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Deficits in locomotor behaviour and thermoregulation produced by intrahypothalamic dopamine injections
Behavioural Brain Research, 12 (1984) 137-143
Elsevier
137
BBR 00346
DEFICITS IN LOCOMOTOR BEHAVIOUR AND THERMOREGULATION PRODUCED BY INTRAHYPOTHALAMIC DOPAMINE INJECTIONS GREGORY L. WILLIS and GRAEME C. SMITH
Monash University Department of Psychological Medicine, Prince Henry's Hospital, St. Kilda Road, Melbourne, Victoria 3004 (Australia) (Received December 2nd, 1983) (Revised version received March 19th, 1984) (Accepted March 19th, 1984)
Key words: amine accumulation - catecholamine depletion - open field activity - lateral hypothalamus - dopamine - rat
When nigrostriatal dopamine neurones degenerate, a loss of functional dopamine in the striatum occurs and is accompanied by increased dopamine in the degenerating axons which traverse the hypothalamus. While the behavioural deficits which occur after nigrostriatal degeneration have been attributed to the loss of functional dopamine neurotransmission, evidence produced by us suggests that the increased levels of amines in the degenerating axons may be neuroactive and participate in the production of these behavioural deficits. To test this hypothesis further, albino rats were injected bilaterally with 200 nmol of dopamine in a location just rostral to the diencephalon/mesencephalonborder, where amine accumulation is commonly observed following lateral hypothalamic damage. The effect of these injections upon open field performance, thermoregulation and motor reflex control was determined 40 min after dopamine injection. In a second study, pargyline (15 mg/kg, i.p.) was administered 30 min before intracerebral dopamine to determine whether this treatment would increase the severity of motor and thermoregulatory deficits which occurred after dopamine injections alone. Deficits in locomotion, rearing and the ability to regulate body temperature were seen after the dopamine injections while motor reflex control in these animals was similar to that seen in vehicle-injected controls. The behavioural deficits displayed by pargyline pretreated, dopamine injected animals were slightly but not significantly more severe than those displayed by animals receiving dopamine injections alone. Fluorescent histochemical assessment of injection sites revealed that dopamine injection produced an increase in fluorescence or "amine accumulation" at the site of injection but this was considerably less than that seen after catecholamine degeneration. These results add further support to the hypothesis that amines which accumulate in degenerating neurones may be neuroactive and may thereby participate in the production of behavioural deficits attributed previously to the loss of functional dopamine neurotransmission.
INTRODUCTION
When the ascending nigrostriatal dopamine (DA) system degenerates a loss of functional neurotransmitter is seen in the axon terminals of the nucleus caudatus putamen (NCP) while the axons which traverse the lateral hypothalamus (LH) become enlarged with accumulated amines9,1 !,15. It is generally held that the reduced neurotransmitter content is the degenerative phenomenon which underlies the changes in thermoregulationT M and locomotory and cons u m m a t o r y b e h a v i o u r s 6"9'16 which are seen in experimental animals bearing DA-depleting lesions. 0166-4328/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Recently, however, it has been suggested that the hypothalamic accumulations may be areas of functional neurotransmitter produced during degeneration, which may also participate in the production ofbehavioural deficits previously attributed only to the loss of functional neurotransmitter in the terminal fields9,1°-15. That this accumulated neurotransmitter may be neuroactive and possibly affecting adjacent catecholamine (CA) receptors is supported by the finding that the injection of CA and CA agonists into the LH produces reductions in food intake which are similar to those produced by degenerative lesions2-4. If amine accumulation is participating in the
138 production of behavioural deficits then intrahypothalamic injections of CA into areas where accumulation is seen might also be expected to produce thermoregulatory and motor impairment similar to that displayed by animals with DA degeneration. It was the object of this study to examine the effects of intrahypothalamic DA injections on thermoregulation and motor performance. MATERIALS AND METHODS
Thirty-two male Sprague-Dawley rats ranging in weight from 250 to 300 g were housed individually in wire mesh cages and allowed ad libitum access to rat pellets (Clarke King, Melbourne) and tap water. A 12 h light/dark cycle was maintained throughout the experiment with lights on at 07.00 h. Room temperature was maintained at 2 2 ° C ( + 2 ° C ) .
Surgery All animals were anaesthetized with 84 mg/kg of alphaxalon i.p. and then placed in a stereotaxic instrument. Cannulae were aimed at the posterior aspect of the lateral hypothalamus (PLH), bilaterally (coordinates A = - 0 . 8 mm, L = + 1.9 mm D = - 6.1 mm). The cannulae were in the plane of Pellegrino et al. 7 and the injection needle extended 2 mm beyond the cannula tip. Animals were allowed at least 2 weeks to recover from surgery before commencing the experiment and were handled daily and were allowed ad libitum access to food and water throughout the study.
Injections A 200 nmol solution of 3,4-dihydroxyphenylethylamine hydrochloride (Dopamine, Sigma) w a s mixed in distilled water containing 0.02 mg/ml of ascorbic acid to retard oxidation. This dose was chosen on the basis of previous work 2. Control injections were made with isotonic saline solutionscontaining 0:02 mg/ml of ascorbic acid. Eachanimal roeeived 1/~1 of the drug or 1 #1 of the control solution on each ~ d e of the brain. Intraperitoneal injections were naade with 15 mg/ml solutions of pargyline hydrochloride
(Sigma) or with isotonic saline and all solutions were administered in the volume of 1 ml/kg. The dose of pargyline employed was chosen on the basis of previous work which demonstrates that this dose potentiates the satiety-inducing effects ofintracerebral DA injections 2. All i.p. injections were made 30 min before intracerebral injections.
Procedure In the first study 5 rats received bilateral intracranial injections of DA while another 5 received a bilateral injection of isotonic saline. A crossover design was employed and 2 days after this the drug- and vehicle-injected groups were reversed and the performance of all animals on the motor tests was assessed and rectal temperature was measured. Therefore, each animal served as his own control. Forty-five minutes after the intracerebral injections the performance of all animals was assessed on open-field and motorreflex tests as described previously 11'~4. The number of squares crossed and the number of rearings during a 5-min test period were recorded. After this the latency to retract an elevated limb, to step up or down from a raised platform and to ambulate from within a prescribed area were also measured. Rectal temperature was taken 1,5 h after intracranial injections. The paradigm employed in the second study utilizing 10 more animals was the same as that described in experiment 1 except that all animals were pretreated with 15 mg/kg ofpargyline 30 min before receiving the intracerebral injections. At the completion of the experiments, all animals were sacrificed and their brains removed, stored in 10~o formalin and then sectioned and stained using cresyl violet and luxol fast blue. The areas of necrosis resulting from injection were plotted on plates from the atlas of Pellegrino et al.7. In the third study 12 animals were cannulated bilaterally as described earlier and then allowed at least 10 days to recover from surgery. They then received a unilateral 1/~1 injection of either 50, 100 or 200 nmol of DA and received a 1 #1 injection of vehicle on the contralateral side (n = 4 animals per group). Half of the animals in each group were sacrificed for fluorescence histochemistry 2 h after
139 Motor Reflex Tests (no pretreatment)
injection while the other half were sacrificed 24 h after injection. Statistical comparisons were made between vehicle injection days and DA injection days for all measures using Student's t-test or two-way ANOVA with repetitions (Cannon, BX-10 statistical package).
15
r-]
Vehicle
B
DA (200nM/~ul)
10 RESULTS
As shown in Fig. 1 the open-field behaviour of DA-injected rats was significantly depressed when compared to vehicle-injected controls. The number of squares crossed during the 5 min test period was significantly less in the unpretreated and pargyline pretreated animals ( d f = 1,36; F = 40.8; a = 0.001) when compared to their respective controls. The number of rearings was also greatly reduced in those animals receiving DA injections with and without pargyline pretreatment (df = 1,36; F - - 23.6; a -- 0.001). Pargyline pretreatment decreased slightly the number of rearings exhibited after DA injection when compared to those animals receiving no pretreat-
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Open Field Behaviours
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PRETREATMENT
PRETREATMENT
NO PRETREATMENT
Fig. 1. The open-field behaviour of animals receiving bilateral injection of dopamine, or vehicle into the posterior lateral hypothalamus. While half of the animals were pretreated with 15 mg/kg of pargyline the others were not. The T-bars represent the standard error of the mean. The number of squares represents the level of significance (mmm, a = 0.001).
"1o c o o o
0
, LATENCY TO
~ LATENCY TO
LATENCY TO
RETRACT A t IMB
STEP D O W N
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Fig. 2. The latency to retract a limb, to step down and to ambulate in animals injected bilaterally with dopamine or vehicle. The T-bars represent the standard error of the mean.
ment and this difference was significant (df = 1,36; F = 89.5; ~ = 0.001). The performance of all DA-injected groups on the 3 motor reflex tests was not significantly impaired by the DA injection. As shown in Figs. 2 and 3, the latency to retract a limb, to step up or down and to ambulate were in some cases increased over that seen after control injections but 2-way ANOVA revealed that these differences were not significant. Intracerebral injections of DA produced a significant drop in rectal temperature 1.5 h after injection as compared to the vehicle-injected controis (df = 1,36; F = 89.5; ~ = 0.001). Pargylinepretreated, DA-injected animals also displayed a significant reduction in rectal temperature when compared to their vehicle-injected counterparts (~ = 0.001). Pargyline pretreatment alone did not reduce significantly the parameter of body temperature. Fluorescent histochemical examination of the injection sites of DA-injected rats revealed a
140 Motor Reflex T e s t s
reliable increase in fluorescence at both 1 and 24 h after DA injection (Fig. 5). Increased fluorescence was observed around the site of injection in the PLH just rostral to the midbrain DA cell bodies. While some increases in fluorescence were seen in animals treated with 50 nmol solutions of DA, the intensity of this fluorescence was generally not as great as that seen in the 100 and 200 nmol dose. Histological verification of the injection sites of the animals used in the behavioural studies confirmed that the sites of DA injection were in the proximity of the medial forebrain bundle in the PLH just rostral to the substantia nigra (Fig. 6).
I-"1 Vehicle 15
B
DA (200ni/pl)
=.
10B
"O C O (9 00
DISCUSSION
5-
_
LATENCY TO
LATENCY TO
LATENCY TO
RETRACT A LIMB
STEP D O W N
AMBULATE
Fig. 3. The latency to retract a limb, to step down and to ambulate in animals injected bilaterally with dopamine or vehicle 30 min after pretreatment with 15 mg/kg ofpargyline. The T-bars represent the standard error of the mean. Rectal Temperature (90min after 40
,,,
intracerebral
DA)
]
Vehicle
B
DA ( 2 0 0 n M / j u l )
39 38 37 36
N 35 ~- 3 4
~ 3a ~
32 31 No Treatmeant
Pargyline Pretreatment
Fig. 4. The mean rectal temperature of animals receiving bilateral intracerebral injections of dopamine or vehicle with and without pargyline pretreatment (15 mg/kg) 30 min prior to intracerebral injection. The T-bars represent the standard error of the mean ( m l , ~ = 0.001).
These results demonstrate that hypothermia and deficits in motor performance can be produced by injections of DA into the PLH. Given that these deficits are similar to those produced by DA-degenerating lesions which produce accumulations proximal to the site of injection in the PLH, lends indirect support to the hypothesis that CA observed by fluorescence histochemistry at sites of accumulation may be neuroactive and may thereby participate in the production ofbehavioural deficits previously attributed only to the loss of functional neurotransmitter in the striatum. Decreases in food intake can be seen when the same dose of DA as that employed in the present study is injected into the perifornical hypothalamus 2-4. This anatomical location of injection is very close to the site where degeneration-associated amine accumulations are found in animals bearing DAdepleting lesions and which suffer severe deficits in food and water intake 8"~°'11'13'15. In a more recent study 14 we have found that the injection of the CA receptor antagonists haloperidol and propranolol directly into areas of accumulation in such animals can reverse the deficits in food intake and motor performance. Furthermore, with only 3 such injections long-tei-i~arecovery can develop, indicating that a change in the sensitivity of CA receptors affected by the degeneration-induced neurotransmitter release may be the responsible mechanism. The results of this work taken together with that from other studies sup-
141
Fig. 5. Fluorescence pictomicrographs of amine fluorescence near the site of dopamine injection in the posterior aspect of the lateral hypothalamus (PLH). A: normal fluorescence of the PLH near the site where 1 #1 of vehicle was injected. B: increased fluorescence near the site of injection of 1 #1 of 100 nmol of dopamine ( x 65). (Publisher's reduction factor 0.76).
142
Fig. 6. A histological plate7 depicting injection sites and necrosis which resulted from the injectionof isotonic saline and dopamine(100 nmol)into the lateral hypothalamus.IC, internal capsule; CPU, nucleus caudatus putamen; CC, corpus collosum. ports the hypothesis that DA released from sites of accumulation may be affecting hypothalamic CA systems which regulate satiety and which may be activated by the peripheral administration of CA precursors and enzyme inhibitors which produce anorexia2"12'16. While we have not yet verified that the amine accumulated in degenerating neurons is DA, the results from the present study and from previous work 14 suggest that DA may be principally involved. However, since other CA systems containing noradrenaline (NA) run in close proximity to the ascending DA systems, NA may also be present at the site of accumulationS'l°'ll. It has been reported previously that the severity ofhypothermia which occurs 1.5 h after 6-OHDA treatment correlates well with the volume of accumulation which is seen in the hypothalamus 1°. The larger the volume of accumulation the more severe the hypothermia. On the basis of this observation it was concluded that the neurotransmitter may be released at these sites rather than from DA terminals in the striatum or preoptic area of the hypothalamus and which is thereby producing the hypothermic response 1'5'9. While the results from the present study lend support to our previous hypothesis, it is not clear that the lesion-induced hypothermia results from activation of DA systems involved specifically in the homeostatic control of thermoregulation 1. It is interesting to note that while severe deficits
in locomotor behaviour were seen in the DAinjected animals, their performance on the 3 motor reflex tests was normal. It could be that the mechanisms governing more finely tuned aspects of motor control, as detected by these tests, are controlled by the nigrostriatal system and were not affected by the intracerebral DA injection. It could be that only locomotory and consummatory behaviours are altered by the accumulation of amines in the hypothalamus. It is also possible that the quantity of neuroactive DA after these intrahypothalamic injections was not as great as that occurring after DA-depleting lesions~°'l~'~5 Our fluorescent histochemical observations confirm that the increased fluorescence is much less than that observed in impaired animals with DAdepleting lesions of the hypothalamus. While the volumes of accumulation can extend for several millimetres when CA neurones degenerate9-~ ~, the largest area of increased fluorescence seen after these intracerebral DA injections extended for no more than 500/~m. In consideration of the good relationship between the volume of accumulation and the severity of behavioural deficit, the amount of amine present after DA injection is probably not sufficient to produce the most severe deficitsl°'l 1.16 Fluorescence histochemical examination revealed that the DA injection produced accumulation-like effects at the same dose as that which was employed to produce locomotory impairment. Increased fluorescence was still discernible at the DA-injected sites 24 h after injection even though the DA induced deficits were observed only a few hours after DA injection. In conclusion, the results from this work and from other studies s'l 1-15 indicate that many of the deficits in thermoregulation, food intake and locomotory behaviour which occur after DAdepleting treatments can also be produced by the intracerebral injection of DA. Furthermore, that CA precursors and agonists which are administered peripherally can also produce deficits in locomotory behaviour, consummatory behaviour and thermoregulation, similar to those seen after DA degeneration or intracerebrat DA, stands in support of the hypothesis that the hypothalamus may be the site of action of these compounds.
143 REFERENCES 1 Cox, B., Kerwin, R. and Lee, T.F., Dopamine receptors in the central thermoregulatory pathways of the rat, J. Physiol. (Lond.), 282 (1978) 471-483. 2 Leibowitz, S.F. and Rossakis, C., L-Dopa feeding suppression: effect on catecholamine neurons of the perifornical lateral hypothalamus, Psychopharmacology, 61 (1979) 273-280. 3 Leibowitz, S.F. and Rossakis, C., Mapping of brain dopamine- and epinephrine-sensitive sites which cause feeding suppression in the cat, Brain Res., 172 (1979) 101-113. 4 Leibowitz, S.F. and Rossakis, C., Pharmacological characterisation of perifornical hypothalmic dopamine receptors mediating feeding inhibition in the rat, Brain Res., 172 (1979) 115-130. 5 Nakamura, K. and Thoenen, H., Hypothermia induced by intraventricular administration of 6-hydroxydopamine in rats, Europ. J. Pharmacol., 16 (1971) 46-54. 60ltmans, G.A. and Harvey, J.A., LH syndrome and brain catecholamine levels after lesions of the nigrostriatal bundle, Physiol. Behav., 8 (1972) 69-78. 7 Pellegrino, L., Pellegrino, A. and Cushman, A., A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York, 1979. 8 Singer, G. and Willis, G.L., Biochemical and pharmacological basis for the lateral hypothalamic syndrome, Brain Res. Bull., 2 (1977) 485-489.
9 Ungerstedt, U., Adipsia and aphagia after 6-hydroxydopamine-induced degeneration of the nigro-striatal dopamine system, Acta physiol, scand., 367 ( 1971 ) 95-122. 10 Willis, G.L. and Singer, G., The failure of alpha-methylpara-tyrosine to produce supersensitivity in 6-OHDA lesioned rats, Pharmac. Biochem. Behav., 12 (1980) 371-376. 11 Willis, G.L. and Smith, G.C., The behavioural effects of intrahypothalamic multistage versus single injection of 6-hydroxydopamine, Brain Res., 245 (1982) 345-352. 12 Willis, G.L. and Smith, G.C., Anorexic properties of three monoamine oxidase inhibitors, Pharmac. Biochem. Behav., 17 (1982) 1135-1139. 13 Willis, G.L. and Smith, G.C., Short-term effects ofintrahypothalamic colchicine, Brain Res., 276 (1983) 119-125. 14 Willis, G.L. and Smith, G.C., Intrahypothalamic injections ofdopamine (DA) receptor antagonists reinstate locomotory and consummatory control after DAdepleting lesions, Neurosci. Lett., Suppl. 15 (1984) $64. 15 Willis, G.L., Smith, G.C. and McGrath, B.P., Deficits in locomotor behaviour and motor performance after central 6-hydroxydopamine or peripheral L-Dopa, Brain Res., 766 (1983) 279-286. 16 Zigmond, M.J. and Stricker, E.M., Recovery of feeding and drinking by rats after intraventricular 6-hydroxydopamine or lateral hypothalamic lesions, Science, 182 (1973) 717-720.
Elsevier
137
BBR 00346
DEFICITS IN LOCOMOTOR BEHAVIOUR AND THERMOREGULATION PRODUCED BY INTRAHYPOTHALAMIC DOPAMINE INJECTIONS GREGORY L. WILLIS and GRAEME C. SMITH
Monash University Department of Psychological Medicine, Prince Henry's Hospital, St. Kilda Road, Melbourne, Victoria 3004 (Australia) (Received December 2nd, 1983) (Revised version received March 19th, 1984) (Accepted March 19th, 1984)
Key words: amine accumulation - catecholamine depletion - open field activity - lateral hypothalamus - dopamine - rat
When nigrostriatal dopamine neurones degenerate, a loss of functional dopamine in the striatum occurs and is accompanied by increased dopamine in the degenerating axons which traverse the hypothalamus. While the behavioural deficits which occur after nigrostriatal degeneration have been attributed to the loss of functional dopamine neurotransmission, evidence produced by us suggests that the increased levels of amines in the degenerating axons may be neuroactive and participate in the production of these behavioural deficits. To test this hypothesis further, albino rats were injected bilaterally with 200 nmol of dopamine in a location just rostral to the diencephalon/mesencephalonborder, where amine accumulation is commonly observed following lateral hypothalamic damage. The effect of these injections upon open field performance, thermoregulation and motor reflex control was determined 40 min after dopamine injection. In a second study, pargyline (15 mg/kg, i.p.) was administered 30 min before intracerebral dopamine to determine whether this treatment would increase the severity of motor and thermoregulatory deficits which occurred after dopamine injections alone. Deficits in locomotion, rearing and the ability to regulate body temperature were seen after the dopamine injections while motor reflex control in these animals was similar to that seen in vehicle-injected controls. The behavioural deficits displayed by pargyline pretreated, dopamine injected animals were slightly but not significantly more severe than those displayed by animals receiving dopamine injections alone. Fluorescent histochemical assessment of injection sites revealed that dopamine injection produced an increase in fluorescence or "amine accumulation" at the site of injection but this was considerably less than that seen after catecholamine degeneration. These results add further support to the hypothesis that amines which accumulate in degenerating neurones may be neuroactive and may thereby participate in the production of behavioural deficits attributed previously to the loss of functional dopamine neurotransmission.
INTRODUCTION
When the ascending nigrostriatal dopamine (DA) system degenerates a loss of functional neurotransmitter is seen in the axon terminals of the nucleus caudatus putamen (NCP) while the axons which traverse the lateral hypothalamus (LH) become enlarged with accumulated amines9,1 !,15. It is generally held that the reduced neurotransmitter content is the degenerative phenomenon which underlies the changes in thermoregulationT M and locomotory and cons u m m a t o r y b e h a v i o u r s 6"9'16 which are seen in experimental animals bearing DA-depleting lesions. 0166-4328/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Recently, however, it has been suggested that the hypothalamic accumulations may be areas of functional neurotransmitter produced during degeneration, which may also participate in the production ofbehavioural deficits previously attributed only to the loss of functional neurotransmitter in the terminal fields9,1°-15. That this accumulated neurotransmitter may be neuroactive and possibly affecting adjacent catecholamine (CA) receptors is supported by the finding that the injection of CA and CA agonists into the LH produces reductions in food intake which are similar to those produced by degenerative lesions2-4. If amine accumulation is participating in the
138 production of behavioural deficits then intrahypothalamic injections of CA into areas where accumulation is seen might also be expected to produce thermoregulatory and motor impairment similar to that displayed by animals with DA degeneration. It was the object of this study to examine the effects of intrahypothalamic DA injections on thermoregulation and motor performance. MATERIALS AND METHODS
Thirty-two male Sprague-Dawley rats ranging in weight from 250 to 300 g were housed individually in wire mesh cages and allowed ad libitum access to rat pellets (Clarke King, Melbourne) and tap water. A 12 h light/dark cycle was maintained throughout the experiment with lights on at 07.00 h. Room temperature was maintained at 2 2 ° C ( + 2 ° C ) .
Surgery All animals were anaesthetized with 84 mg/kg of alphaxalon i.p. and then placed in a stereotaxic instrument. Cannulae were aimed at the posterior aspect of the lateral hypothalamus (PLH), bilaterally (coordinates A = - 0 . 8 mm, L = + 1.9 mm D = - 6.1 mm). The cannulae were in the plane of Pellegrino et al. 7 and the injection needle extended 2 mm beyond the cannula tip. Animals were allowed at least 2 weeks to recover from surgery before commencing the experiment and were handled daily and were allowed ad libitum access to food and water throughout the study.
Injections A 200 nmol solution of 3,4-dihydroxyphenylethylamine hydrochloride (Dopamine, Sigma) w a s mixed in distilled water containing 0.02 mg/ml of ascorbic acid to retard oxidation. This dose was chosen on the basis of previous work 2. Control injections were made with isotonic saline solutionscontaining 0:02 mg/ml of ascorbic acid. Eachanimal roeeived 1/~1 of the drug or 1 #1 of the control solution on each ~ d e of the brain. Intraperitoneal injections were naade with 15 mg/ml solutions of pargyline hydrochloride
(Sigma) or with isotonic saline and all solutions were administered in the volume of 1 ml/kg. The dose of pargyline employed was chosen on the basis of previous work which demonstrates that this dose potentiates the satiety-inducing effects ofintracerebral DA injections 2. All i.p. injections were made 30 min before intracerebral injections.
Procedure In the first study 5 rats received bilateral intracranial injections of DA while another 5 received a bilateral injection of isotonic saline. A crossover design was employed and 2 days after this the drug- and vehicle-injected groups were reversed and the performance of all animals on the motor tests was assessed and rectal temperature was measured. Therefore, each animal served as his own control. Forty-five minutes after the intracerebral injections the performance of all animals was assessed on open-field and motorreflex tests as described previously 11'~4. The number of squares crossed and the number of rearings during a 5-min test period were recorded. After this the latency to retract an elevated limb, to step up or down from a raised platform and to ambulate from within a prescribed area were also measured. Rectal temperature was taken 1,5 h after intracranial injections. The paradigm employed in the second study utilizing 10 more animals was the same as that described in experiment 1 except that all animals were pretreated with 15 mg/kg ofpargyline 30 min before receiving the intracerebral injections. At the completion of the experiments, all animals were sacrificed and their brains removed, stored in 10~o formalin and then sectioned and stained using cresyl violet and luxol fast blue. The areas of necrosis resulting from injection were plotted on plates from the atlas of Pellegrino et al.7. In the third study 12 animals were cannulated bilaterally as described earlier and then allowed at least 10 days to recover from surgery. They then received a unilateral 1/~1 injection of either 50, 100 or 200 nmol of DA and received a 1 #1 injection of vehicle on the contralateral side (n = 4 animals per group). Half of the animals in each group were sacrificed for fluorescence histochemistry 2 h after
139 Motor Reflex Tests (no pretreatment)
injection while the other half were sacrificed 24 h after injection. Statistical comparisons were made between vehicle injection days and DA injection days for all measures using Student's t-test or two-way ANOVA with repetitions (Cannon, BX-10 statistical package).
15
r-]
Vehicle
B
DA (200nM/~ul)
10 RESULTS
As shown in Fig. 1 the open-field behaviour of DA-injected rats was significantly depressed when compared to vehicle-injected controls. The number of squares crossed during the 5 min test period was significantly less in the unpretreated and pargyline pretreated animals ( d f = 1,36; F = 40.8; a = 0.001) when compared to their respective controls. The number of rearings was also greatly reduced in those animals receiving DA injections with and without pargyline pretreatment (df = 1,36; F - - 23.6; a -- 0.001). Pargyline pretreatment decreased slightly the number of rearings exhibited after DA injection when compared to those animals receiving no pretreat-
~= •
Open Field Behaviours
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PRETREATMENT
NO PRETREATMENT
Fig. 1. The open-field behaviour of animals receiving bilateral injection of dopamine, or vehicle into the posterior lateral hypothalamus. While half of the animals were pretreated with 15 mg/kg of pargyline the others were not. The T-bars represent the standard error of the mean. The number of squares represents the level of significance (mmm, a = 0.001).
"1o c o o o
0
, LATENCY TO
~ LATENCY TO
LATENCY TO
RETRACT A t IMB
STEP D O W N
AMBULATE
Fig. 2. The latency to retract a limb, to step down and to ambulate in animals injected bilaterally with dopamine or vehicle. The T-bars represent the standard error of the mean.
ment and this difference was significant (df = 1,36; F = 89.5; ~ = 0.001). The performance of all DA-injected groups on the 3 motor reflex tests was not significantly impaired by the DA injection. As shown in Figs. 2 and 3, the latency to retract a limb, to step up or down and to ambulate were in some cases increased over that seen after control injections but 2-way ANOVA revealed that these differences were not significant. Intracerebral injections of DA produced a significant drop in rectal temperature 1.5 h after injection as compared to the vehicle-injected controis (df = 1,36; F = 89.5; ~ = 0.001). Pargylinepretreated, DA-injected animals also displayed a significant reduction in rectal temperature when compared to their vehicle-injected counterparts (~ = 0.001). Pargyline pretreatment alone did not reduce significantly the parameter of body temperature. Fluorescent histochemical examination of the injection sites of DA-injected rats revealed a
140 Motor Reflex T e s t s
reliable increase in fluorescence at both 1 and 24 h after DA injection (Fig. 5). Increased fluorescence was observed around the site of injection in the PLH just rostral to the midbrain DA cell bodies. While some increases in fluorescence were seen in animals treated with 50 nmol solutions of DA, the intensity of this fluorescence was generally not as great as that seen in the 100 and 200 nmol dose. Histological verification of the injection sites of the animals used in the behavioural studies confirmed that the sites of DA injection were in the proximity of the medial forebrain bundle in the PLH just rostral to the substantia nigra (Fig. 6).
I-"1 Vehicle 15
B
DA (200ni/pl)
=.
10B
"O C O (9 00
DISCUSSION
5-
_
LATENCY TO
LATENCY TO
LATENCY TO
RETRACT A LIMB
STEP D O W N
AMBULATE
Fig. 3. The latency to retract a limb, to step down and to ambulate in animals injected bilaterally with dopamine or vehicle 30 min after pretreatment with 15 mg/kg ofpargyline. The T-bars represent the standard error of the mean. Rectal Temperature (90min after 40
,,,
intracerebral
DA)
]
Vehicle
B
DA ( 2 0 0 n M / j u l )
39 38 37 36
N 35 ~- 3 4
~ 3a ~
32 31 No Treatmeant
Pargyline Pretreatment
Fig. 4. The mean rectal temperature of animals receiving bilateral intracerebral injections of dopamine or vehicle with and without pargyline pretreatment (15 mg/kg) 30 min prior to intracerebral injection. The T-bars represent the standard error of the mean ( m l , ~ = 0.001).
These results demonstrate that hypothermia and deficits in motor performance can be produced by injections of DA into the PLH. Given that these deficits are similar to those produced by DA-degenerating lesions which produce accumulations proximal to the site of injection in the PLH, lends indirect support to the hypothesis that CA observed by fluorescence histochemistry at sites of accumulation may be neuroactive and may thereby participate in the production ofbehavioural deficits previously attributed only to the loss of functional neurotransmitter in the striatum. Decreases in food intake can be seen when the same dose of DA as that employed in the present study is injected into the perifornical hypothalamus 2-4. This anatomical location of injection is very close to the site where degeneration-associated amine accumulations are found in animals bearing DAdepleting lesions and which suffer severe deficits in food and water intake 8"~°'11'13'15. In a more recent study 14 we have found that the injection of the CA receptor antagonists haloperidol and propranolol directly into areas of accumulation in such animals can reverse the deficits in food intake and motor performance. Furthermore, with only 3 such injections long-tei-i~arecovery can develop, indicating that a change in the sensitivity of CA receptors affected by the degeneration-induced neurotransmitter release may be the responsible mechanism. The results of this work taken together with that from other studies sup-
141
Fig. 5. Fluorescence pictomicrographs of amine fluorescence near the site of dopamine injection in the posterior aspect of the lateral hypothalamus (PLH). A: normal fluorescence of the PLH near the site where 1 #1 of vehicle was injected. B: increased fluorescence near the site of injection of 1 #1 of 100 nmol of dopamine ( x 65). (Publisher's reduction factor 0.76).
142
Fig. 6. A histological plate7 depicting injection sites and necrosis which resulted from the injectionof isotonic saline and dopamine(100 nmol)into the lateral hypothalamus.IC, internal capsule; CPU, nucleus caudatus putamen; CC, corpus collosum. ports the hypothesis that DA released from sites of accumulation may be affecting hypothalamic CA systems which regulate satiety and which may be activated by the peripheral administration of CA precursors and enzyme inhibitors which produce anorexia2"12'16. While we have not yet verified that the amine accumulated in degenerating neurons is DA, the results from the present study and from previous work 14 suggest that DA may be principally involved. However, since other CA systems containing noradrenaline (NA) run in close proximity to the ascending DA systems, NA may also be present at the site of accumulationS'l°'ll. It has been reported previously that the severity ofhypothermia which occurs 1.5 h after 6-OHDA treatment correlates well with the volume of accumulation which is seen in the hypothalamus 1°. The larger the volume of accumulation the more severe the hypothermia. On the basis of this observation it was concluded that the neurotransmitter may be released at these sites rather than from DA terminals in the striatum or preoptic area of the hypothalamus and which is thereby producing the hypothermic response 1'5'9. While the results from the present study lend support to our previous hypothesis, it is not clear that the lesion-induced hypothermia results from activation of DA systems involved specifically in the homeostatic control of thermoregulation 1. It is interesting to note that while severe deficits
in locomotor behaviour were seen in the DAinjected animals, their performance on the 3 motor reflex tests was normal. It could be that the mechanisms governing more finely tuned aspects of motor control, as detected by these tests, are controlled by the nigrostriatal system and were not affected by the intracerebral DA injection. It could be that only locomotory and consummatory behaviours are altered by the accumulation of amines in the hypothalamus. It is also possible that the quantity of neuroactive DA after these intrahypothalamic injections was not as great as that occurring after DA-depleting lesions~°'l~'~5 Our fluorescent histochemical observations confirm that the increased fluorescence is much less than that observed in impaired animals with DAdepleting lesions of the hypothalamus. While the volumes of accumulation can extend for several millimetres when CA neurones degenerate9-~ ~, the largest area of increased fluorescence seen after these intracerebral DA injections extended for no more than 500/~m. In consideration of the good relationship between the volume of accumulation and the severity of behavioural deficit, the amount of amine present after DA injection is probably not sufficient to produce the most severe deficitsl°'l 1.16 Fluorescence histochemical examination revealed that the DA injection produced accumulation-like effects at the same dose as that which was employed to produce locomotory impairment. Increased fluorescence was still discernible at the DA-injected sites 24 h after injection even though the DA induced deficits were observed only a few hours after DA injection. In conclusion, the results from this work and from other studies s'l 1-15 indicate that many of the deficits in thermoregulation, food intake and locomotory behaviour which occur after DAdepleting treatments can also be produced by the intracerebral injection of DA. Furthermore, that CA precursors and agonists which are administered peripherally can also produce deficits in locomotory behaviour, consummatory behaviour and thermoregulation, similar to those seen after DA degeneration or intracerebrat DA, stands in support of the hypothesis that the hypothalamus may be the site of action of these compounds.
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