GABAergic denervation of rat substantia nigra: functional and pharmacological properties

Brain Research, 183 (1980) 217-223

217

© E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

GABAergic denervation of rat substantia nigra: functional and pharmacological properties

K A R E N G A L E a n d M I C H A E L J. I A D A R O L A

Georgetown University, Schools of Medicine and Dentistry, Department of Pharmacology, 3900 Reservoir Road, N. I4I., Washington, D.C. 20007 (U.S.A.) (Accepted O c t o b e r 4th, 1979)

Key words: G A B A e r g i c t e r m i n a l s - - s u b s t a n t i a nigra - - denervation - - rotational behavior

The concentration of GABA in substantia nigra (SN) is among the highest found in any region of the brain4,13. The major portion of the GABA content in the SN is associated with nerve terminals afferent to this nucleus, many of which arise from cell bodies located in the caudate-putamen and globus pallidus4,7,1a, 16. There has been increasing interest in the role of the striatonigral GABA pathways, and an anatomically associated projection containing substance P (see refs. 7 and 15) for the regulation of activity of nigral efferent pathwaysl,3,8, 9. Lesion techniques have often been employed to study this problem, largely in a effort to ascertain which functions remain intact after removal of the GABA-containing projections to SN 1,8,8,9. After these lesions, which were designed to interrupt neural connections between SN and forebrain nuclei, decreases of approximately 50-60 ~ (based on mean values) in GABA concentrations or glutamic acid decarboxylase (GAD) activity in SN were foundl,Z,9,16,18,19. However, since these studies did not attempt to select for complete lesions on the basis of either biochemical measurements or histological assessment of lesion damage, the values obtained most likely include data from animals with sub-total destruction. In contrast, a few studiesS, 13,20, in which the investigators selected for complete lesions, suggest that afferents to SN from forebrain may account for more than 75 ~o of the GABA in this nucleus. In order to ascertain the total extent of the forebrain contribution to nigral GABA content, we developed a modified hemitransection technique which allowed us to produce reliably complete destruction of fibers between SN and striatum without otherwise damaging nigral tissue. As a result, our data confirm that the forebrain contribution to nigral GABA content is greater than 75 ~ . At the same time, we provide evidence that a small percentage of intact GABA terminals remaining after incomplete lesions may have considerable functional impact. In a preliminary series of hemitransections between SN and striatum, we obser-

218

Fig. 1. a: stainless steel wire (18 g) was bent into a loop and attached to a stereotaxic electrode carrier, so that the plane formed by the two parallel sides of the loop was perlzendicular to the midline. Male Sprague-Dawley rats weighing 300-330 g were anesthetized with Equithesin (0.5-0.7 ml i.p.) and placed in a stereotaxic head holder with the incisor bar 5 mm above the interaural line. The electrode carrier was inclined on the anterior-posterior plane at a 6.3° angle from vertical so that the lower tip of the loop was directed rostrally. The probe entered the cortex at AP 2.8 mm (see ref. 22), was lowered approximately 9.0 mm from dura, until it hit the base of the skull, and was moved 5 mm laterally from the midline by manipulating the stereotaxic carrier. The lateral movement was repeated 3-4 times, adjusting the vertical depth as necessary in order to follow the contours of the base of the skull, b: view of ventral surface of brain. The transection could be seen to extend through the cerebral peduncles at the base of the brain (arrow), just caudal to the premammillary nucleus, approximately 1.0 mm anterior to SN.

ved substantial variability in the completeness of the lesion and, as a consequence, variability in the loss of G A B A in SN. This variability resulted in what appeared to be a b i m o d a l distribution, with m a n y animals exhibiting 5 0 - 6 0 ~ decreases in G A B A c o n t e n t in SN a n d others exhibiting 80-90 ~ decreases. Gross e x a m i n a t i o n of hemitransections in which only a 50-60 ~ loss of nigral G A B A was obtained suggested that it was necessary to transect fibers travelling ventrally in the cerebral peduncles in order to achieve a more complete loss of nigral G A B A content. F o r this purpose, we devised a stainless steel loop (Fig. la) which could be stereotaxically lowered to the base of the rat's skull a n d then m a n i p u l a t e d in a medial-lateral plane in order to bluntly sever fibers r u n n i n g in a rostro-caudal direction (Fig. lb). The design of this probe allowed it to be r u n across the base of the skull with a m i n i m u m of vascular damage. A n i m a l s were allowed 7-10 days postoperative survival before sacrifice for m e a s u r e m e n t of G A B A . G A B A levels were measured in SN of 36 hemitransected rats;

219 TABLE I Effect o f hemitransection (a) alone and (b) with isoniazid on GABA concentration in S N

Rats were hemitransected as described in Fig. 1 and sacrificed 7-10 days later. GABA measurements were made in SN from lesioned hemispheres, contralateral intact hemisl~heres and from unorerated rats. For measurement of GABA, rats were decapitated and their brains were rapidly removed and placed on ice for micro-dissection of SN as previously described 7. A single SN contained 300-400/tg protein. No significant differences between intact and lesioned SN with respect to wet weight and protein content were observed. GABA was extracted from the tissues with 0.4 N perchloric acid and assayed with the method of Okada et al. 21. Proteins were measured according to Lowry et al. 17. In a separate group of 10 lesioned rats sacrificed by focused microwave irradiation (4.0 sec), it was determined that the method of sacrifice did not influence the per cent loss of GABA due to the lesions. Isoniazid was dissolved in distilled H20 and injected i.p., 50 min prior to sacrifice; control rats received an equivalent volume of H20. Each value in (b) represents the mean 4- S.E. of at least 8 rats. In a separate group of 6 lesioned rats sacrificed by focused microwave irradiation, isoniazid treatment resulted in a 42 % decrease in the GABA content of the intact SN (control = 85 nmol/mg prot.; isoniazid treated = 49 nmol/mg prot.) and a 36 ~ decrease in GABA in the SN from the lesioned side (control = 18 nmol/mg prot. ; isoniazid treated = 11.5 nmol/mg prot.). Rats (n)

(a) GABA remaining (% of intact) 09)

Control Isoniazid

5

11

10

5

5

6-9

10-14

15-19

20-24

25-30

GABA (nmol/mg protein) Unoperated

Intact

Lesioned

102 :~ 7 48 d: 6*

110 d_ 8 51 d- 6*

21 :~ 3 13 ± 2*

* Denotes significance (P < 0.01) when compared with vehicle-injected control tats. for each a n i m a l the per cent of G A B A r e m a i n i n g o n the lesioned side was calculated using the contralateral SN of the same a n i m a l as control. The per cent of G A B A r e m a i n i n g i n the SN of the hemitransected side ranged from 6 ~o to 30 ~ ; the m e a n value was 19 ~ (Table la). I n the majority of lesioned a n i m a l s (26 out of 36, or 72 ~ ) , the G A B A c o n t e n t fell below 20 ~ of control. I n order to determine whether the G A B A r e m a i n i n g in the deafferented SN was subject to further reduction, one g r o u p of hemitransected animals received isoniazid (400 mg/kg, i.p. 50 m i n prior to sacrifice) in order to i n h i b i t G A B A synthesis 24. lsoniazid caused a 50 ~ reduction of G A B A levels in the intact SN (Table I b ; see also ref. 11) w h e n c o m p a r e d with vehicle-injected controls. As shown in Table 1, the isoniazid t r e a t m e n t was able to reduce the G A B A c o n t e n t of the deafferented SN to a value which was approximately 40 ~ lower t h a n the c o n c e n t r a t i o n f o u n d in vehicleinjected lesioned rats. These results were o b t a i n e d in rats sacrificed by either decapitation or focused microwave i r r a d i a t i o n (see Table I legend). I n a previous study it was d e m o n s t r a t e d that surgical h e m i t r a n s e c t i o n between SN a n d f o r e b r a i n did n o t cause a loss of [ a H ] G A B A b i n d i n g sites i n S N when measured one or more weeks postoperatively 12. This suggests that these lesions

220 destroy prejunctional GABAergic terminals while sparing postsynaptic components which are GABA-receptive. To verify whether functional G A B A receptors remain in SN after our complete hemitransections, a behavioral response to intranigrally injected muscimol was measured. Muscimol, a potent G A B A receptor agonist, has been previously demonstrated to induce contralateral circling behavior after direct unilateral application into SN, an effect not mediated by nigrostriatal dopaminergic pathways 23. In rats which had 80-90 ~ loss of nigral G A B A content after hemitransections, we observed pronounced contralateral circling responses to intranigral muscimol (50 ng in 0.5/~1 saline) similar to those obtained from unlesioned control rats (Table II). The presence of this behavior after our complete hemitransections between SN and forebrain indicates that it does not depend u p o n ipsilateral forebrain projections but instead is mediated by descending nigral efferents which remain intact after our lesions. Since isoniazid treatment appeared to influence the G A B A content o f the deafferented SN we were interested in determining whether isoniazid could have a functional impact in this system. Again, a behavioral response to unilateral intranigral microinjection was examined. W h e n locally-injected unilaterally into an intact SN, isoniazid caused ipsilateral circling behavior which reached a maximum intensity 30 min after injection, coincident with a decrease in G A B A content in the injected SN (Table III). To investigate the effects of isoniazid on a GABA-denervated SN, a group o f hemitransected rats (7 days post-operative) received local injections ofisoniazid directly into the SN o f the transected hemisphere and were observed for circling behavior. After behavioral observations were made, animals were allowed 3 days survival before sacrifice for measurement o f G A B A . In animals with 20 ~ or less G A B A remaining in

TABLE I1 Circling behavior (total turns in 3 rain) after intranigral injections in hemitransected rats

All intranigral injections were made stereotaxically (see legend to Table lid into SN in the hemisphere in which transections were made 7 days before. Isoniazid was administered as described in legend of Table III; muscimol (0.2 nmol in 0.5 ~1 saline) was infused over 3 min. After removing the rats from the stereotaxic apparatus the behavioral response to muscimol appeared within 5 min and persisted for at least 1 h. Scoring period for circling behavior was between 25 and 30 rain after termination of drug infusion. Values represent the mean number of total complete turns in a 3 min period for a group of 4-6 rats. In order that the per cent of GABA remaining in the lesioned side reflected the effect of the lesion and not the drug treatment, 3 days elapsed between drug treatment and sacrifice for measurement of GABA. Ipsi, ipsilateral; contra, contralateral. S.E.s were < 10 ~ of mean values. GABA remaining ( % o f intact)

10-20 25-30 100 (intact)

Intranigral injection Saline

lsoniazid

Muscimol

ipsi

contra

ipsi

contra

ipsi

contra

4.0 3.5 2.0

1.0 2.0 3.0

5.0 14.5 29.0

2.0 0.5 1.0

0 0 0

50.0 55.0 53.0

221 TABLE III

Circling behavior after intranigral isoniazid correlated with depletion o f GABA in S N Isoniazid (1.0 #mol) dissolved in distilled H~O was infused unilaterally into SN (1.0 pl over 5 min) via a stereotaxically-positioned stainless steel cannula (28 g) while the rat was under ether anesthesia. Coordinates ~2 for injection were: AP + 2.6 mm, Lat 2.0 mm, DV --2.8 mm. Time after infusion of isoniazid was measured from the end of the injection period. Circling behavior was measured as the total number of complete (360 °) turns made in each direction (defined with respect to side of injection) over a 3 min period during which the rat was placed on a large table top free of obstacles. Within 5 min after removal from the stereotaxic apparatus, all animals were awake and able to locomote. Rats were sacrificed immediately after the 3 min observation period. The data represent mean values obtained from 6 animals. See Table I legend for methods of sacrifice and GABA assay. S.E.M. for behavioral data < 1 0 ~ of mean values.

Time (min) a]?er infusion o f isoniazid, l Itmol

GABA (nmol/mg prot.) Control side

Treated side

10 20 30

101 i 8 98 4- 6 97 ± 5

92 ± 5 70 -- 8 40 ± 3

Treated % - control 91 71 39

Turns in 3 min ipsi

eontra

1 8 29

2 0 1

SN, isoniazid injections into the deafferented SN failed to induce either postural asymmetry or circling behavior (Table II). On the other hand, animals which had 25-30 ~ GABA remaining in SN showed ipsilateral circling in response to intranigral isoniazid (Table II). The intensity of the circling response appeared directly related to the amount of GABA remaining in SN, with the partially lesioned animals showing a significantly attenuated response to isoniazid when compared with unlesioned controls. Thus, it appears that the functional effect of isoniazid in SN requires the presence of a minimum amount of intact afferent GABA terminals projecting from the forebrain. The small amount of GABA remaining in SN after even the most complete hemitransection is probably derived largely from glial cells and neuronal perikarya in the SN. In other experiments 14 we found no significant decrease in nigral GABA content after hemitransections placed posterior to SN. It is therefore unlikely that the SN receives any GABA-containing projections ascending from caudal brain regions. The possibility that GABA-containing terminals associated with GABAergic neurons intrinsic to SN may account for some of the residual GABA in the deafferented SN cannot be excluded. However, if GABAergic interneurons do exist in SN they evidently account for a very small portion of the normal GABA content of this nucleus. In many lesion studies the extent of damage to nigral GABAergic afferent projections has been assessed using G A D activity as a marker. The possibility that estimates using this enzyme and estimates made on the basis of GABA concentration may be at variance with one another must be considered. However, it has been demonstrated that nigral GABA content varies linearly with nigral G A D activity after lesions 20 and it appears that the G A D content of SN can also be decreased to less than 20 ~ of control by properly placed hemitransections 13. Fonnum and co-workers 5 have

222 demonstrated decreases in nigral G A D ranging from 72 to 87 % after hemitransections which (based on histological verification) disconnect the SN from both globuspallidus and the tail of the caudate-putamen. We are able to corroborate this anatomical relationship since the rostrocaudal angle of our transection probe results in lesions which fall between the caudal extent of the striatum and the rostral tip of SN. The observation that as little as 25 % G A B A remaining in SN can sustain a significant behavioral response to isoniazid raises the possibility that other functional effects of G A B A may be preserved after sub-total destruction of GABA terminals afferent to SN. For example, several investigators have attempted to assess the role of nigral GABA afferents for regulating nigrostriatal dopaminergic neurons. In certain cases this has involved determining the extent to which dopamine-antagonists can still elicit an increase in dopamine metabolites after either kainic acid lesions of the striatum 3 or mechanical lesions of G A B A projections to SN 9. These investigators found that the response to dopamine-antagonists was preserved after their lesions, suggesting that the GABA projections were not necessary for this response. However, the destruction of GABAergic afferents in these experiments was sub-total (according to our estimates) and it is therefore possible that the small amount of GABA terminals remaining could have contributed to the response. Compensatory mechanisms such as an increase in number of postsynaptic G A B A receptors which has been observed after hemitransections between SN and forebrain 12, may in part account for the maintenance of function after apparently major destruction of presynaptic G A B A terminals. This possibility must be kept in mind when attempting to interpret results of studies which utilize lesions of G A B A projections to SN in order to determine the functional importance of these pathways. In summary, our data demonstrate that in order to abolish a functional response (isoniazid-induced circling behavior) mediated by nigral GABA terminals, the GABA content of the SN must be reduced by at least 80 %. This was accomplished by blunt transection of all fibers connecting the SN with the ipsilateral forebrain. These lesions did not destroy GABA-receptive nigral efferents which mediate the behavioral circling response to intranigral muscimoi. This work was supported by U.S. Public Health Service Grants DA 02206, NS 12566, G M 07443-03 and M H 32359. K.G. is a recipient of a Faculty Development Award from the Pharmaceutical Manufacturers Association Foundation.

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