-DOPA in DA-depleted rats. Rats were tested in an operant chamber (30 rein) on day 6, and this day of testing served as a behavioral baseline for subsequent drug testing that occurred on day 7 postsurgery. Sixty minutes prior to being placed in the operant chamber on day 7 of postsurgical testing, DA-depleted rats received either O.lYoascorbate vehicle, 20.0 or 40.0 mg/kg L-DOPA. Prior to the 1P injections, rats that received 6-OHDA injections into the VLS were assigned to systemic drug treatment group, and the groups were counterbalanced based on the number of lever presses emitted on day 6. Thus, lever pressing deficits were comparable among the VLS DA depleted rats receiving either O.1% ascorbate vehicle, 20.0 mg/kg or 40.0 mg/kg L-DOPA. Following this assignment, rats that were treated with intra-VLS 6-OHDA received 1P injections of either O.lYOascorbate (n = 7), 20.0 mg/kg L-DOPA (n = 8), or 40.0 mg\kg L-DOPA (n= 9). Rats treated with intra-VLS ascorbate vehicle injections (n = 9) received 0.1% ascorbate vehicle 1P on day 7. These rats were not analyzed statistically as a part of the L-DOPA study, but in order to provide comparisons with the DA-depleted rats, their lever pressing data are included in the caption for Fig. 3 (see below).

2.5. L-DOPA treatment 2.7. Data analysis L-DOPA was obtained from Sigma (St Louis, MO) and was dissolved in O.lYOascorbic acid solution and injected at 6.0 ml/kg 1P. Control rats were injected with O.lYO ascorbate at 6.0 ml/kg IP. The two L-DOPA doses used in the present study were 20.0 mg/kg and 40.0 mg/kg. These dosages were chosen because pilot data indicated that in VLS DA-depleted rats there was a substantial increase in lever pressing after injections of 40.0 mg/kg L-DOPA (expressed as a percent of the previous baseline day, 268.0% + 94.3; n = 7). Injections of higher doses failed to produce larger increases in responding (60.0 mg/kg L-DOPA, 181.4~0+ 95.3; n = 3; or 80.0 mg/kg L-DOPA, 80.0%; n = 1). 2.6. Experimental procedure Rats were trained on the FR5 schedule for 2 weeks prior to surgery. These rats received injections of either ascorbate vehicle (n = 9) or 6-OHDA (n = 24) into the VLS as described above. All rats that received VLS 6OHDA injections had to receive wet mash in order to maintain their body weight. Some rats with VLS DA depletions also required tube feeding for a few days after surgery. All supplementary feeding was conducted after completion of the daily operant test session. The first phase of the study was designed to determine the behavioral effects of VLS DA depletions. Ascorbate vehicle- and 6-OHDA-treated rats were tested on days 3–5 postsurgery in an operant chamber (30 rnin/day for 3 days). The second phase of the study was conducted on days 6 and 7

Neurochemical data were analyzed using analysis of variance (ANOVA), and post-hoc comparisons were made using Tukey’s test. Control rats were compared to DA-depleted rats on various behavioral measures over days 3–5 of postsurgical testing. The number of lever presses on days 3-5 of postsurgical testing were analyzed by a 2 X 3 (group X day) factorial ANOVA with repeated measures on the day factor to test for differences between the DA-depleted group and control group. Several additional parameters of responding also were analyzed (see subsection Behavioral Procedures). Average response duration and average response initiation times were analyzed, and average response initiation time was also partitioned into two components: the average length of fast initiation times and the average length of pauses. Other measures of responding included total pause time, the relative number of very high rate responses and the relative number of pauses. The latter two measures were expressed as a percent of total responses in order to correct for differences between animals in terms of number of responses; these measures have been shown to be highly sensitive to the effects of DA depletions [11,49]. The various response measures were analyzed using the Mann–Whitney U-test (average response initiation, average length of fast initiation times, average length of pauses, total pause time, relative number of very high rate responses, relative number of pauses; see subsection Behavioral Procedures for details). The Pearson-product moment correlation coefficient was used to establish relations between DA levels

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and lever pressing parameters across days 3–5 and to establish relationships between the behavioral measures. To study the effects of L-DOPA, several statistical analyses were performed. For each parameter of responding, two types of statistical analyses were used to characterize the effects of L-DOPA: (a) for each treatment group, data obtained on the drug treatment day (day 7) were compared with data from the same group obtained on the pre-dmg baseline day (day 6), and (b) data obtained on the drug treatment day were corrected in terms of the pre-drug baseline, and these data were compared across all three treatment groups (i.e., vehicle, 20.0 and 40.0 mg\kg LDOPA). Analyses of all behavioral data on days 6 and 7 were conducted on only those rats that received intra-VLS 6-OHDA. The paired t-test was used to compare the number of lever presses for each L-DOPA drug treatment on days 6 (undrugged) and 7 (drugged) of postsurgical testing. In order to correct for pre-drug differences in responding, the number of lever presses emitted on day 7 was also expressed as a percentage of the number of lever presses emitted on day 6 postsurgery. Because these data violated the homogeneity of variance assumption of ANOVA, the nonparametric Kruskal-Wallis ANOVA test was used, and specific comparisons between L-DOPA

Table 1 Mean ( + S.E.M., below the mean) DA content (ng DA/mg tissue) in nucleus accumbens, medial neostriatum, dVLS and vVLS

Vehicle: Vehicle (n= 9) 6-OHDA: All groups (n= 24)

Brain region Nucleus accumbens

Medial striatum

dVLS

vVLS

7.46 (0.50)

14.09 (1.77)

14.71 (1.70)

9.77 (0.77)

6.33 (0.34)

9.83 * (0.98)

2,01 ‘ # (0.68)

0.89 *# (0.27)

* P <0.05, different from vehicle.# P <0.05, different from medial striatum.

treatment groups were made with the Mann–Whitney Utest. The average response initiation time, average length of fast initiation times, average length of pauses, total pause time, relative number of very high rate responses, and the relative number of pauses were compared for each drug treatment between days 6 and 7 of postsurgical testing with the Wilcoxon signed-rank test. In order to correct for variability in pre-drug performance, difference scores (day 7 minus day 6) were calculated for the average response initiation time, average length of fast initiation times, average length of pauses, total pause time, relative number of very high rate responses, and the relative number of pauses. These values were compared across the drug treatment groups using the Kruskal-Wallis test followed by the Mann–Whitney U-test.

3. Results 3.1. Neurochemical analysis of dopamine depletions

Fig. 1. The tissue sample sites used in the present experiment (hatched circles indicate placement of tissue punches; top, nucleus accumbens; middle, medial striatum; bottom, dVLS and vVLS); closed triangles indicate locations of 6-OHDA injections.

Fig. 1 shows the VLS target site used in the present study. The mean (i- S.E.M.) tissue levels of DA in control and 6-OHDA-injected rats for each condition are shown in Table 1. For these analyses, animals in all three 6-OHDA groups were combined (n = 24) because there were no differences between these groups. There was a significant overall effect of 6-OHDA treatment on DA levels (F1,31= 94.1, P < 0.001), a significant site difference (F~,?3= 24.4, P < 0.001) and a significant group X site interaction (F’3,g~ = 169.3, P < 0.001). There were no significant effects of 6-OHDA injection on DA levels in the nucleus accumbens. Post-hoc analyses with the Tukey test indicated that DA levels in medial striatum, vVLS and dVLS differed in 6-OHDA-treated rats compared to vehicle-treated control rats. Among all 24 rats that received 6-OHDA, DA levels in vVLS and dVLS did not differ from each other, but both sites had significantly lower DA levels than the medial striatum.

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3.2. Behavioral effects of ventrolateral striatal dopamine depletions VLS DA depletions substantially reduced FR5 lever pressing. ANOVA revealed that the number of lever presses emitted across days 3–5 of postsurgical testing was significantly impaired in DA-depleted rats (F1,~l = 67.08, P < 0.0001), as shown in Fig. 2. The effects of VLS DA depletions on the detailed temporal parameters of responding for days 3–5 after surgery are shown in Table 2. The Mann–Whitney U-test (P < 0.05) demonstrated that the average response initiation time, average length of pauses, total pause time, relative number of very high rate (0–125 ms) responses, and the relative number of pauses emitted by DA-depleted rats were significantly different from control rats on days 3–5 after surgery. Rats that received 6-OHDA differed from controls on the measure of average length of fast initiation times on days 4 and 5. Average (+ S.E.M) response durations (in seconds) for days 3-5 were as follows: vehicle, 0.71 ( + 0.21) on day 3, 0.48 (+0.03) on day 4,0.46 (+0.04) on day 5; 6-OHDA, 0.53 (+0.04) on day 3, 0.51 (t O.04) on day 4, 0.50 (+0.04) on day 5. There were no significant differences in average response duration between the two groups, although both groups had longer durations after surgery when compared to the pre-surgical baseline. There was a significant correlation between number of lever presses on days 3-5 and DA levels in both the dVLS and vVLS (r= 0.76 and 0.91, respectively, P < 0.0001). The number of lever press responses was correlated with the average length of pauses and the average response initiation time across days 3–5 for rats receiving intra-VLS 6-OHDA injections (r= 0.67, P < 0.001; r = 0.50, P <0.01, respectively). There was a significant correlation between the average response initiation time and DA levels in the dVLS (r= 0.44, P < 0.05). 3.3. Behavioral effects of L-DOPA treatment Fig. 3 shows the number of lever press responses for days 6 (baseline) and 7 (drug test) after surgery for rats EVER PRESSES 1000

750 500 250 0

3

4

5

DAYS =

CONTROL

=

6-OHDA

Fig. 2. Mean ( + S.E.M.) number of lever presses for control and 6OHDA-treated rats during days 3-5 of postsurgical testing (* P <0.05, different from control).

Table 2 Mean ( f S.E.M.) of various behavioral measures obtained from ascorbate vehicle and 6-OHDA-treated rats on days 3–5 after surgery Vehicle (n= 9) Average response initiation time (s): Day 3 3.14 (1.22) Day 4 2.62 (0.97) Day 5 1.72 (0.35) Auerage length of fast responses (s): Day 3 0.58 (0.06) Day 4 0.58 (0.07) Day 5 0.57 (0.06) Average length ofpauses (s): Day 3 9.35 (2.05) Day 4 10.45 (3.03) Day 5 7.58 (0.85] Total pause time (s): Day 3 1079.70 (85.07] Day 4 941.44 (109.93) Day 5 943.39 (85.16) Very high rate responses (%): Day 3 31.63 (5.78) Day 4 28.76 (7.19) Day 5 39.40 (5.60) Pauses (%): Day 3 23.28 (4.23) Day 4 16.58 (2.90) Day 5 15.56 (2.43)

6-OHDA (n = 24) 21.56 “ (6.99) 14.53 * (2.42) 16.05 * (4.29) 0,71 (0.06) 0.89 ‘ (0.07) 0.83 * (0.05) 38.52 “ (7.85) 31.44 “ (4.68) 34.49 ‘ (6.75) 1487.94 “ (43.59) 1522.79 ‘ (30.31) 1442.55 * (45.66) 16.32 “ (2.40) 5.57 * (0.74) 17.11 * (2.40) 45.90 “ (3.45) 43.54 * (2.67) 40.04 * (2.93)

* P <0.05, different from vehicle,

that received intra-VLS 6-OHDA. The number of lever press responses on the drug treatment day (day 7 after surgery) was significantly higher than the pre-drug baseline day (day 6) only among those DA depleted rats that received 40.0 mg/kg L-DOPA (t= 2.76, df = 8, P < 0.05).The number of lever presses emitted on the drug test day (day 7 after surgery) was recalculated as a percentage of the number of responses on day 6 (baseline) for rats receiving intra-VLS 6-OHDA (expressed as mean Yoof baseline +S.E.M.; vehicle, 103.17+ 7.5; 20.0 mg/kg L-DOPA, 134.19 + 15.6; 40.0 mg/kg L-DOPA, 194.08 ~ 46.1). There was art overall significant difference in terms of the percent of lever presses on day 7 relative to day 6 among the three DA depleted groups (Kruskal-Wallis test,

M.S. Cousins, J.D. Salamone/Brain Research 732 (1996) 186-194

EVER PRESSES 500

*

400 300 200 100

191

These observations indicated that the vast majority of lever presses in both control and DA-depleted rats were triggered by forelimb contact with the lever. In a few cases, there was also orofacial or snout contact with the lever, and on rare occasions snout contact alone deflected the lever. There were never any cases observed in which a rat pressed the lever using a part of the body other than the forelimb or head region.

0 6

7

DAYS _

VEH

n

20.0 mg/kg L-DOPA

=

40.0 mg/kg L-DOPA

Fig. 3. Mean ( ~ S.E.M.) number of lever presses for 6-OHDA-treated rats on days 6 (pre-dnrg baseline) and day 7 (drugged) of postsurgical testing ( * P <0.05, different from day 6). The mean ( ~ S.E.M.) number of lever presses for rats that received intra-VLS vehicle was 945.22 (~ 311.45) on day 6 and 979.89 (+224.40) on day 7 (data not shown on figure).

H = 7.8, P < 0.05). The Mann–Whitney U-test indicated that DA-depleted rats treated with systemic 40.0 mg/kg L-DOPA showed significantly higher responding on day 7 (as a % of day 6) than DA-depleted rats treated with systemic vehicle (U= 6.0, P < 0.05). The effects of L-DOPA on the detailed temporal parameters of responding are shown in Table 3. The Wilcoxon signed-rank test (T) showed that the average response initiation time was significantly shorter on the drug test day (day 7) after injection of either 20.0 or 40.0 mg/kg L-DOPA (T(8) = 2, P <0.05 and T(9) = 1, P <0.01, respectively) as compared to the pre-drug baseline day (day 6). Injection of 40.0 mg/kg L-DOPA also resulted in a significant decrease in the total pause time (T(9) = 17, P < 0.05) on the drug test day as compared to the pre-drug baseline day. Table 3 also shows the effects of various drug treatments expressed as the differences between days 6 and 7 performance. There was an overall significant effect of L-DOPA on average response initiation time (Kruskal-Wallis test, H= 7.8, P < 0.05). DA-depleted rats that received either 20.0 or 40.0 mg/kg L-DOPA showed a significant decrease in the average response initiation time as compared to vehicle-injected rats (U = 10, P < 0.05; U = 6, P <0.01, respectively). On the drug test day, correlational analyses of all DA-depleted rats showed that number of responses was significantly correlated with the average response initiation time, relative number of pauses, and the average length of pauses (r= –0.79, P < 0.0001; r = –0.68, P < 0.001; r = –0.84, P < 0.0001, respectively). 3.4. General observations As well as recording objective measures of lever pressing described above, some rats were directly observed during behavioral sessions and a few rats were videotaped.

Table 3 Mean (~ S.E.M.) of various behavioral measures on days 6 (pre-drug baseline) and 7 (drug test) for rats that received intra-VLS 6-OHDA and either 0.0, 20.0 or 40.0 mg/k.q L-DOPA on day 7 L-DOPA (mg/kg) O.o(n = 7) 20.O(n= 8) Average response initiation time (s): Day 6 10.53 8.11 (1.61) (3.31) Day 7 11.34 6.36 # (3.76) (1.46) A 0.81 – 1.76 ‘ (0.99) (0.55) Auerage length offast initiation times (s): Day 6 0.93 0.81 (0.11) (0.09) Day 7 0.99 0.70 (0.08) (0.08) A 0.61 –0.104 (0.15) (0.07) Average length ofpauses (s): Day 6 24.83 22.99 (6.42) (3.92) Day 7 25.54 18.69 (6.79) (3.26) A 0.71 –4.30 (3.05) (2.20) Total pause time (s): Day 6 1409.21 1464.95 (125.39) (90.94) 1360.53 Day 7 1379.50 (144.39) (87.51] ,+ 24.83 –85.45 (49.72) (6.42) Veq high rate responses (%): 13.84 Day 6 19.01 (3.28) (3.56) 13.76 17.87 Day 7 (2.96) (3.02) – 1.14 (2.61) ;auses(%): ‘;;; Day 6 36.21 32.02 (513) (3.68) Day 7 35.34 28.70 (3.44) (6.77) . –3.31 –0.97 (2.56) (5.15)

40.o(n = 9) 20.28 (12.45) 6.98 #

(2.22) -13.31 * (10.52) 0.77 0.08 0.87 (0.20) 0.09 (0.16) 34.23 (14.96) 17.29 (4.90) –24.21 (12.27) 1508.23 (67.99) 1371.19 # (105.82) – 137.04 (57.17) 24.44 (4.79) 24.83 (4.59) 0.39 (2.84) 41.09 (6.02) 33.46 (4.11) -7.63 (5.41)

‘Behavioral measure, expressed as the difference between day 6 and 7 (day 7 minus day 6). * P <0.05, different from vehicle; # ~ <0,05, different from day 6.

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4. Discussion VLS DA depletions dramatically impaired lever pressing on days 3–5 of postsurgical testing. The total number of lever presses was substantially reduced in DA-depleted rats. Detailed analyses of the temporal parameters of lever pressing revealed that decreases in responding in DA-depleted rats were not due to increases in response duration; rather, the lever pressing deficits were related to substantial increases in average response initiation time, Increases in the interresponse time and response initiation time have been observed previously in rats with either moderate or severe VLS DA depletions [11,49]. In the present study, average response initiation time was partitioned into two components: average length of fast initiation times and average length of pauses. Rats with VLS DA depletions showed significant increases in both components of the initiation time, indicating that there was a general slowing of response initiation. Increases in both the relative number and average length of pauses resulted in an increase in total pause time in DA-depleted rats. Additionally, DA-depleted rats responded at a slower local rate even during periods in which they were pressing the lever. These two deficits (i.e., a reduced local rate of responding and an increase in total pause time) rendered the DA-depleted rats severely impaired in terms of the overall number of responses emitted. Thus, a detailed analysis of the temporal characteristics of lever pressing indicates that VLS DA depletions impair a number of different indices of response initiation. Administration of L-DOPA to DA-depleted rats did not restore responding to normal levels. Nevertheless, this drug did produce a small but statistically significant improvement in some aspects of responding in DA-depleted rats. L-DOPA significantly increased the number of lever presses and decreased the average response initiation time in DA-depleted rats. Both doses of L-DOPA reduced average response initiation time but only 40.0 mg/kg L-DOPA increased the total number of lever presses. As noted above, several additional measures of response initiation were obtained in the present study, including the average length of fast initiation times, relative number of very high rate responses and pauses, as well as the average length of each pause and total pause time. Although these measures were highly sensitive to the effects of VLS DA depletions, L-DOPA had little or no consistent effect on these more specific response parameters. Clearly, the ability of LDOPA to increase responding in DA-depleted rats is not dependent upon a highly specific effect on any particular aspect of responding. In this regard, it is important to emphasize that the two simplest and most global measures of responding (i.e., total number of responses and average initiation time) were the behavioral measures that were most sensitive to the effects of L-DOPA. Thus, L-DOPA may be having modest effects on a number of different response measures, or may be affecting different aspects of

response initiation in different animals, yet the broadest aggregate measures of responding such as total responses and average initiation time were capable of detecting the effect of L-DOPA. Severe VLS DA depletions have been shown previously to increase the average response duration [11], a finding that was not supported in the present study. There are several differences between the two studies that may account for the present finding. First, the previous study compared response duration across 3 weeks of postsurgical testing. In that study, rats with severe VLS DA depletions showed small but significant increases in response duration, and it also was observed that controls had subtle average duration deficits during the first week of postsurgical testing as compared to the presurgical testing. In the present study, only the first 3 days of postsurgical testing were used for statistical comparisons, and it is possible that there was no apparent effect of DA depletion on response duration because the control group did not have enough time to recover normal response durations after surgery. Additionally, a higher dose of pargyline was used in the present study, which may have contributed to the increased response durations in the present control group. Thus, the lack of difference between DA-depleted rats and control rats in terms of response duration seems largely attributable to the fact that the control procedure also increased response durations in the first few days after surgery. Because the lateral striatum of the rat receives dense afferents from primary sensory and motor neocortex [9,43,60,62], it is possible that this neostriatal subregion represents the rodent homologue of the putamen. Evidence indicates that the putamen of primates and rodents is somatotopically organized, with the forepaw and orofacial region being organized in more ventral portions of the striatum [1,39,43]. Thus, it is possible that VLS DA depletions have such a profound effect on lever pressing because DA in this region is involved in skilled motor or sensorimotor functions of the head and forepaws. This interpretation is in agreement with the large body of evidence showing that lateral striatal DA depletions profoundly disrupt forepaw use [15,17,19,27,46,50], and also is corroborated by our own observations that the vast majority of lever pressing responses were observed to result from forelimb or snout contact with the lever. The deficits in lever pressing produced by VLS DA depletions are not due to a general decrease in motor activity, however, as it has been shown that VLS DA depletions do not reduce locomotion or rearing [12,27]. Parkinsonian patients have impairments involving orofacial, hand and upper limb motor control. Patients with Parkinson’s disease have difficulties swallowing and also have speech disturbances [7,14,23,54]. In his classic review, Marsden [36] suggested that parkinsonian patients have impairments in the automatic execution of learned motor acts. In fact. difficulties with fine hand movements

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are one of the warning signals for the onset of Parkinson’s disease, and it has been suggested that the speed of finger tapping could be used as a test for assessing parkinsonian symptoms [2]. Patients with Parkinson’s disease have slower reaction times [16] and slower arm movements than normal, and have difficulties coordinating both arms [32]. Many of these motor disturbances are partially reversible with L-DOPA [3,10,58], for example, impairments in the timing of repetitive wrist movements are improved by L-DOPA administration [28,41]. Therefore, it is evident that many of the motor impairments observed in Parkinson’s disease involve skilled movements of the orofacial and arm region. Although Parkinson’s disease is characterized by disturbances in skilled motor control of the arm and orofacial region, rodent models of this syndrome typically do not quantify motor activity other than locomotion [6,20,34,42,47,57]. Ljungberg and Ungerstedt [35] did report that 40.0 mg\kg L-DOPA and 0.1 mg/kg apomorphine increased feeding behavior in aphagic DA-depleted rats. These findings, along with the present results, indicate that L-DOPA cart improve aspects of motor activity other than locomotion in DA-depleted rats. In summary, VLS DA depletions severely impaired lever pressing in rats. Administration of 40.0 mg/kg LDOPA to DA-depleted rats increased the number of responses. L-DOPA also decreased average response initiation time and produced a slight decrease in total pause time, indicating that L-DOPA increased the overall speed of responding in terms of the initiation of lever pressing. Thus, it seems reasonable to suggest that the skilled motor deficits produced by VLS DA depletions may serve as a useful animal model of some aspects of Parkinson’s disease. Although L-DOPA is the most widely used therapy [13], the need for testing additional antiparkinsonian drugs is exemplified by the fact that L-DOPA therapy is not without its disadvantages. Many motor disturbances still exist following L-DOPA administration [3,4,28,44,58]. The therapeutic effects of L-DOPA can wear off, or dyskinetic movements may result [22]. Future research involving the motor deficits induced by VLS DA depletions in rats can be used to study additional antiparkinsonian treatments such as selective D1 and D2 agonists, anticholinergic drugs and excitatory amino acid antagonists [21,26,30,31,33,40]. Acknowledgements This work was supported by a grant from the University of Connecticut Psychology Department. Many thanks to Juliet Aberman for her assistance with the manuscript. References [1] Alexander, G.E., Delong, M.R. Microstimulation of the primate neostriatum. 2. Somatotopic organization of striatal microexcitable

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zones and their relation to neuronal response properties, J. Neurophysiol., 53 (1985) 1417-1430. [2] Barbeau, A. Parkinson’s disease: clinical features and etiopathology.In Vinken, Bruyn and Klawans (Eds.), Handbook of Clinical Neurology, Vol. 5 (49): Extrapyramidal Disorders, Elsevier, Amsterdam, 1986, pp. 87-152. [3] Baroni, A., Benvenuti, F., Fantini, L., Pantaleo, T., Urbani, F. Human ballistic arm abduction movements: effects of L-DOPA treatment in Parkinson’s disease, Neurology, 34 (1984) 868–876. [4] Benecke, R., Rotbwell, J.C., Dick, J.P.R., Day, B.L., Marsden, C.D. Simple and complex movements off and on treatment in patients with Parkinson’s disease, J. Neurol. Neurosurg. Psychiatry, 50 (1987) 296-303. [5] Bernheimer, H., Birkmayer, W., Homykiewicz, O., Jellinger, K., Seitelberger, F. Brain dopamine and the syndromes of Parkinson and Huntington: clinical, morphological and neurochemical correlations, J. Neurol. Sci., 20 (1973) 415-455. [6] Brautigam, K., Sohr, R., Morgenstem, R. 3-0-Methyl-DOPA is not involved in the development of behavioral supersensitivity after repeated L-DOPA administration in 6-OHDA lesioned rats, Behau. Brain Res., 63 (1994) 41-45. [7] Caligiuri, M.P. Labial kinematics during speech in patients with parkinsonian rigidity, Brain, 110 (1987) 1033-1044. [8] Camarata, P.J., Parker, R.G., Park, SK., Haines, S.J., Turner, D.A., Chae, H., Ebner. T.J. Effects of l-metbyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP)-induced hemiparkinsonism on the kinematics of a two-dimensional, multijoint arm movement in the rhesus monkey, Neuroscience, 48 (1992) 607-619. [9] Collins, R.C. Kindling of neuroanatomic pathways during recurrent focal penicillin seizures, Brain Res., 150 (1978) 503-517. [10] Cotzias, G.C., Papavasiliou, P.S., Gellene, R. Modification of parkinsonism — chronic treatment with L-DOPA, N. Engl. J. Med., 280 (1969) 337-345. [11] Cousins, M.S., Salamone, J.D. Involvement of ventrolateral striatal dopamine in movement initiation and execution: a microdialysis and behavioral investigation, Neuroscience, 70 (1996) 849-859. [12] Cousins, M.S., Sokolowski, J.D., Srdamone, J.D. Different effects of nucleus accumbens and ventrolateral striatal dopamine depletions on instrumental response selection in the rat, Pharmacol. Biochem. Behav, 46 (1993) 943-951. [13] Creasey, H.M., Broe, G. Parkinson’s disease, J. Australia, 159 (1993) 249-252. [14] Doty, R.L., Rikkm, M., Deems, D.A., Reynolds, C., Stellar, S. The olfactory and cognitive deficits of Parkinson’s disease: evidence for independence, Ann. Neurol., 25 (1989) 166–171. [15] Dunnett, S.B., Iversen, S.D. Sensorimotor impairments following localized kainic acid and 6-hydroxydopamine lesions of the neostriatum, Brain Res., 248 (1982) 121–127. [16] Evarts, E.V., Teravainen, H., Caine, D.B. Reaction time in Parkinson’s disease, Brain, 104 (1981) 167–186. [17] Evenden, J.L., Robbins, T.W. Effects of unilateral 6-hydroxydopamine lesions of the caudate-putamen on skilled forelimb use in the rat, Behau. Brain Res., 14 (1984) 61–68. [18] Factor, S.A., Weiner, W.J. The current clinical picture of Parkinson’s disease. In F. Hefty and W.J. Weiner (Eds.), Progress in Parkinson Research, Plenum Press, New York, 1988, pp. 1-9. [19] Fairley, P.C., Marshall, J.F. Dopamine in the lateral caudate-putamen of the rat is essential for somatosensory orientation, Behau. Neurosci., 100 (1987) 652-663. [20] Fink, J.S., Smith, G.P. L-DOPA repairs deficits in locomotor exploration produced by denervation of catecholamine terminal fields in the forebrain of rats, J. Comp. Physiol. PsychoL, 93 (1979) 66–73. [21] Greenamyre, J.T. Glutamate-dopamine interactions in the basal ganglia: relationship to Parkinson’s disease, J. Neural. Transm., 91 (1993) 255-269. [22] Hardie, R.J., Lees, A.J., Stem, G.M. On-off fluctuations in Parkinson’s disease, Brain, 107 (1984) 487–506.

194

M.S. Cousins, J.D. Salamone/Brain Research 732 (1996) 186-194

[23] Hartelius, L., Svennson, P, Speech and swallowing symptoms associated with Parkinson’s disease and multiple sclerosis: a survey, Folia Phoniatr. L.ogop., 46 (1994) 9-17. [24] Heindel, W.C., Salmon, D,P., Shults, C.W., Walicke, P.A., Butters, N. Neuropsychological evidence for multiple memory systems: a comparison of Alzheimer’s, Huntington’s and Parkinson’s disease patients, J. Neurosci., 9 (1989) 582-587, [25] Homykiewicz, O. Dopamine and its physiological significance in brain function. In G.H. Browne (Ed.), The Structure and Function of Nervous Tissue, Academic Press, New York, 1972. [26] Hubbard, C.A., Trugman, J.M. Reversal of reserpine-induced catalepsy by selective D1 and D2 dopamine agonists, Mouement Disord., 8 (1993) 473-478. [27] Jicha, G., Salamone, J.D. Vacuous jaw movements and feeding deficits in rats with ventrolateral striatal dopamirredepletions: possible model of parkinsonian symptoms, J. Neurosci., 11 (1991) 822-3829. [28] Johnson, M.T.V, Mendez, A., Kipnis, A.N., Silverstein, P., Zwiebel, F., Ebner, T.J. Acute effects of levodopa on wrist movement in Parkinson’s disease. Kinematics, volitional EMG modulation and reflex amplitude modulation, Brain, 117 (1994) 1409–1422. [29] Klockgether, T., Dichgans, J. Visual control of arm movement in Parkinson’s disease, Mouement Disord., 9 (1994) 48-56. [30] Klockgether, T., Turski, L., Honore, T., Zhang, Z., Gash, D., Kurlan, R., Greenamyre, J.T. The AMPA receptor antagonist NBXQ has antiprrrkinsonian effects in monoamine-depleted rats and MPTP-treated monkeys, Ann. Neurol., 30 (1991) 717-723. [31] Lange, K.W., Riederer, P. Glutamatergic drugs in Parkinson’s disease, Lt~eSci., 55 (1994) 2067–2075. [32] Lazarus, A., Stelmach, G.E. Interlimb coordination in Parkinson’s disease, Mouement Disord., 7 (1992) 159-170. [33] Lees, A.J. Dopamine agonists in Parkinson’s disease: a look at apomo~hine, Fund. Clin, Pharmacol., 7 (1993) 121–128. [34] Ljungberg, T., Ungerstedt, U. Reinstatement of eating by dopamine agonists in aphagic dopamine denervated rats, Physiol. Behau., 16 (1974) 277-283. [35] Longstreth, W.T., Nelson, L., Linde, M., Munoz, D. Utility of sickness impact profile in Parkinson’s disease, J. Geriatr. F’sychiatry Neurol., 5 (1992) 142–148. [36] Marsden, C.D. The mysterious motor function of the basal ganglia: the Robert Wartenberg lecture, Neurology, 32 (1982) 514-539. [37] Marshall, J.F., Richardson, J.S., Teitelbaum, P. Nigrostriatal damage and the lateral hypothalamic syndrome, J. Comp. Physiol. Psychol., 87 (1974) 808-830. [38] Marshall, J.F., Levitan, D., Sticker, E.M. Activation-induced restoration of sensorimotor functions in rats with dopamine-depleting brain lesions, J. Comp. Physiol. PsychoZ,, 90 (1976) 536–546. [39] McGeorge, A.J., Faull, R.L.M. The organization of the projection from the cerebral cortex to the striatum in the rat, Neuroscience, 29 (1989) 503-537. [40] Morelli, M., Fenu, S., Pinna, A., DiChiara, G. Opposite effects of NMDA receptor blockade on dopaminergic D1- and D2-mediated behavior in the 6-hydroxydopamine model of turning: relationship with c-@s expression, J. PharmacoL Exp. Ther., 260 (1992) 402– 408. [41] Pastor, M.A., Jahanshahi, M., Artieda, J., Obese, J.A. Performance of repetitive wrist movements in Parkinson’s disease, Brain, 115 (1992) 875-891. [42] Paul, M.L., Graybiel, A.M., David, J.C., Robertson, H.A. D1-like and D2-like dopamine receptors synergistically activate rotation and c-fos expression in the dopamine-depleted striatum in a rat model of Parkinson’s disease, J. Neurosci., 12 (1992) 3729-3742. [43] Pisa, M., Schranz, J.A. Dissociable motor roles of the rat’s striatum conform to somatotopic model, Behau. Neurosci., 102 (1988) 429– 440.

[44] Pullman, S.L,, Watts, R.L., Juncos, J,L., Chase, T.N., Sanes, J.N. Dopaminergic effects on simple and choice reaction time performance in Parkinson’s disease, Neurology, 38 (1988) 249–254. [45] Rinne, J.O. Nigral degeneration in Parkinson’s disease, Mouement Disord., 8 (1993) s31–s35, [46] Sabol, K.E., Neill, D.B., Wages, S.A., Church, W.H., Justice, J.B. Dopamine depletion in a striatal subregion disrupts performance of a skilled motor task in the rat, Brain Res,, 335 (1985) 33–43. [47] Sakai, K., Gash, D,M. Effect of bilateral 6-OHDA lesions of the substantial nigra on locomotor activity in the rat, Brain Res., 633 (1994) 144-150. [48] Salamone, J.D. Strategies for drug development in the treatment of dementia. In P. Winner (Ed.), Behavioral Models in Psychopharmacology, Cambridge Univ. Press, Cambridge, UK, 1990, pp. 598–613. [49] Salamone, J.D., Kurth, P.A., McCullough, L.D., Sokolowski, J.D., Cousins, M.S. The role of brain dopamine in response initiation: effects of haloperidol and regionally specific dopamine depletions on the local rate of instrumental responding, Brain Res., 628 (1993) 218-226. [50] Salamone, J.D,, Mahan, K., and Rogers, S. Ventrolateral striatal dopamine depletions impair feeding and food handling in rats, PharmacoL Biochem. Behau., 44 (1993) 605-610. [51] Salamone, J,D., Zigmond, M.J., Sticker, E.M. Characterization of the impaired feeding behavior in rats given haloperidol or dopamine-depleting brain lesions, Neuroscience, 39 (1990) 17-24. [52] Schallert, T,, Whishaw, I.Q., Rarnirez, V.D., Teitelbaum, P, Compulsive, abnormal walking caused by anticholinergics in akinetic, 6-hydroxydopamine-treated rats, Science, 199 (1978) 1461-1463. [53] Stricker, E.M., Zigmond, M.J. Recovery of function after damage to catecholamine-containing neurons: a neurochemical model for the lateral hypothalamic syndrome. In J.M. Sprague and A.N. Epstein (Eds.), Progress in Psychobiology and Physiological Psychology, Academic Press, New York, 1976. [54] Svensson, P., Henningson, C., Karlsson, S. Speech motor control in Parkinson’s disease: a comparison between a clinical assessment protocol and a quantitative analysis of mandibular movments, Folia Phoniatr, Base, 45 (1993) 157-164. [55] Taylor, J.R., Lawrence, D.E., Redmond, J.D., Elsworth, J.D., Roth, R.H., Nocols, D.E., Mailman, R.B. Dihydrexidine, a full doparnine DI agonist, reduces MPTP-induced parkirrsonism in monkeys, Eur. J. PharmacoL, 199 (1991) 389. [56] Ungerstedt, U, Aphagia and adipsia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system, Acta PhysioL Scand., 82 Suppl. 367 (1971) 95-122. [57] Uretsky, NJ., Schoenfeld, R.I. Effect of L-DOPA on the locomotor activity of rats pretreated with 6-hydroxydopamine, Nature, 234 (1971) 157-159. [58] Velasco, F., Velasco, M. A quantitative evaluation of the effects of L-DOPA on Parkinson’s disease, Neuropharmacology, 12 (1973) 89-99. [59] Verrmrlen,R.J., Drukarch, B., Sahadat, M.C.R., Goosen, C., Welters, E.C,, Stoof, J.C. The selective dopamine D1 receptor agonist, SKF 81297, stimulates motor behavior of MPTP-lesioned monkeys, Eur. J. Pharmacol., 235 (1993) 143-147, [60] Webster, K.E. Cortico-striate interrelations in the aJbino rat, J, Anat., 95 (1961) 523-543, [61] Whishaw, I.Q., O’Connor, W.T., Dunnett, S.B. The contribution of motor cortex, nigrosriatal dopamine and caudate-putamen to skilled forepaw use in the rat, Brain, 109 (1986) 805-843. [62] Wise, S.P., Jones, E.G. Cells of origin and terminal distribution of descending projections of the rat somatic sensory cortex, J. Comp. Neurol., 175 (1977) 129-158, [63] Zigmond, M.J., Stricker, E.M., Berger, T.W. Parkinsonism: insights from animal models utilizing neurotoxic agents, In Animal Models of Dementia, Alan R. Liss, New York, 1987, pp. 1–38.