neurochemical indexes of nigrostriatal dopaminergic function were compared between sedentary control rats (n = 12) and daily spontaneous running (DSR) ...
Daily spontaneous and neurochemical DEAN
E. DLUZEN,
Departments Rootstown,
running indexes BINJUN
LIU,
alters behavioral of nigrostriatal function CHAO-YIN
of Anatomy and Physiology, Ohio 44272-0095
Dluzen, Dean E., Binjun Liu, Chao-Yin Chen, and Stephen E. DiCarlo. Daily spontaneous running alters behavioral and neurochemical indexes of nigrostriatal function. J. Appl. Physiol. 78(4): 1219-1224, 1995.-Behavioral and neurochemical indexes of nigrostriatal dopaminergic function were compared between sedentary control rats (n = 12) and daily spontaneous running (DSR) male rats (n = IO). Nine weeks of DSR did not significantly alter body, heart, pituitary, or testes weights. DSR and control animals did differ in performance on a sensorimotor beam walking task, with DSR rats showing significantly shorter times required to crossthe beam (60 t 17 vs. 119 t 14 s; P < 0.02) as well as fewer slips off the beam (3.0 t 0.8 vs. 6.2 t 1.1; P < 0.05). DSR animals also engagedin significantly greater durations of social investigation than control rats (43 t 5 vs. 25 2 3 s; P < 0.01) when tested in a social investigation memoryrecognition test. Basal dopamine release rates from superfused corpus striatal tissue fragments of DSR rats were about one-half those obtained from control animals (18 t 5 vs. 34 t 6 pg. mg-’ . min-‘; P < 0.05), whereas responsesof these striatal tissue fragments to a depolarizing concentration of potassium were virtually identical (45 t 10 vs. 47 t 8 pg mg-’ min-I). These data indicate that a relatively limited intensity of DSR insufficient to alter cardiovascular function can exert substantial effects on behavioral and neurochemical indicators of nigrostriatal dopaminergic activity. l
CHEN,
Northeastern
l
dopamine; corpus striatum; exercise; sensorimotor; social investigation
AND
STEPHEN
Ohio Universities,
E. DICARLO College of Medicine,
From these reports, two important points emerge. First, there exists a strong association between NSDA function and cognitive components of motor behavior. Second, the predominant means to study this relationship has involved pharmacological treatments and lesions with behavioral analyses and in some cases has been combined with measurements of striatal dopamine release. Relatively little work has focused on attempts to noninvasively modulate NSDA functioning and examine the resultant effects on behavioral and/or neurochemical parameters. There is evidence that raising rats in an enriched enviroment substantially alters the NSDA system as indicated by changes in spontaneous and amphetamine-stimulated locomotor activity compared with impoverished control animals (1). Moreover, rats subjected to a 4-wk forced swimming exercise training program showed marked differences in locomotor activity as well as yawning and stereotypy in response to apomorphine compared with nonexercised control animals (2). Therefore, it appears that subjecting animals to specific enviromental and/or training programs can exert a considerable influence on subsequent NSDA functioning. In this report we determined whether animals allowed to spontaneously exercise when given free access to a running wheel would also show alterations in NSDA function. The effects of this daily running activity were evaluated on sensorimotor as well as social investigatory memory-recognition behavioral tests reflecting NSDA function and striatal dopamine release as determined with in vitro superfusion.
NIGROSTRIATAL DOPAMINERGIC (NSDA) system is a motor-sensorimotor integration area within the brain that has been associated with the pathology of Parkinson’s disease. This relationship between the NSDA sys- 11MATERIALS AND METHODS tem and motor behavior has been substantiated by reAnimals. Male Sprague-Dawley rats were weaned at 3 wk sults from numerous experimental paradigms utilizing of age and randomly assignedto a control group (n = 12) or animal models. For example, pharmacological agents a daily spontaneousrunning (DSR) group (n = 10). Control that can activate the NSDA system, such as amphetrats remained housed in standard laboratory cages(45 x 24 amine or apomorphine, produce clear changes in motor x 20 cm), whereas the DSR rats had free accessto a running behavior (23,29). When these treatments are performed wheel for the duration of the exercise phase of the experiin the unilaterally 6-hydroxydopamine-lesioned animal, ment. Running distances (in m) were recorded daily, and they yield an effective and extensively used animal rotamean weekly distances were calculated for each animal durtion model with which to study NSDA function (12). ing the 9 wk of exercise. Both groups were maintained under Complementing these findings are data showing that identical 12:12-h light-dark cycles (lights on at 0600) with increases in striatal dopamine release occur when rats food and water available ad libitum. BehavioraL tests. At the completion of the spontaneousrunare engaging in a motor behavior task (9,11). Although these findings demonstrate a robust relationship be- ning or sedentary control period, all animals were subjected to two behavioral tests, a sensorimotor task and a social intween NSDA function and motor behavior, this system vestigation memory-recognition test. The choicefor these two does not appear to be devoted exclusively to motor activtests was basedon their involvement with the NSDA system. ity, since drinking (24) and social investigatory behavFor the sensorimotor task, rats were placed on the “open” iors (20) have also been associated with increased striaend of a 1.7-cm-wide 95-cm-long beam and the time required tal dopamine release. When these data are compiled, it for the rats to walk from the “open” to the opposite end of appears that the striatal dopamine system functions as the beam (leading into their home cage) was recorded. In a complex integrator of sensory, associative, and af- addition to the amount of time required to traverse the beam, fective processes involving motor behavior (25). the number of slips off the beam was recorded for each aniTHE
0161-7567/95
$3.00 Copyright 0 1995 the American
Physiological
Society
1219
1220
DAILY
RUNNING
AND NIGROSTRIATAL
mal. The purpose of this test was to evaluate whether DSR would exert any effects on functioning of the NSDA system as indicated by the rat’s sensorimotor performance on this task. For the social investigation memory-recognition task, an ovariectomized stimulus rat was placed in the home cage of the male rat for a 5min period and the amount of time (in s) involved with social investigation directed to the stimulus female was recorded (t&Z 1). At the end of this 5-min test period the female was removed, and after a 30-min (intertrial interval) period the same female was placed in the male’s home cage for a second 5-min period (triaZ 2) and the amount of time involved with social investigation directed to the female was again recorded. When males were tested twice with the same stimulus, the amount of investigation time typically decreased during the second presentation, which has been interpreted as indicative of a memory-recognition response (30). Because it has been reported that social stimuli increase striatal dopamine release (17, ZO), this behavioral test was included to assess whether DSR would influence a social/ cognitive behavioral response that is related to NSDA function. Both tests were conducted by a naive observer who was blind to the experimental group. All tests were initiated at >48 h after the last DSR session. Assays. At the conclusion of behavioral testing (>24 h), all rats were weighed and killed by decapitation. At autopsy the heart, testes, and pituitary were removed and weighed. The heart was removed and weighed to evaluate whether a significant cardiovascular effect was achieved in the DSR rats as evident by an increase in heart weight or in the heartto-body weight ratio. Because we have reported that male gonadal steroid hormones may alter behavioral and neurochemical responses related to NSDA function (3,4), the testes and pituitary were weighed to determine whether differences in the pituitary-gonadal axis were present between DSR and control rats. The brain was removed, and the corpus striatum (CS) was dissected and prepared for superfusion to determine dopamine release rates in vitro. Effluent superfusion samples were assayed for dopamine by using high-performance liquid chromatography with electrochemical detection (ESA, Bedford, MA). Biogenic amines were separated on a model C-18 Biophase 5-pm sphere analytic column (Bioanalytical Systems, West Lafayette, IN). The mobile phase consisted of (in mM) 50 sodium acetate, 27.4 citric acid, 10 sodium hydroxide, 0.1 sodium octyl sulfate and 0.1 EDTA and 7% methanol in filtered distilled water. The final pH of 4.5 was achieved with the addition of sodium hydroxide, and the mobile phase was filtered (0.45 pm, Millipore filter) before use. Standards for dopamine were diluted in the superfusion medium, and doses of 7.8, 15.5, 31, 62, 125, and 250 pg/20 ~1 were used to construct a standard curve. The sensitivity for dopamine, as defined by a response three times above baseline noise, was between 7.8 and 15.5 pg. Tissue dissection. For dissection of CS, a coronal cut was made through the optic chiasm and CS was removed within the perimeter of the corpus callosum from the anterior portion of this dissection. In this way, the anterior-dorsal half of the bilateral CS was used for assessment of dopamine release rates. The CS was further dissected into -0.5 x 0.5 x 0.5.mm tissue fragments before placement into superfusion chambers. In vitro superfusion. The superfusion medium consisted of a modified Krebs-Ringer phosphate (KRP) buffer composed of (in mM) 120 NaCl, 4.8 KCl, 0.8 CaC12, 1.2 MgS04, 10.2 Na2HP04 and 1.8 NaH2P04 and 0.18% glucose at pH 7.4. The KRP medium was filtered (0.45 pm, Millipore filter) before use. The superfusion chamber consisted of the barrel of a lml plastic tuberculin syringe cut off at the 0.3-ml level and was attached to a 22-gauge stainless steel syringe needle.
FUNCTION
This assembly was placed in a temperature-controlled water bath that maintained the superfused CS at 37°C. The CS tissue fragments were suspended on cellulose filter paper within the chamber and were contained within 100 ,wl of medium volume. The chamber was sealed with a stopper containing two needles, one supplying filtered humidified air to the chamber and the other serving as an exit port for the collection of effluent samples. Superfusion medium was delivered through the bottom of the chamber at a flow rate of 25 $/min, and effluent samples were collected at lO-min intervals in plastic tubes maintained on ice. At the end of the superfusion, CS tissue fragments were removed and weighed. The superfusion system for determination of in vitro catecholamine release has been extensively used and validated in our laboratory (2 1). Superfusionprotocol. In this report, both the basal (spontaneous) and potassium-stimulated dopamine release rates were evaluated from the superfused CS tissue fragments of control and DSR animals. After placement of the CS into the superfusion chamber, the tissue fragments were superfused for a 30-min equilibration period during which no samples were collected. Subsequently, effluent samples were collected at lo-min intervals for a total of eight collection intervals. Two basal samples (collection intervak 1 and 2) were followed by a potassium-stimulation response challenge. For the potassium-stimulation challenge, KRP superfusion medium containing a depolarizing concentration of potassium (30 mM) was infused during collection intervals 3 and 4, after which the normal KRP medium was resumed for the remainder of the superfusion. Media with the depolarizing concentration of potassium contained a reduced concentration of NaCl to maintain an osmolarity of 290 mosM. StatisticaL anaZyses. For the sensorimotor behavioral test data, separate t-tests were used to compare differences in performance between control and DSR animals for the times to cross the beam and the number of slips off the beam. The data from the social investigation memory-recognition task were analyzed with a two-by-two two-way analysis of variance (treatment condition x trial). All tissue weights were analyzed by individual t-tests. For the dopamine release rate data, separate t-tests were used to compare differences between control and DSR animals for the basal release (as calculated from the mean values obtained in collection interuaZs 1 and 2) and potassium-stimulated release (as calculated from the mean values obtained in collection interuaZs 3-5). P < 0.05 was required for results to be considered statistically significant. RESULTS
The mean weekly running distance (m/day) for the 10 DSR rats is shown in Fig. 1. Running distance steadily increased after the 1st 3 wk, plateaued by week 6, and remained fairly constant until the end of the daily running period at week 9. The results from the behavioral sensorimotor and social investigation memory-recognition tests performed at the conclusion of the daily running or sedentary control period are shown in Figs. 2 and 3, respectively. DSR animals performed significantly better than control animals for both measures of sensorimotor task performance. Specifically, DSR rats required significantly less time to cross the beam (P < 0.02; Fig. 2A) and were characterized by significantly fewer slips off the beam (P < 0.05; Fig. 2.B) compared with control animals. Social investigation memory-recogni-
DAILY
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FIG. 1. Daily spontaneous male rats (n = 10) permitted
period. Data are means t SE
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tion scores of DSR animals also differed markedly from that of control animals (Fig. 3). The investigation times of DSR rats were significantly greater (P < 0.01) than those of control animals. On completion of these behavioral tests (>24 h), all animals were killed. Body and organ weights were determined from each animal, and these data are shown in Table 1. Although body weights of DSR animals were lower than those of control animals, these differences failed to achieve statistical significance. Heart, testes, and nituitarv weights were also not different between DSR&and control rats, with the overall mean weights of these organs being virtually identical between-the two groups. A summary of the basal and potassium-stimulated dopamine release rate data is shown in Fig. 4. Basal dobamine release rates from superfused striatal tissue ofDSR rats were significantly-lower (P < 0.05) than values from control animals. -In contrast to basal release, values of the potassium-stimulated dopamine release rate were virtually identical between the two groups and no statistically significant differences were obtained. DISCUSSION
The present results demonstrate indexes related to NSDA function
that a number of are modulated by
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DSR. It had been shown previously that a forced swimming exercise regimen significantly altered NSDA-associated behaviors as evidenced by changes in locomotor activity, yawning, and sterotypy in response to an apomorphine challenge (2). The significance of the present results lies in the demonstration that, given the opportunity , animals w ill spontaneously “se1f-exercise” with the result of this behavior being a significant change in both behavioral and neurochemical responses related to the NSDA system. The quantity of this DSR in the animals of the present experiment was quite limited. This statement is based on the relatively truncated distances traveled by these animals compared with previous reports u sing this paradigm (28) as well as by the absence of a significant increase in the heart weight of these DSR rats. With regard to this latter parameter it should be noted that body weights failed to differ significantly and, therefore, the heartto-body weight ratios were also not different between DSR and control animals, indicating an absence of a cardiovascular training response. These cardiovascular results can be contrasted to the significant increases in heart-to-body weight ratios obtained from animals that were given forced swimming exercise training and showed changes in NSDA activity (2). There was also not any apparent effect of our DSR regimen on the gonadal system, wi th both testes and pituitary weights of DSR and control animals showing virtually identical values. Consequently, the results we obtained cannot be attributable to any changes in gonadal steriod hormones, which can significantly modulate the NSDA system (3,4). The salient conclusion that emerges when these running distance and bioassay weight data are collated is that the DSR rats can be characterized as animals that engaged in low levels of spontaneous running insufficient to alter their cardiovascular system while significantly modulating NSDA function. The effects of this low-intensity spontaneous running can be distinguished from the more intense stress-induci wz modulatory effects on the NSDA system obtained from a forced swimming training regimen (2). In an attempt to achieve a relatively comprehensive index of the effects of this daily running on NSDA function, we evaluated both behavioral and neurochemical determinations associated with this system. Significant effects of daily running were obtained for both
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