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Microelectrophoretic application of kainic acid into the globus pallidus: Disturbances in feeding behavior
Brain Research Bulletin, Vol. 28, pp. 151-756, Printedin the USA. AUrights reserved.
0361-9230/92
1992
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Copyright 0 1992 Pergamon Press Ltd.
Microelectrophoretic Application of Kainic Acid Into the Globus Pallidus: Disturbances in Feeding Behavior P. SANDOR, A. HAJNAL, G. JANDO, Z. KARADI AND L. LeNzkRD’ NeurophysiologyResearch Group of the Hungarian Academy of Sciences at Institute of Physiology UniversityMedical School, P&s, H-7443 P&s, Hungary Received 15 October 199 1 SANDOR, P., A. HAJNAL, G. JAN&, Z. KAaDI AND L. LfiNWRD. Microelectrophoretic application of kainic acid into the globus pallidus: Disturbances in feeding behavior. BRAIN RES BULL 28(S) 75 l-756, 1992.-Body weight changes, food and water intake, and sensorimotor disturbances of male rats were studied after bilateral kainic acid-(KA) induced lesions of the globus pallidus (GP). To minimize the extent of damages, KA was applied electrophoretically by means of glass micropipettes (tip diameter of the pipettes was lo- 15 pm). The neuron-specific damages of the GP resulted in aphagia and adipsia and rapid body weight decrease. Lesioned animals showed permanent motor disturbances but only temporary difficulties in the orientation toward sensory stimuli. Our data show that the selective destruction of the GP neurons results in a complex disorder that has motivational, (sensori)motor, and metabolic components. Globus pallidus
Kainic acid microelectrophoresis
Feeding deficits
Motor disturbances
echolaminergic (CA) fibers of passage without involving local cellular elements (39). It was suggested, therefore, that the NSDS is essential for the organization of hunger-motivated behavior (even for the metabolic and sensorimotor regulation) (31,39,46). Previous results of our laboratory suggested, however, that the sensory neglect seen after pallidal lesions is due to the damage of fibers of the mesolimbic dopaminergic system (MLDS) pas-sing through the GP to reach different neostriatal areas (23,24). According to this interpretation, lesion of the NSDS may cause only motor disturbances whereas destruction of the MLDS results in perceptual dysfunctions (22). Kainic acid (KA) and ibotenic acid (IB), the neurotoxic analogs of glutamate, destroy neuronal cell bodies intrinsic to the injected area but leave passing fibers intact (10,18,52). By means of these neuroexcitotoxins as lesioning agents, it is possible to examine the effects of selective destruction of local neurons. Recently, several laboratories proved that injection of KA into different brain areas, although sparing passing fibers, produces feeding disturbances or sensorimotor deficits (1,5,&l 1,2 1, 25,41,48,5 I). Bilateral injection of KA into the striatopallidal region causes enhanced aversion to tastes and aphagia (5). Damage to the pallidum with IB produces changes of both daily locomotor activity and learned running response for food (3,32). Because intracerebral hydraulic microinjection of KA or IB causes neural degenerations at distant brain areas from the in-
IT is well established that the globus pallidus (GP) plays essential roles in neural mechanisms controlling food and water intake. Bilateral electrolytic lesions of the GP, destroying both cellular elements and passing fibers, result in aphagia and adipsia and rapid body weight loss and early death of animals (22,26,28,34). Most animals die without artificial tube feeding and even those that recover to spontaneous feeding or drinking show regulatory deficits in different physiological challenges and exhibit abnormal blood glucose concentration (12,26,3 I ,34,36). These results suggest the GP is involved in the metabolic regulation (12,13,20,26). On the other hand, the GP has been reported to play an important role in the sensorimotor control of movements. GP-lesioned animals display bizarre posture and have trouble using their forelimbs and mouth during eating or drinking (19,20,22,28,43). After electrolytic GP lesion, sensory neglect develops, that is, rats exhibit impairments in orientation to different sensory stimuli (22,27). It is suggested that all these symp toms contribute to the feeding disturbances seen after the destruction of GP (22,27). Symptoms similar to those seen after pallidal electrocoagulation can also be observed after 6-hydroxydopamine (6-OHDA) injections into the GP, lateral hypothalamus (LH), or substantia nigra (SN) (8,9,20,3 1,40). The ascending nigrostriatal dopaminergic system (NSDS) originating from the SN passes through these structures (15,30). 6-OHDA causes the interruption of cat-
’ Requests for reprints should be addressed to L.&z16tin&d, M.D., Ph.D., D.Sc., Neurophysiology Research Group of the Hungarian Academy of Sciences at Institute of Physiology, University Medical School, Szigeti str. 12., P&s, H-7643 WCS,Hungary.
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jetted site, these results were criticized. In addition, long-lasting epileptic activity (up to 40-60 days), enhanced motor excitability, and disturbances in autonomic activity also appear as side effects of these injections (7,16,35,47,5 1). These side effects of KA and IB can directly or indirectly affect motor activity, as well as feeding behavior, of animals. Our previous results demonstrated that the side effects of KA can be eliminated by electrophoretic application of KA and that KA microelectrophoresis is a useful technique in the study of feeding behavior (2 1,25). In the present experiments, body weight and food and water intake of rats were studied after bilateral KA-induced lesion of the GP. KA was applied electrophoretically by means of glass micropipettes to minimize the side effects of this excitotoxin. It was examined whether well-localized selective destruction of GP neurons can produce 1) feeding deficits and 2) sensorimotor disturbances influencing eating and drinking. METHODS
Animals and Procedures Fifty-four adult, male CFY rats (LATI, Godollo) weighing 294-341 g at the beginning of the experiments were used. For 9 days before surgery, all animals were kept in a temperatureand light-controlled room (2 1 f 2°C 12 L: 12 D cycle, light on at 6:00 a.m.). They were housed individually and standard laboratory food pellets and tapwater were available ad lib. Daily food and water consumptions and body weight were measured to the nearest g and ml, respectively. Preoperative measurements were carried out for 8 days. At the eighth day (1 day before operation), mean body weight and food and water consumption of rats were: 328.5 + 19.2 g, 21.05 + 3.54 g, and 33.5 + 4.78 g, respectively. Based on data of preoperative body weight records, animals were distributed into two evenly formed groups [for details, see our previous publications (21,25)]: 27 KA and 27 sham-operated controls (CO). Surgery Double-barrelled glass micropipettes pulled from Pyrex glass tubes were filled with the following solutions: 1) one of the two barrels was filled with KA (Sigma Chemical Co., St. Louis, MO, KA dissolved in distilled water and titrated to pH 6.9-7.2 with NaOH); 2) the second barrel served for the ejection of Pontamine Sky Blue (PSB, GURR, saturated PSB in 0.15 M NaCl and 0.5 M Na acetate) to mark the lesioned area. Stereo&c coordinates for the GP according to DeGroot (6) were: A, 6.6; L, 3.3; V, -0.5. Bilateral operations were carried out under Calypso1 anesthesia (ketaminum, Richter, Budapest, 100 mg/kg, IP). After drilling a small hole in the skull and opening the dura mater under microscopic control, pipettes were carefully lowered to the target area. KA was released electrophoretically by means of a constant current device (EMG 4767). Because current intensity was monitored by a microammeter, changes in conditions of electrodes (damage of the pipette tip, blocking, etc.) could be controlled. KA was applied at the following parameters (2 1,25): concentration, 80 mM; tip diameters of pipettes, 10-l 5 pm; current intensity, 5-8 PA for 4 min. After ejection of KA, PSB was also applied into the same loci (I 5- 18 WAfor 5 min). After the current was terminated, pipettes remained in place for 5 min. In the CO rats only PSB (with identical parameters) was applied into the GP.
appearance of spontaneous feeding. The artificial diet consisted of one part sugar, two parts egg yolk and milk, salt, and vitamin B (Polybe, Richter) (26). All animals were weighed daily before feeding and were tube fed with 8- 10 ml of the aforementioned diet and 8 ml tap water. Those animals that died or had to be killed were dissected to control the state of their abdomen and mediastinum. No injuries or pathological changes were found in these rats. Behavioral Tests The following behavioral tests were carried out during the postoperative period (days 1, 2, 5, 13, and 2 1) (14,27). Motor capability. Coordination and use of limbs. 1) Proprioceptive placing reflex (the accuracy of the forelimbs of the rats when stepped on a table) was explored; 2) to assess visual placing, suspended rats were moved downward and the flexion of the forelimbs when reaching the experimental table’s surface was examined; 3) limb use when climbing on a wire grid was observed. Muscular tone. Each limb was extended and flexed by the experimenter to assess the resistance to displacement in either direction. Orientation tests. Orientation to various kinds of sensory stimuli. 1) Visual modality (4 X 4 cm white-black squared paper card) was assessed; 2) to explore somesthesia, vibrissae and the anterior, medium, and posterior lateral surface of the body were touched bilaterally using a wooden cotton probe. Perioral stimulation. The lower lips of animals on both sides were pressed lightly with a wooden probe. The biting and grasping of the probe with the forepaw were recorded. Responses were rated on a three-point scale as follows: 0, no reaction; 1, medium reaction; 2, normal response. Open-jeld activity (second and ninth postoperative days). Animals were placed for 3 min in a 60 X 60 X 40 cm grey painted wooden cage marked off into 12 X 12 cm squares. The number of crossing of squares in 3 min was recorded and the numbers of crossings in K&treated rats were compared to the corresponding values of sham-operated controls. Statistical Analysis For statistical analysis of data, analysis of variance (ANOVA), t-test with paired comparison, and Mann-Whitney U-test were used (45,53). Because of the high mortality rate of K&treated animals, data of body weight and food and water intake were evaluated by Friedman’s ANOVA and Mann-Whitney U-test (45) and in the corresponding figures (Figs. l-3) the median values of KA and control (CO) groups are represented. Histology After completing experiments, animals were given an overdose of pentobarbital (Nembutal) and perfused with isotonic saline followed by 10% formalin. For histological analysis, sections were stained with cresyl violet. In a few cases, for visualization of cell bodies and myelinated fibers the combined KltiverBarrera method was used ( 17). RESULTS
Postsurgical Maintenance
Body Weight, Food and Water Intake
Because no animals ate or drank after GP lesions and their weight dropped, feeding and hydration by a stomach tube began on the second postoperative day and maintained until the first
Aphagic-adipsic period. Bilateral electrophoretic KA lesions of the GP resulted in serious feeding deficits. After operation, every lesioned animal showed immediate aphagia (first to fifth
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FIG. 1. Food intake of rats after bilateral electrophoretic KA lesion of the GP. The median curves. Food intake is expressed in percent of preoperative values (Food W). Arrow signifies day of operation (day 0 on the abscissa); *significant differences. KA, KA-lesioned rats; CO, shamoperated control animals. Aphagia in KA-lesioned group.
(see Figs. 1 and 2). Body weight loss of lesioned animals was rapid between the first and fifth postoperative days (Friedman’s ANOVA, p < 0.0 1; Fig. 3A; because of the high mortality rate of KA-lesioned rats, the median values of KA and CO groups are represented in Figs. l-3). From the second day, KA-lesioned rats were artificially fed with liquid diet to promote survival and prevent the consequences of aphagia and adipsia (see the Method section). Despite this procedure, 15% of the KA-treated rats died during this early postoperative period (Fig. 3B). Hypophagic-hypodipsic and recovery periods. Seventeen KAlesioned rats (63% of the KA-treated rats) returned to the spontaneous drinking and feeding. The first signs of reappearance of drinking and eating were observed between the fourth to ninth days. Water intake of KA rats remained under the values of controls for 9 days after the beginning of spontaneous drinking (Friedman’s ANOVA, fourth to ninth days, p < 0.01; tenth to twelfth days, p < 0.05). On the other hand, the aphagic period was followed by 3 days of long hypophagic epoch (Friedman’s ANOVA, fifth to eighth days, p < 0.01). Animals started to regain their body weight after beginning spontaneous eating and drinking. Regain was slow: Their weight did not reach the body weight of control rats until the twenty-sixth postoperative day (Friedman’s ANOVA, sixth to fourteenth days, p < 0.0 1; fifteenth to twenty-sixth days, p c 0.05). Six KA rats (22% of the KAlesioned rats) did not eat spontaneously and needed tube feeding. These animals lost weight continuously until death, which ensued when they were under 60% of their initial body weight. days) and adipsia (first to third days), respectively
Spontaneous Behavior
Spontaneous behavior of GP-lesioned animals was characteristic: Rats did not move in the home cage and were inattentive to placement of food pellets or any diet into their cage. Rats did not show grooming; their unclean hair fell out. In several cases, penile erection also developed (20). During the first several days, lesioned animals refused to swallow the liquid diet or eat food pellets when put directly into their mouths. Vomiting or swallowing could not be elicited when the tube used for artificial feeding was introduced into their mouth or pharynx. Behavioral Tests Muscle tone and motor capability. Initially, limbs of the lesioned rats were flaccid. After the second to third postoperative
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FIG. 2. Water intake of rats after bilateral electrophoretic KA lesion of GP. The median curves. Water intake is expressed in percent of preoperative values (Water %). Arrow signifies day of operation (day 0 on the abscissa); * and + significant differences. Abbreviations are the same as in Fig. 1. Adipsia followed by hypodipsia in KA-lesioned rats.
days, rigidity was observed. Increased muscle resistance to flexion or extension of limbs was seen; however, muscle power was decreased (Mann-Whitney U-test, KA/CO third to fourteenth days, p < 0.0 1). GP animals had additional difficulties during climbing on a wire grid platform (Mann-Whitney U-test, KA/CO first to thirteenth days, p < 0.01): Their limbs often fell extended between rungs. They did not extend their forelimbs when dropped suddenly and their proprioceptively elicited placing reaction was also abnormally slow compared to control animals (Mann-
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FIG. 3. (A) Body weight changes after bilateral KA electrophoresis into the GP. Median body weight values are expressed in percent of preoperative body weight (BW %). Arrow signifies day of operation (day 0 on the abscissa); * and + significant differences. Abbreviations are the same as in Fig. 1. Note the significant body weight decrease of KA-lesioned rats from the first to the twenty-fifth postoperative days. (B) Survival of IL&treated rats in percent of preoperative number of KA animals. (Day 0 is the day of operation.) Thirty-seven percent of the lesioned rats died despite artificial tube feeding during the first 2 weeks of the experiment.
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Whitney U-test, KA/CO first to eighth days, p < 0.05). Disturbances in motor functions did not improve remarkably before the end of the second week. Orientation tests. During the early postoperative period (first to second days), KA rats had difficulties orienting to visual or tactile stimuli (Mann-Whitney U-test, first to second days KA/ CO, p < 0.05). The appearance of forced grasping reflex was observed in several cases. When the lips were touched with a wooden probe, animals did not bite it (Mann-Whitney U-test, first to second days KA/CO, p < 0.05). After the second day, no deficits in orientation were observed. Openfield. After operation, open-field activity ofGP animals decreased considerably (t-test with paired comparison, second day KA/CO, p < 0.001; see Fig. 4). Recovery of exploration activity began during the second postoperative week, but the number of crossed squares by KA rats still did not reach the values of controls (t-test with paired comparison, ninth day KA/ CO, p < 0.05). Nevertheless, improvement of motor functions was significant during this period (ANOVA with repeated measures, KA on the second day&A on the ninth day, p < 0.005). During walking and on stills, the gait of animals was abnormal. Their backs were hunched and hindlimbs were hyperextended. They walked abnormally slow.
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FIG. 4. Open-field activity on the second and ninth postoperative days after electrophoretic application of KA into the GP. KA, KA-lesioned rats; CO, sham-operated control animals. Identical symbols represent significant differences.
tivity did not develop in lesioned animals (based on behavioral
Histology
Due to the limited extent of the electrophoretic lesions of GP, the area of neuronal loss was less appreciable. Because PSB was applied electrophoretically after KA into the same area, blue somata of neurons with shrinkage and blue debris of cellular elements could be found in the target area, which helped to identify the location of lesions. Identification was successful in 28 of 29 KA-treated animals. Micropipettes were terminated symmetrically, bilaterally in the ventral part of the GP. The lesioned area was well limited (diameter, 400-800 pm) and confined to the GP. Incomplete cellular loss and moderate gliosis was also observed in the lesioned zone. In distant brain areas (i.e., in the thalamus, hippocampus, amygdala, and lateral hypothalamus) or at the site of pipette tracks, no apparent sign of cellular loss was observed. DISCUSSION
Our results revealed that bilateral selective destruction of neurons in the GP results in dramatic changes in body weight regulation and causes motor incapabilities. Despite artificial tube feeding, almost half (40%) the lesioned animals died within the first 2 postoperative weeks. KA-treated rats had disturbances using their limbs. They also showed temporary deficits in orientation toward various kinds of sensory stimuli (up to l-2 days). Exploratory activity of KA-lesioned animals decreased in open field throughout the first 2 postoperative weeks. Electrophoretic Application Method
The neurotoxin KA was applied electrophoretically by means of glass micropipettes to decrease toxic side effects (i.e., extensive brain damage, seizures, motor excitability, and disturbances in autonomic activity) (2 1,25). It has been proven that injection of KA with hydraulic pressure caused distant lesions from the site of application (44). This effect might be due to the high diffusion capacity of the drug or additional, nonspecific effects of KA. Namely, the long-lasting epileptic activity produced by KA can be due to metabolic changes or hypoxia in distant brain regions, which may destruct the neurons in these areas (i.e., the hippocampus) (4). In our experiments, permanent epileptic ac-
observations and EEG records, unpublished data). Our histological analysis showed no detectable cellular loss in distant regions (i.e., in the hippocampus). Lesions were well localized, had limited extent (400-800 pm), and the fine glass micropipettes did not cause any nonspecific lesion. Our present and previous findings, thus, clearly show the advantages of this application method of KA compared to the microinjection method (2 1,25). Body Weight Changes and Motor Disturbances
Our present results demonstrated that weight loss and feeding suppression could be produced by KA-induced neuron-selective microlesions of the GP cells. These symptoms were similar to those seen after electrolytic- or 6-OHDA-induced damages of the pallidum (20,25,26,28,29,34). Because both electrolytic and 6-OHDA lesions disrupted the NSDS and MLDS, it was concluded that the feeding disturbances were caused by disruption of these dopaminergic pathways (25,27,39,50). By contrast, previous findings indicated that the dopaminergic pathways en passage remained intact after KA lesions of the lateral hypothalamus and striatopallidal area (7,2 1,52). We can conclude, therefore, that feeding deficits can develop after KA lesion of the GP without involvement of dopaminergic pathways. It has been suggested that after electrolytic or 6-OHDA lesions GP rats had persisting deficits in metabolic regulation (12,3 1,36). We did not examine the metabolic status (i.e., oxygen consumption) of animals in the present experiments; however, the existence of metabolic disturbances can be supposed: 1) lesioned rats lost weight despite the artificial tube feeding; 2) body weight of KA-lesioned animals did not reach that of controls until the end of the third postoperative week, although food and water consumption of KA-lesioned rats did not differ from those of controls. Concerning the development of motor disturbances observed in the present experiment, our results are in good agreement with previous findings that concluded that pallidal neurons play an important role in regulation of movements (2,3,42,54). KAlesioned animals appeared to move slowly and with substantial difficulties. The rats exhibited difficulties in limb use during feeding and drinking. It has been demonstrated (49) that GPstimulation-induced movements of the hindlimbs are more in-
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tense in rats deprived of food or water than in satiated animals.
KA rats seemed to be “apathetic.” Nevertheless, tail-pinching could evoke quick escape reaction. It suggests that motor deficits caused by lesions of the GP can be due to disturbances of neural systems involved in the regulation of sensorimotor integration. It is supported by other results that the pallidum may play a role in the regulation of movements only in goal-directed and food-rewarded situations (33,37,38). Both electrolytic and 6-OHDA lesions of the GP resulted in long-lasting deficits in orientation toward sensory stimuli (i.e., sensory neglect). After KA destruction of the pallidum, however, similar deficits were observed only at the very early postoperative period (during the first 2 days). The existence of sensory neglect after KA lesion of the GP is therefore questionable. Namely, the lack of orientation toward sensory stimuli could have been due to the appearance of the serious motor incapabilities and
to the condition of animals at the early postoperative stage. This interpretation is in good agreement with our previous results that sensory neglect develops when the MLDS crossing through the pallidum is damaged (22,27). The complex syndrome seen after bilateral KA microlesion of the GP supports our previous findings that the pallidum is one of the important sites in the brain for the regulation of body weight and food and water intake, as well as for the feeding associated sensorimotor and metabolic integration. ACKNOWLEDGEMENT
This work was supported by the Hungarian Academy of Sciences with Grants-in Aid OTKA 1404 and partly by an ETT T-565/ 1990 grant of the Ministry of Health, Hungary (L.L.). P.S. and A. H. were supported by fellowship from the Foundation for the Hungarian Science, Hungarian
Credit Bank.
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43. Schneider, J. S.; Olazabal, U. E. Behaviorally specific limb use deficits following globus pallidus lesions in rats. Brain Res. 308:341-346; 1984. 44. Schwab, J. E.; Fuller, T.; Price, J. L.; Olney, J. W. Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: A histological study. Neuroscience 5:99 I - 1014; 1980. 45. Siegel, S. Nonparametric statistics: For the behavioral sciences. New York: McGraw-Hill; 1956. 46. Solomone, J. D. Behavioral pharmacology of dopamine systems: A new synthesis. In: Willner, P.; Scheel-Kruger, J., eds. The mesolimbic dopamine system: From motivation to action. Chichester: Wiley; 1991:599-613. 47. Sperk, G.; Lassmann, H.; Seitelberger, F.; Homykiewicz, 0. Kainic acid-induced seizures: Dose relationship of behavioural, neurochemical and histopathological changes. Brain Res. 338:289-295; 1985. 48. Snicker, E. M.; Swerdloff, A. F.; Zigmond, M. J. Intrahypothalamic injections of kainic acid oroduce feedina and drinkina deficits in rats. Brain Res. 158:470-473; 1978. 49. Szaho, I.; Sarkasian, J.; LCnlrd, L.; Nemeth, L. Pallidal stimulation in rats: Facilitation of stimulus-bound chewing by pallidal stimulation. Physiol. Behav. 18:361-368; 1977. 50. Ungerstedt, U. Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system. Acta Physiol. Stand. 367(suppl.):95-122; I97 1. 51. Wayner, M. J.; Kantak, K. M.; Barone, F. C.; DeHaven, D. L.; Wayner, M. J., III; Cook, R. C. Effects of LH kainic acid infusions on ingestion and autonomic activity. Physiol. Behav. 27:369-376; 1981. 52. Weinreich, P.; Seeman, P. Effects of kainic acid on striatal dopamine receptors. Brain Res. 198:491-496; 1980. 53. Winer, B. J. Statistical principles in experimental design. New York: McGraw-Hill; 1962. 54. Yiantzoglou, A. A.; Carahalion, N.; Papadopoulos, G.; Spyraki, C. Dopamine mediated circling in rats with globus pallidus lesions. Neuroscience 22(suppl.):638; 1987.
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Microelectrophoretic Application of Kainic Acid Into the Globus Pallidus: Disturbances in Feeding Behavior P. SANDOR, A. HAJNAL, G. JANDO, Z. KARADI AND L. LeNzkRD’ NeurophysiologyResearch Group of the Hungarian Academy of Sciences at Institute of Physiology UniversityMedical School, P&s, H-7443 P&s, Hungary Received 15 October 199 1 SANDOR, P., A. HAJNAL, G. JAN&, Z. KAaDI AND L. LfiNWRD. Microelectrophoretic application of kainic acid into the globus pallidus: Disturbances in feeding behavior. BRAIN RES BULL 28(S) 75 l-756, 1992.-Body weight changes, food and water intake, and sensorimotor disturbances of male rats were studied after bilateral kainic acid-(KA) induced lesions of the globus pallidus (GP). To minimize the extent of damages, KA was applied electrophoretically by means of glass micropipettes (tip diameter of the pipettes was lo- 15 pm). The neuron-specific damages of the GP resulted in aphagia and adipsia and rapid body weight decrease. Lesioned animals showed permanent motor disturbances but only temporary difficulties in the orientation toward sensory stimuli. Our data show that the selective destruction of the GP neurons results in a complex disorder that has motivational, (sensori)motor, and metabolic components. Globus pallidus
Kainic acid microelectrophoresis
Feeding deficits
Motor disturbances
echolaminergic (CA) fibers of passage without involving local cellular elements (39). It was suggested, therefore, that the NSDS is essential for the organization of hunger-motivated behavior (even for the metabolic and sensorimotor regulation) (31,39,46). Previous results of our laboratory suggested, however, that the sensory neglect seen after pallidal lesions is due to the damage of fibers of the mesolimbic dopaminergic system (MLDS) pas-sing through the GP to reach different neostriatal areas (23,24). According to this interpretation, lesion of the NSDS may cause only motor disturbances whereas destruction of the MLDS results in perceptual dysfunctions (22). Kainic acid (KA) and ibotenic acid (IB), the neurotoxic analogs of glutamate, destroy neuronal cell bodies intrinsic to the injected area but leave passing fibers intact (10,18,52). By means of these neuroexcitotoxins as lesioning agents, it is possible to examine the effects of selective destruction of local neurons. Recently, several laboratories proved that injection of KA into different brain areas, although sparing passing fibers, produces feeding disturbances or sensorimotor deficits (1,5,&l 1,2 1, 25,41,48,5 I). Bilateral injection of KA into the striatopallidal region causes enhanced aversion to tastes and aphagia (5). Damage to the pallidum with IB produces changes of both daily locomotor activity and learned running response for food (3,32). Because intracerebral hydraulic microinjection of KA or IB causes neural degenerations at distant brain areas from the in-
IT is well established that the globus pallidus (GP) plays essential roles in neural mechanisms controlling food and water intake. Bilateral electrolytic lesions of the GP, destroying both cellular elements and passing fibers, result in aphagia and adipsia and rapid body weight loss and early death of animals (22,26,28,34). Most animals die without artificial tube feeding and even those that recover to spontaneous feeding or drinking show regulatory deficits in different physiological challenges and exhibit abnormal blood glucose concentration (12,26,3 I ,34,36). These results suggest the GP is involved in the metabolic regulation (12,13,20,26). On the other hand, the GP has been reported to play an important role in the sensorimotor control of movements. GP-lesioned animals display bizarre posture and have trouble using their forelimbs and mouth during eating or drinking (19,20,22,28,43). After electrolytic GP lesion, sensory neglect develops, that is, rats exhibit impairments in orientation to different sensory stimuli (22,27). It is suggested that all these symp toms contribute to the feeding disturbances seen after the destruction of GP (22,27). Symptoms similar to those seen after pallidal electrocoagulation can also be observed after 6-hydroxydopamine (6-OHDA) injections into the GP, lateral hypothalamus (LH), or substantia nigra (SN) (8,9,20,3 1,40). The ascending nigrostriatal dopaminergic system (NSDS) originating from the SN passes through these structures (15,30). 6-OHDA causes the interruption of cat-
’ Requests for reprints should be addressed to L.&z16tin&d, M.D., Ph.D., D.Sc., Neurophysiology Research Group of the Hungarian Academy of Sciences at Institute of Physiology, University Medical School, Szigeti str. 12., P&s, H-7643 WCS,Hungary.
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jetted site, these results were criticized. In addition, long-lasting epileptic activity (up to 40-60 days), enhanced motor excitability, and disturbances in autonomic activity also appear as side effects of these injections (7,16,35,47,5 1). These side effects of KA and IB can directly or indirectly affect motor activity, as well as feeding behavior, of animals. Our previous results demonstrated that the side effects of KA can be eliminated by electrophoretic application of KA and that KA microelectrophoresis is a useful technique in the study of feeding behavior (2 1,25). In the present experiments, body weight and food and water intake of rats were studied after bilateral KA-induced lesion of the GP. KA was applied electrophoretically by means of glass micropipettes to minimize the side effects of this excitotoxin. It was examined whether well-localized selective destruction of GP neurons can produce 1) feeding deficits and 2) sensorimotor disturbances influencing eating and drinking. METHODS
Animals and Procedures Fifty-four adult, male CFY rats (LATI, Godollo) weighing 294-341 g at the beginning of the experiments were used. For 9 days before surgery, all animals were kept in a temperatureand light-controlled room (2 1 f 2°C 12 L: 12 D cycle, light on at 6:00 a.m.). They were housed individually and standard laboratory food pellets and tapwater were available ad lib. Daily food and water consumptions and body weight were measured to the nearest g and ml, respectively. Preoperative measurements were carried out for 8 days. At the eighth day (1 day before operation), mean body weight and food and water consumption of rats were: 328.5 + 19.2 g, 21.05 + 3.54 g, and 33.5 + 4.78 g, respectively. Based on data of preoperative body weight records, animals were distributed into two evenly formed groups [for details, see our previous publications (21,25)]: 27 KA and 27 sham-operated controls (CO). Surgery Double-barrelled glass micropipettes pulled from Pyrex glass tubes were filled with the following solutions: 1) one of the two barrels was filled with KA (Sigma Chemical Co., St. Louis, MO, KA dissolved in distilled water and titrated to pH 6.9-7.2 with NaOH); 2) the second barrel served for the ejection of Pontamine Sky Blue (PSB, GURR, saturated PSB in 0.15 M NaCl and 0.5 M Na acetate) to mark the lesioned area. Stereo&c coordinates for the GP according to DeGroot (6) were: A, 6.6; L, 3.3; V, -0.5. Bilateral operations were carried out under Calypso1 anesthesia (ketaminum, Richter, Budapest, 100 mg/kg, IP). After drilling a small hole in the skull and opening the dura mater under microscopic control, pipettes were carefully lowered to the target area. KA was released electrophoretically by means of a constant current device (EMG 4767). Because current intensity was monitored by a microammeter, changes in conditions of electrodes (damage of the pipette tip, blocking, etc.) could be controlled. KA was applied at the following parameters (2 1,25): concentration, 80 mM; tip diameters of pipettes, 10-l 5 pm; current intensity, 5-8 PA for 4 min. After ejection of KA, PSB was also applied into the same loci (I 5- 18 WAfor 5 min). After the current was terminated, pipettes remained in place for 5 min. In the CO rats only PSB (with identical parameters) was applied into the GP.
appearance of spontaneous feeding. The artificial diet consisted of one part sugar, two parts egg yolk and milk, salt, and vitamin B (Polybe, Richter) (26). All animals were weighed daily before feeding and were tube fed with 8- 10 ml of the aforementioned diet and 8 ml tap water. Those animals that died or had to be killed were dissected to control the state of their abdomen and mediastinum. No injuries or pathological changes were found in these rats. Behavioral Tests The following behavioral tests were carried out during the postoperative period (days 1, 2, 5, 13, and 2 1) (14,27). Motor capability. Coordination and use of limbs. 1) Proprioceptive placing reflex (the accuracy of the forelimbs of the rats when stepped on a table) was explored; 2) to assess visual placing, suspended rats were moved downward and the flexion of the forelimbs when reaching the experimental table’s surface was examined; 3) limb use when climbing on a wire grid was observed. Muscular tone. Each limb was extended and flexed by the experimenter to assess the resistance to displacement in either direction. Orientation tests. Orientation to various kinds of sensory stimuli. 1) Visual modality (4 X 4 cm white-black squared paper card) was assessed; 2) to explore somesthesia, vibrissae and the anterior, medium, and posterior lateral surface of the body were touched bilaterally using a wooden cotton probe. Perioral stimulation. The lower lips of animals on both sides were pressed lightly with a wooden probe. The biting and grasping of the probe with the forepaw were recorded. Responses were rated on a three-point scale as follows: 0, no reaction; 1, medium reaction; 2, normal response. Open-jeld activity (second and ninth postoperative days). Animals were placed for 3 min in a 60 X 60 X 40 cm grey painted wooden cage marked off into 12 X 12 cm squares. The number of crossing of squares in 3 min was recorded and the numbers of crossings in K&treated rats were compared to the corresponding values of sham-operated controls. Statistical Analysis For statistical analysis of data, analysis of variance (ANOVA), t-test with paired comparison, and Mann-Whitney U-test were used (45,53). Because of the high mortality rate of K&treated animals, data of body weight and food and water intake were evaluated by Friedman’s ANOVA and Mann-Whitney U-test (45) and in the corresponding figures (Figs. l-3) the median values of KA and control (CO) groups are represented. Histology After completing experiments, animals were given an overdose of pentobarbital (Nembutal) and perfused with isotonic saline followed by 10% formalin. For histological analysis, sections were stained with cresyl violet. In a few cases, for visualization of cell bodies and myelinated fibers the combined KltiverBarrera method was used ( 17). RESULTS
Postsurgical Maintenance
Body Weight, Food and Water Intake
Because no animals ate or drank after GP lesions and their weight dropped, feeding and hydration by a stomach tube began on the second postoperative day and maintained until the first
Aphagic-adipsic period. Bilateral electrophoretic KA lesions of the GP resulted in serious feeding deficits. After operation, every lesioned animal showed immediate aphagia (first to fifth
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FIG. 1. Food intake of rats after bilateral electrophoretic KA lesion of the GP. The median curves. Food intake is expressed in percent of preoperative values (Food W). Arrow signifies day of operation (day 0 on the abscissa); *significant differences. KA, KA-lesioned rats; CO, shamoperated control animals. Aphagia in KA-lesioned group.
(see Figs. 1 and 2). Body weight loss of lesioned animals was rapid between the first and fifth postoperative days (Friedman’s ANOVA, p < 0.0 1; Fig. 3A; because of the high mortality rate of KA-lesioned rats, the median values of KA and CO groups are represented in Figs. l-3). From the second day, KA-lesioned rats were artificially fed with liquid diet to promote survival and prevent the consequences of aphagia and adipsia (see the Method section). Despite this procedure, 15% of the KA-treated rats died during this early postoperative period (Fig. 3B). Hypophagic-hypodipsic and recovery periods. Seventeen KAlesioned rats (63% of the KA-treated rats) returned to the spontaneous drinking and feeding. The first signs of reappearance of drinking and eating were observed between the fourth to ninth days. Water intake of KA rats remained under the values of controls for 9 days after the beginning of spontaneous drinking (Friedman’s ANOVA, fourth to ninth days, p < 0.01; tenth to twelfth days, p < 0.05). On the other hand, the aphagic period was followed by 3 days of long hypophagic epoch (Friedman’s ANOVA, fifth to eighth days, p < 0.01). Animals started to regain their body weight after beginning spontaneous eating and drinking. Regain was slow: Their weight did not reach the body weight of control rats until the twenty-sixth postoperative day (Friedman’s ANOVA, sixth to fourteenth days, p < 0.0 1; fifteenth to twenty-sixth days, p c 0.05). Six KA rats (22% of the KAlesioned rats) did not eat spontaneously and needed tube feeding. These animals lost weight continuously until death, which ensued when they were under 60% of their initial body weight. days) and adipsia (first to third days), respectively
Spontaneous Behavior
Spontaneous behavior of GP-lesioned animals was characteristic: Rats did not move in the home cage and were inattentive to placement of food pellets or any diet into their cage. Rats did not show grooming; their unclean hair fell out. In several cases, penile erection also developed (20). During the first several days, lesioned animals refused to swallow the liquid diet or eat food pellets when put directly into their mouths. Vomiting or swallowing could not be elicited when the tube used for artificial feeding was introduced into their mouth or pharynx. Behavioral Tests Muscle tone and motor capability. Initially, limbs of the lesioned rats were flaccid. After the second to third postoperative
p
FIG. 2. Water intake of rats after bilateral electrophoretic KA lesion of GP. The median curves. Water intake is expressed in percent of preoperative values (Water %). Arrow signifies day of operation (day 0 on the abscissa); * and + significant differences. Abbreviations are the same as in Fig. 1. Adipsia followed by hypodipsia in KA-lesioned rats.
days, rigidity was observed. Increased muscle resistance to flexion or extension of limbs was seen; however, muscle power was decreased (Mann-Whitney U-test, KA/CO third to fourteenth days, p < 0.0 1). GP animals had additional difficulties during climbing on a wire grid platform (Mann-Whitney U-test, KA/CO first to thirteenth days, p < 0.01): Their limbs often fell extended between rungs. They did not extend their forelimbs when dropped suddenly and their proprioceptively elicited placing reaction was also abnormally slow compared to control animals (Mann-
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FIG. 3. (A) Body weight changes after bilateral KA electrophoresis into the GP. Median body weight values are expressed in percent of preoperative body weight (BW %). Arrow signifies day of operation (day 0 on the abscissa); * and + significant differences. Abbreviations are the same as in Fig. 1. Note the significant body weight decrease of KA-lesioned rats from the first to the twenty-fifth postoperative days. (B) Survival of IL&treated rats in percent of preoperative number of KA animals. (Day 0 is the day of operation.) Thirty-seven percent of the lesioned rats died despite artificial tube feeding during the first 2 weeks of the experiment.
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Whitney U-test, KA/CO first to eighth days, p < 0.05). Disturbances in motor functions did not improve remarkably before the end of the second week. Orientation tests. During the early postoperative period (first to second days), KA rats had difficulties orienting to visual or tactile stimuli (Mann-Whitney U-test, first to second days KA/ CO, p < 0.05). The appearance of forced grasping reflex was observed in several cases. When the lips were touched with a wooden probe, animals did not bite it (Mann-Whitney U-test, first to second days KA/CO, p < 0.05). After the second day, no deficits in orientation were observed. Openfield. After operation, open-field activity ofGP animals decreased considerably (t-test with paired comparison, second day KA/CO, p < 0.001; see Fig. 4). Recovery of exploration activity began during the second postoperative week, but the number of crossed squares by KA rats still did not reach the values of controls (t-test with paired comparison, ninth day KA/ CO, p < 0.05). Nevertheless, improvement of motor functions was significant during this period (ANOVA with repeated measures, KA on the second day&A on the ninth day, p < 0.005). During walking and on stills, the gait of animals was abnormal. Their backs were hunched and hindlimbs were hyperextended. They walked abnormally slow.
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FIG. 4. Open-field activity on the second and ninth postoperative days after electrophoretic application of KA into the GP. KA, KA-lesioned rats; CO, sham-operated control animals. Identical symbols represent significant differences.
tivity did not develop in lesioned animals (based on behavioral
Histology
Due to the limited extent of the electrophoretic lesions of GP, the area of neuronal loss was less appreciable. Because PSB was applied electrophoretically after KA into the same area, blue somata of neurons with shrinkage and blue debris of cellular elements could be found in the target area, which helped to identify the location of lesions. Identification was successful in 28 of 29 KA-treated animals. Micropipettes were terminated symmetrically, bilaterally in the ventral part of the GP. The lesioned area was well limited (diameter, 400-800 pm) and confined to the GP. Incomplete cellular loss and moderate gliosis was also observed in the lesioned zone. In distant brain areas (i.e., in the thalamus, hippocampus, amygdala, and lateral hypothalamus) or at the site of pipette tracks, no apparent sign of cellular loss was observed. DISCUSSION
Our results revealed that bilateral selective destruction of neurons in the GP results in dramatic changes in body weight regulation and causes motor incapabilities. Despite artificial tube feeding, almost half (40%) the lesioned animals died within the first 2 postoperative weeks. KA-treated rats had disturbances using their limbs. They also showed temporary deficits in orientation toward various kinds of sensory stimuli (up to l-2 days). Exploratory activity of KA-lesioned animals decreased in open field throughout the first 2 postoperative weeks. Electrophoretic Application Method
The neurotoxin KA was applied electrophoretically by means of glass micropipettes to decrease toxic side effects (i.e., extensive brain damage, seizures, motor excitability, and disturbances in autonomic activity) (2 1,25). It has been proven that injection of KA with hydraulic pressure caused distant lesions from the site of application (44). This effect might be due to the high diffusion capacity of the drug or additional, nonspecific effects of KA. Namely, the long-lasting epileptic activity produced by KA can be due to metabolic changes or hypoxia in distant brain regions, which may destruct the neurons in these areas (i.e., the hippocampus) (4). In our experiments, permanent epileptic ac-
observations and EEG records, unpublished data). Our histological analysis showed no detectable cellular loss in distant regions (i.e., in the hippocampus). Lesions were well localized, had limited extent (400-800 pm), and the fine glass micropipettes did not cause any nonspecific lesion. Our present and previous findings, thus, clearly show the advantages of this application method of KA compared to the microinjection method (2 1,25). Body Weight Changes and Motor Disturbances
Our present results demonstrated that weight loss and feeding suppression could be produced by KA-induced neuron-selective microlesions of the GP cells. These symptoms were similar to those seen after electrolytic- or 6-OHDA-induced damages of the pallidum (20,25,26,28,29,34). Because both electrolytic and 6-OHDA lesions disrupted the NSDS and MLDS, it was concluded that the feeding disturbances were caused by disruption of these dopaminergic pathways (25,27,39,50). By contrast, previous findings indicated that the dopaminergic pathways en passage remained intact after KA lesions of the lateral hypothalamus and striatopallidal area (7,2 1,52). We can conclude, therefore, that feeding deficits can develop after KA lesion of the GP without involvement of dopaminergic pathways. It has been suggested that after electrolytic or 6-OHDA lesions GP rats had persisting deficits in metabolic regulation (12,3 1,36). We did not examine the metabolic status (i.e., oxygen consumption) of animals in the present experiments; however, the existence of metabolic disturbances can be supposed: 1) lesioned rats lost weight despite the artificial tube feeding; 2) body weight of KA-lesioned animals did not reach that of controls until the end of the third postoperative week, although food and water consumption of KA-lesioned rats did not differ from those of controls. Concerning the development of motor disturbances observed in the present experiment, our results are in good agreement with previous findings that concluded that pallidal neurons play an important role in regulation of movements (2,3,42,54). KAlesioned animals appeared to move slowly and with substantial difficulties. The rats exhibited difficulties in limb use during feeding and drinking. It has been demonstrated (49) that GPstimulation-induced movements of the hindlimbs are more in-
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tense in rats deprived of food or water than in satiated animals.
KA rats seemed to be “apathetic.” Nevertheless, tail-pinching could evoke quick escape reaction. It suggests that motor deficits caused by lesions of the GP can be due to disturbances of neural systems involved in the regulation of sensorimotor integration. It is supported by other results that the pallidum may play a role in the regulation of movements only in goal-directed and food-rewarded situations (33,37,38). Both electrolytic and 6-OHDA lesions of the GP resulted in long-lasting deficits in orientation toward sensory stimuli (i.e., sensory neglect). After KA destruction of the pallidum, however, similar deficits were observed only at the very early postoperative period (during the first 2 days). The existence of sensory neglect after KA lesion of the GP is therefore questionable. Namely, the lack of orientation toward sensory stimuli could have been due to the appearance of the serious motor incapabilities and
to the condition of animals at the early postoperative stage. This interpretation is in good agreement with our previous results that sensory neglect develops when the MLDS crossing through the pallidum is damaged (22,27). The complex syndrome seen after bilateral KA microlesion of the GP supports our previous findings that the pallidum is one of the important sites in the brain for the regulation of body weight and food and water intake, as well as for the feeding associated sensorimotor and metabolic integration. ACKNOWLEDGEMENT
This work was supported by the Hungarian Academy of Sciences with Grants-in Aid OTKA 1404 and partly by an ETT T-565/ 1990 grant of the Ministry of Health, Hungary (L.L.). P.S. and A. H. were supported by fellowship from the Foundation for the Hungarian Science, Hungarian
Credit Bank.
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