Rotatory

Behavior

Induced

the Substantia JORGE S. ~VE~WEZ, BILLIE Medical

Rrscarch

by Glycine Nigra

IV. I;IKN,

Ccntcr, Bvookhuocu U/~tota, L.I., New York

of the

Injected Rat

AND KAREN E. DAHL National 11973

into

’

Laboratory,

Implantation of crystalline glycine and dopamine into the substantia nigra ‘of the rat caused the rat to turn away from the injected side. This response is similar to that observed after electrical stimulation of the substantia nigra, or after amphetamine administration when the contralateral substantia nigra has been previously damaged with 6-hydroxydopamine. Rotatory behavior was not observed after injection of glycine into the caudate nucleus, whereas dopamine did induce this effect, as previously demonstrated.

INTRODUCTION y-Aminobutyric acid (GABA) has been found in high concentrations in the substantia nigra of man (12) and other animals (7, 20)) and glycine has been found in relatively high concentrations in the spinal cord (3). There is evidence that GABA functions as an inhibitory neurotransmitter in the mammalian central nervous system. It has been proposed (8, 14, 19, 21, 27) that GABA is released by caudate-nigral fibers after the monosynaptic inhibitory action of such fibers in the substantia nigra (27) has been blocked by picrotoxin (22). Furthermore, the amounts of GABA and its synthesizing enzyme, glutamic acid decarboxylase, in the substantia nigra was markedly decreased after destruction of the striatum (13, 16). Some evidence also indicates that glycine may play a role as an inhibiting transmitter in the entopeduncular nucleus ( 19) and in the mammalian spinal cord (5), but this depressant effect has not been observed in the substantia nigra (17). 1 This work was supported by the National Institutes of Health (11131-01)) Energy Research and Development Administration, and, in part, by The Charles E. Merrill Trust. The authors wish to express their thanks to Drs. G. C. Cotzias and Paul S. Papavasiliou for their helpful discussion during the preparation of our manuscript and to Frank Amendola for skillful technical assistance. 171 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

SUBSTANTIA

NIGRA

173

Since pathological changes in the substantia nigrd constitute the most constant feature of Parkinson’s disease, it is of physiological and clinical interest to explore the effects of these amino acids in the substantia nigra of experimental animals. Unilateral electrothermic ( 1) or chemical (24) lesions of the compact zone of the substantia nigra in the rat are followed by degeneration of the nigrostriatal pathways and a consequent imbalance between the two sides of the brain that is probably responsible for the rotation behavior observed after administering drugs that stimulate the intact side (e.g., amphetamine) or the lesion side (e.g., apomorphine) (26). Turns occur away from the stimulated side. The purpose of the present experiments is to learn whether GABA or glycine injected locally into the substantia nigra can induce a transitory imbalance similar to that induced by the permanent lesion. MATERIALS

AND

METHODS

AGnals and Stereotaxic Injectiom. Fifty male Sprague-Dawley rats weighing 150 -t- 5 g were lightly anesthesized with a mixture of halothane, oxygen, and nitrous oxide administered through a nose mask. Crystalline GABA, glycine, dopamine, or palmitic acid was tamped into a stainless steel cannula 0.2 mm in diameter and introduced stereotaxically into ‘the brain. When the tip of the cannula had reached the desired position in the right side, the compound (about 100 pg) was delivered by pushing a stylet clown the cannula. The coordinates were: A 2.4 mm ; L 1.8 mm ; V 2.4 mm according to Konig & Klippel’s stereotaxic atlas. The effects of these substances were tested in groups of six or seven rats. In four other groups of six rats, crystals of each of these compounds were injected in the right caudate nucleus. The coordinates were A 7.8 mm ; L 2.5 mm ; V 4.0. Scoring System. Prior to the operation all animals were tested for turning ability after the injection of apomorphine (1 mg/kg, ip), or without any drug, in a rotometer (26) consisting of an aluminum hemisphere with a mechanism for counting each full turn the animal makes by means of a switch connected to a thin wire fitted as harness over the animal’s chest. Immediately following injection of the crystals, the rats were placed in the rotometer and tested for 60 min; 2 hr later they were given apomorphine, (1 mg/kg, ip) and again tested for 60 min. These tests were repeated on each of the next 4 days. The results were cumulatively recorded and stored as digital teletype printout every minute for each side for 60 min. Two months later the rats were killed, and the brains were fixed by a perfusion technique ; the needle track and the histological features of the

substantia nigra neuroils lvcrc slutlird ill 1)araffin sections stainctl \vitll cresyl violet. Drugs Used. r-Amino-n-butyric acid (GABA), Sigma ; aminoacetic acid (glycine), Nutritional Biochem. Corp. ; 3-hydroxytyramine*HCl (dopamine), Carbiochem. Corp. ; palmitic acid, Eastman Organic Chemicals. Dopamine was used as a dopaminergic agent; palmitic acid as a control crystalline substance that does not possess neurotransmitter properties. RESULTS The turning of the SO rats before the injection of crystals comprised a few turns to either side during the 60 min of observation. The turning behavior after apomorphine ( 1 “g/kg, ip) was similar (Table 1). Injection info the Substa~lfia Nigra. The rats injected with dopamine or glycine crystals in the right side substantia nigra showed a sustained turning behavior away from the injected side beginning 10 to 15 min after anesthesia was discontinued and lasting for 50 to 60 min. They were on11 a few turns in the opposite direction. The difference between left and right turns was significant (P < 0.04 for dopamine and P < 0.0017 for glycine) . The turning behavior after injection of GABA or palmitic acid was similar to that observed before the injection of the crystals. Apomorphine (1 mg/kg, ip) given 2 hr after the injection of crystals resulted in a turning behavior in all four groups similar to the preoperative behavior, with no significant difference between the number of turns to one side or the other. Injection into the Caudate Nucleus. Only dopamine induced a sustained turning behavior away from the injected side with a significant difference between left and right turns (P < 0.05). The turning behavior observed immediately after injection of GABA, glycine, or palmitic acid was similar to that of the preinjection period and remained unchanged, after administration of apomorphine (1 mg/kg. ip) 2 hr later, HistoZog31. The whole substantia nigra was studied in serial sections stained with cresyl violet. This structure extends from rostra1 to caudal 1.9 mm (23), and the point of the injection was in its rostra1 half at the pars compacta, extending to the boundary with pars reticulata. A necrotic lesion of about 0.14 to 0.20 mm diameter was observed around the injection tip. The neuronal population of the substantia nigra and adjacent nuclei outside the necrotic lesion was intact. DISCUSSION The injection of even small amounts of fluid under pressure produces fairly extensive diffusion (lS), while the injections of drugs in crystalline

SUUSTANTIA

NIGRA

‘I-ABLE

1

TURNING RESPONSE AFTER INJECTION SUBSTANTIA NIGRA AND CAUDATE Drug injected into right substantia nigra None None Dopamine Dopamine + Glycine Glycine + GABA GABA + Palmitic acid Palmitic acid

Treatmenta (1 rdg, ip)

OF CRYSTAL INTO THE NUCLEUS OF RATS

Number of rats

-

Mean of total no. turns

50 50 6 6 6 6 6 6 7 7

Apomorphine Apomorphine* Apomorphine* Apomorphine* +

177

Apomorphine*

Left

Right

Mean of total duration (min)

15 17 62 20 181 33 24 35 8 15

17 16 4* 10 3** 17 31 28 12 20

60 60 50 50 53 53 52 52 40 40

-

-

Drug injected into right caudate nucleus Dopamine Dopamine + Glycine Glycine f GABA GABA + Palmitic acid Palmitic acid

-

7 I 6 6 6 6 6 6

Apomorphine* Apomorphine* Apomorphine* +

Apomorphine*

50 21 5 8 6 20 5 14

a* 21 9 15 9 22 10 19

57 57 40 40 50 50 55 55

0 Apomorphine

was injected

2 hr after

drug

implantation.

* P < 0.05. ** P < 0.01.

form, with the extracellular fluid serving as solvent (9), makes it difficult to estimate the dose delivered. However, dye-diffusion and autoradiographic studies have shown that under certain conditions, the diffusion of intracerebral crystals is limited to a sphere about 1 mm in diameter ( 10, 17, IS), and this has been confirmed by behavioral and electrophysiological data and autoradiographic analysis. Ernst and Smelik (6) used the crystal injection technique to demonstrate the site of action of dopamine receptor stimulation by apomorphine. This site was later confirmed with biochemical techniques (2).

178

iUI