Quinolinic acid elevates striatal and pallidal met-enkephalin levels: the role of enkephalin synthesis and release

Brain Research, 562 (1991) 117-125 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/911503.50 ADONIS 000689939117027J

117

BRES 17072

Quinolinic acid elevates striatal and pallidal Met-enkephalin levels: the role of enkephalin synthesis and release Bianca B. Ruzicka, Robert Day* and Khem Jhamandas Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont. (Canada) (Accepted 28 May 1991)

Key words: Striatum; Globus pallidus; Met-enkephalin-like immunoreactivity; Huntington's chorea; Excitatory amino acid; Quinolinic acid; Proenkephalin mRNA; Release

Huntington's chorea (HC) is characterized, in part, by a substantial deficit in the striatal and pallidal enkephalin levels. Recently, an attempt was made to replicate this deficit by focally injecting quinolinic acid (QUIN), an excitotoxin, into the rat striatum. However, at 7 days post-injection, QUIN produced a dose-related and bilateral increase in the striatal and pallidal levels of mct-enkephalin-like immunoreactivity (ME-i.r.), an effect which was attenuated in the presence of excitatory amino acid (EAA) receptor antagonists. In the present study, the action of QUIN was investigated further. To determine whether the QUIN (72 nmol)-induced elevations in ME-i.r. reflected the enhanced synthesis of the peptide, the striatal levels of proenkephalin mRNA were assayed 7 days following a unilateral injection of QUIN into the rat striatum. QUIN significantly depleted (50%) the proenkephalin mRNA level in the injected, but not the contralateral striatum when compared to that in the saline-injected animals. To determine whether the QUIN-induced increases in ME-i.r. were due to an impaired release of the peptide, the release of ME-i.r. from the striatal or pallidal slices obtained from animals 7 days after a saline- or QUIN-injection, was measured. The 30 mM K÷-stimulated ME-i.r. release from the saline-injected and contralateral striatum represented an 8-fold increase above the spontaneous release level, while this stimulus induced a 6-fold increase in the ME-i.r. release from both the QUINinjected and contralateral striatum. In the globus pallidus ipsilateral and contralateral to the saline-injection, 30 mM K + produced a 14- and an 18-fold increase in the ME-i.r. release, respectively. Similarly, stimulation of the ipsilateral and contralateral globus pallidus from the QUIN-injected rats resulted in an 18- and a 10.5-fold increase in the ME-i.r. release, respectively. The K +-evoked release responses observed in the QUIN-injected animals did not differ from those observed in the saline-injected animals. To determine whether the QUINinduced changes in the ME-i.r. were time-dependent, the striatal and pallidal ME-i.r. levels were measured in rats sacrificed at different times (2, 6, 12, 24, 48 h and 4, 7 and 14 days) following an intrastriatal QUIN-injection. QUIN produced a time-dependent increase in the striatal and the pallidal ME-i.r. levels. The peak effect, observed in both brain regions at 7 days following the QUIN-injection, represented increases in the striatal and the pallidal ME-i.r. levels of 200% and 255%, respectively, when compared to saline-treated control animals. Thus, the intrastriatal injection of QU1N produced a bilateral and time-dependent elevation in the striatal and the pallidal ME-i.r. levels, an effect not associated with an increase in the striatal proenkephalin mRNA level or an impaired release of the peptide. Although the QUINinduced increases in the striatal and the pallidal ME-i.r. do not appear to resemble the enkephalin deficit associated with a progressed stage of HC, they may more closely model the changes in enkephalin observed in the presymptomatic stages of the disease. INTRODUCTION The striatum and the globus pallidus, which contain the highest enkephalin levels in the brain 17, are anatomically linked by an enkephalinergic striatopallidal pathway 8a°. A substantial degeneration of this pathway leads to one of the major neurochemical deficits associated with H u n t i n g t o n ' s chorea (HC), an inherited disease characterized by both behavioral and motor disturbances 12"24. Recently, attempts have been made to replicate some of these deficits in experimental animals by focal injections of excitotoxins, substances which activate excitatory amino acid ( E A A ) receptors, into the stria-

tum 1-3"6"7"25"29"31. Thus, the intrastriatal microinjection of kainic acid (KA) has been found to deplete biochemical markers for the striatal y-aminobutyrate ( G A B A ) - , acetylcholine (ACh)- and substance P-containing neurons, neurochemical deficits which correspond to those observed in H E 7'25. In contrast, the excitotoxin injection failed to deplete the striatal levels of methionine-enkephalin-like immunoreactivity (ME-i.r.) 16. Indeed, a single unilateral injection of K A into the striatum was shown to elevate the levels of ME-i.r. in the hippocampus, an effect which occurred bilaterally and was still observed two weeks following the excitotoxin injection is. We have recently demonstrated that the intrastriatal

* Present address: Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Que., Canada, H2W 1R7. Correspondence: K. Jhamandas, Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont., Canada K7L 3N6. Fax: (1) (613) 545-6336.

11X injection of QUIN, N-methyl-o-aspartate *‘,

dramatically

the striatum

an excitotoxin which activates the (NMDA) type of EAA receptor*”

enhanced

the levels of ME-i.r.

and the globus pallidus3’.

in both

This bilateral

ef-

fect, observed at seven days following the injection, was dose-related and was sensitive to EAA receptor antagonists,

suggesting

tor was involved3’. producing

that the activation Additionally,

the maximal

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apparent

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it was suggested

peptide biosynthesis contribute

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to the excitotoxin-induced

peptide

release may

elevations

in the

ME-i.r.“‘. Thus, the first objective of the present study was to examine the effect of an intrastriatal QUIN injection on enkephalin biosynthesis by measuring the striatal level of proenkephalin mRNA. The second objective was to examine the effect of this injection on peptide release by measuring the K+-evoked striatal and pallidal release of ME-i.r. Additionally, on the basis of previous studies in the hippocampus, it has been reported that the excitotoxininduced changes in ME-i.r. are time-dependent”. Kanamatsu et al., (1986) have shown that an intrastriatal injection of KA produces a biphasic response: an early decrease in the hippocampal ME-i.r. levels at 6-12 h post-injection followed by a rebound increase at 24 h post-injection. Thus, an additional objective of this study was to examine the time-course of QUIN-action and to determine whether the time profiles in the QUIN-injetted and uninjected hemispheres were similar. MATERIALS

AND

METHODS

Stereolaxic injections Stereotaxic injections were performed as described previously”. Briefly, male Sprague-Dawley rats (275-350 g) were anesthetized with halothane (Halocarbon, Malton, Ontario; 2% halothane, 98% oxygen via inhalation) and unilaterally injected (1 ~1 over a period of 1 min 42 s) in the striatum3’ with saline or QUIN. The following coordinates were used according to the atlas by Paxinos and Watson: 1.2 mm anterior to bregma, 3.0 mm lateral to bregma and 5.5 mm ventral to the surface of the skull with the incisor bar set at -3.3 mm. Infused drugs were dissolved in 0.9% saline and titrated to pH 7.0-7.3. Following the infusion, the injection cannula was left in place for 2 min to allow diffusion of the infused agents. Following a specified recovery period (see figure legends), the animals were killed for determinations of brain ME-i.r. or proenkephalin mRNA, or release experiments. Extraction of At specified days), animals removed. To striatum, two

ME-i.r. from brain times post-injection (2, 6, 12, 24, 48 h or 4, 7, or 14 were killed by decapitation and the brains quickly produce a coronal section of tissue containing the vertical cuts were made through the brain, one at the

level of the optic chiasm, and the second at a distance 3 mm ros-tral to the first cut. Similarly. two vertical cuts were made to produce a coronal section containing the globus pallidus. but the first cut was located 0.5 mm caudal to the optic chiasm while the second cut was located 2.0 mm caudal to the optic chiasm. The right and left striatal or pallidal sections were separated and extracted for enkephalin by boiling in 0.2 N HCI for 25 min followed by hand homogenization and centrifugation (I h. 8 “C) in a table top microfuge (Eppcndorf model 3200). The supcrnatants were stored at -70 “C until assayed for ME-i.r. using a radioimmunoassay (RIA. see below). Levels of protein in the extract samples were detcrmined using the Lowry protein assay”‘. Lcvcls of ME-i.r. in the various tissue sections were expressed as ng ME-i.r./mg protein present in the tissue. Measurement of proenkephalin mRNA Probes. The rat proenkephalin cRNA probe was generated from a 693 base-pair SphI-SmaI restriction fragment of a proenkephalin cDNA (generous gift of Dr. S. Sabol, NIH) subcloned into pGEM3 (Promega, Madison, WI). The probe was labelled using [“*P]UTP (ICN) according to the method described by Melton et al.” and resulted in a cRNA probe with a specific activity of 115,000 Ci/ mmol. Changes in the levels of cyclophilin mRNA were used as a marker for changes in global mRNA levels’, and were detected by probing with pl B1.5 (generous gift of Dr. J.G. Sutcliffe, Research Inst. of Scripps Clinic), a cDNA clone of the rat mRNA encoding cyclophilin. Norrhern blot analysis. Rats were killed by decapitation 7 days post-QUIN (72 nmol) injection. The left and right striata were dissected and immediately frozen on isopentane/dry ice. Tissue samples were kept frozen at -70 “C until RNA extraction. Total cellular RNA was extracted by the guanidinium isothiocyanate lysis method5 followed by LiCl precipitation4. Total RNA (10 kg) was fractioned on a 1.5% agarose-formaldehyde gel in HEPES (N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid) electrophoresis buffer. The RNA was transferred by capillary action to Nytran membranes (Schleicher and Schuell, Keene, NH) in 10 x SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). The membranes were dried overnight at room temperature (1 g) and the RNA was fixed to the membranes by long-wave U.V. irradiation. Following a prehybridization period of 2 h, the probe was added to a final concentration of at least 10” cpm/mI. The membranes were hybridized at 60 “C for 16-24 h in 50% formamide, 400 mM sodium phosphate (pH 7.2). 1 mM EDTA, bovine serum albumin (1 mg/mI) and 5% sodium dodecyl sulfate (SDS). After hybridization, the blots were washed at 70 “C for 90 min in 0.1 X SSC, 0.1 mM EDTA, 0.5% SDS and then exposed to X-ray film (Kodak XAR) with enhancer screens at -70 “C for 24-48 h. The optical density of each RNA band was determined using a Biorad Model 620 Video-densitometer. The levels of proenkephalin mRNA relative to the level of cyclophilin mRNA present were expressed as the proenkephalin mRNA/cyclophilin mRNA ratio. Release experiments One week post-injection. animals were killed by decapitation and their brains quickly removed. The right and left striatum or globus pallidus was dissected and sliced to 300 pm thickness using a McIlwain tissue chopper. Striatal or pallidal slices obtained from either the right or the left hemisphere were pooled from two animals for each experiment. The slices, continuously aerated with 95% O#% CO,, were equilibrated at 37 “C for a period of 40 min during which changes of buffer were carried out at 10 min intervals. The Krebs-Henseleit buffer, unless specified otherwise, was composed of (mM): NaCl 118, KCI 4.8, KH,PO, 1.2, MgSO, 1.2, CaCI, 2.5, NaHCO, 25, glucose 11, and contained thiorphan (I FM) and bestatin (1 BM) to minimize the enzymatic degradation of the released ME. Tissue slices were then incubated in Krebs buffer (3 ml) for 15 min periods to collect ME released from the tissue. A total of five serial release fractions was collected by transferring a small basket containing the brain slices from one collection vial to

119 another every 15 rain for a total collection time of 75 rain. The effeet of a high K + concentration on the release of ME was examined by exposing the tissue to a buffer containing 30 mM K + (isomolar replacement of NaC1 with KCi) during the third collection period. Collected release fractions were immediately poured into 5 ml polypropylene syringes connected to stopcocks and kept on ice. Fractions were applied onto Sep-pak Cls cartridges (Waters Associates, Milford, MA) that had been previously primed with 20 ml acetone followed by 50 ml glass distilled water. Samples on the cartridge were washed with 4% acetic acid (4 ml) and the enkephalins'ehited with acetone/0.2 N HCl (75:25, v/v, 3 ml). Under these conditions, the recovery of the eluted ME was approximately 90%. Eluates were evaporated in a Savant-speed concentrator (Model H100, Emerston Instruments, Scarborough, Ontario) and reconstituted with sodium phosphate buffer (0.1 M with 0.1% bovine serum albumin, pH 6.4) for analysis of ME-i.r. content by RIA (see below). Upon completion of the experiment, the tissue was gently blotted and weighed to determine its wet weight. The release of MEi.r. was expressed as pg ME-i.r./h/mg tissue weight. The overflow of ME-i.r. (i.e. the release in excess of the baseline release values) was expressed as a percentage of the baseline release.

Radioimmunoassay of methionine-enkephalin RIA's of ME were performed as described previously31. Briefly, anti-ME serum (1:3600 final dilution, 100/~1; INCSTAR, Stillwater MN) was incubated with [lz~I]ME (177--495/~Ci//~g; 100/A) and unlabelled samples (reconstituted release fractions or tissue extract samples diluted 1:35 in assay buffer; 200/A) in a total volume of 400 ?d sodium phosphate buffer (0.1 M with 0.1% BSA, pH 6.4) for 18-24 h at 4 °(2. The reaction was terminated at room temperature by the addition of rabbit v-globulin (2%, 100/M) and saturated ammonium sulfate (500/A). Tubes were vortexed and allowed to sit for 15-25 rain and were then centrifuged at 1200 g for 15 rain (Damon/IEC CRU-5000 centrifuge). Supematants were aspirated and the pellets counted in a Beckman Gamma-4000 counter for 1 rain (counting efficiency = 72%). Each sample was analyzed in duplicate. The interassay variability was 14.8% (n = 73). Crossreactivity of the antiserum was 2.8% with leueine-enkephalin. Sensitivity of the assay was 8 pg of ME-i.r. per assay tube (data not shown).

Drugs and chemicals QUIN and bestatin were obtained from Sigma (St. Louis, MO) D,L-Thiorphan was purchased from Bachem (Pittsburgh, PA). Acetone (HPLC grade) was supplied by BDH Chemicals (Toronto, Ont., Canada) and ME was obtained from the Armand-Frappier Institute (Laval, Que., Canada). All other chemicals were of reagent grade and were supplied by Sigma.

Statistical analysis Groups of data were compared using a one-way analysis of variance followed by a Newman-Keuls test, or a two-tailed Student's t-test, where appropriate. The levels of ME-i.r. or proenkephalin mRNA, and the release of ME-i.r. (in either brain hemisphere) following the intrastriatai infusion of QUIN were compared to the evels of ME-Lr. or proenkephaiin mRNA, and the release of MEi.r. in corresponding brain hemispheres following intrastriatal saline injections.

RESULTS

QUIN effects on proenkephalin m R N A levels Previously, the intrastriatal injection o f Q U I N was found to elevate the striatal and the pallidal ME-i.r. levels at seven days post-injection 31. To d e t e r m i n e w h e t h e r

this elevation reflected the e n h a n c e d synthesis of the p e p t i d e , the striatal levels of p r o e n k e p h a l i n m R N A were m e a s u r e d seven days after the intrastriatal injection of 72 nmol Q U I N , a dose previously found to p r o d u c e the maximal increase in ME-i.r. levels. The results of these experiments are p r e s e n t e d in Fig. 1. T h e concentrations of p r o e n k e p h a l i n m R N A were m a r k e d l y r e d u c e d in both the Q U I N - i n j e c t e d and the contralateral striatum when c o m p a r e d to the corresponding concentrations o b s e r v e d in saline-injected control animals (Fig. 1A). The reduction in the p r o e n k e p h a l i n m R N A in the injected striatum was a p p r o x i m a t e l y 77%, while that in the contralateral striatum was a p p r o x i m a t e l y 50%. H o w e v e r , as shown in Fig. 1B, the Q U I N injection also dramatically decreased the concentrations of cyclophilin m R N A , a m a r k e r of global m R N A levels 9, in b o t h the injected and the contralateral striatum when c o m p a r e d to those in the saline-injected control animals. The m e a n decrease in the cyclophilin m R N A in the injected striatum was approximately 46%, while that in the contralateral striatum was a p p r o x i m a t e l y 52%. H o w e v e r , when expressed relative to the total cyclophilin m R N A present (i.e. the p r o e n k e p h a l i n m R N A / c y c l o p h i l i n m R N A ratio), the p r o e n k e p h a l i n m R N A content in the Q U I N - i n j e c t e d , but not the contralateral striatum was significantly reduced when c o m p a r e d to that in the saline-injected a n imals (Fig. 1C). Thus, the Q U I N - i n d u c e d increases in the ME-i.r. content were not associated with increased striatal p r o e n k e p h a l i n m R N A levels.

QUIN effects on ME-i.r. release To d e t e r m i n e w h e t h e r the Q U I N - i n d u c e d increases in the striatal and the pallidal M E - i . r . levels were due to an impaired release of the p e p t i d e , the K + - e v o k e d release o f the p e p t i d e was e x a m i n e d following a Q U I N injection. A n i m a l s were given an intrastriatal injection of either saline o r Q U I N (72 nmol), sacrificed 7 days later and release experiments were then p e r f o r m e d on the striatal and the pallidal slices o b t a i n e d from these animals. R e p r e s e n t a t i v e striatal and pallidal release profries, o b s e r v e d in tissues o b t a i n e d from u n t r e a t e d animals, are shown in Fig. 2A. In the striatal tissue, the spontaneous release of ME-i.r. (i.e. the release occurring in the absence of K+-stimulation) ranged b e t w e e n 0.5-3.5 pg/h/mg tissue. In the presence of 30 m M K +, the release of ME-i.r. from the striatal slices increased 9-fold a b o v e the spontaneous release values (Fig. 2A). Following the termination of the K+-stimulus, the release values r e t u r n e d to the pre-stimulation levels in the fifth collected release fraction. In the pallidal tissue, the spontaneous release o f ME-i.r. ranged b e t w e e n 3.5-10.5 pg/h/mg tissue. In the presence o f 30 m M K ÷ the M E i.r. release increased 20-fold above the baseline release

120 values (Fig. 2A). A s was seen in the striatal release experiments, upon termination of the K+-stimulus, the ME-i.r. release values returned to prestimulation levels in the fifth release fraction.

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Fig. 1. Effects of intrastriatal quinolinate (QUIN, 72 nmol) injections on the striatal levels of proenkephalin mRNA and cyclophilin mRNA. A: Northern blot analysis of proenkephalin mRNA levels at 7 days following the intrastriatal injection of saline (S) or QUIN (Q). Individual lanes show levels of proenkephalin mRNA in the injected (I) or the uninjected (U) striatum. The mean optical density readings (relative units) were: saline injection (injected striatum, 9.37 -+ 1.71; uninjected striatum, 8.86 -+ 0.83, mean -+ S.E.M., n = 3) and QUIN injection (injected striatum, 2.15 -+ 0.68; uninjected striatum, 4.48 -- 1.29, mean -+ S.E.M., n = 3). B: Northern blot analysis of cyclophilin mRNA levels at 7 days following the intrastriatal injection of saline (S) or QUIN (Q). The blot shown in (A) was stripped and reprobed for cyclophilin mRNA. Individual lanes show levels of cyclophilin mRNA in the injected (I) or the uninjected (U) striatum. The mean optical density readings (relative units) were: saline injection (injected striatum, 8.45 -+ 1.73; uninjected striatum, 7.61 -+ 1.99, mean -+ S.E.M., n = 3) and QUIN injection (injected striatum, 4.55 -+ 2.03; uninjected striatum, 3.67 - 0.89, mean --- S.E.M., n = 3). C: levels of proenkephalin mRNA expressed as a ratio of the cyelophilin mRNA present at 7 days following an intrastriatal injection of saline or QUIN. Levels of mRNA were determined in both the uninjected (open bars) and the injected (hatched bars) striata. Each point is the mean -+ S.E.M. of 3 separate determinations. *P < 0.05 when compared to the corresponding saline-injected striatum. Ap < 0.05 when compared to the corresponding uninjected striatum (one-way ANOVA, Newman-Keul's test).

Similar release profiles (including spontaneous release values) were observed using the striatal and pallidal tissues obtained from the saline- or the Q U I N - i n j e c t e d animals (data not shown). The exposure of the saline-injected or contralateral striatal slices to 30 mM K ~ p r o d u c e d an approximate 8-fold increase in the ME-i.r. release above the spontaneous release values (Fig. 2B). Similarly, 30 m M K + e v o k e d a 6-fold increase in the ME-i.r. release from the Q U I N - i n j e c t e d or the contralateral striatal slices, a response which did not differ statistically from that e v o k e d from the saline-injected and contralateral striatal tissues (Fig. 2B). Thus, the intrastriatal infusion of Q U I N did not influence the spontaneous or the K + - e v o k e d release of ME-i.r. from the striatum in either hemisphere. In the globus pallidus contralateral and ipsilateral to the intrastriatal saline-injection, 30 m M K + induced an 18- and a 14-fold increase in the ME-i.r. release above the baseline release values (Fig. 2C). Similar release responses were observed using the pallidal tissue obtained from the Q U I N - i n j e c t e d animals. The 30 m M K+-stim ulated release of ME-i.r. from the contralateral and ipsilateral pallidal slices represented an 18- and a 10.5-fold increase above the spontaneous release, respectively (Fig. 2C). Neither the ipsilateral nor the contralaterai release response differed statistically from that measured in the corresponding pallidum obtained from the salineinjected animals. Thus, as was seen in the striatal release experiments, the Q U I N injection, when c o m p a r e d to the saline injection, did not influence the spontaneous or the K+-induced pallidal release of ME-i.r from either hemisphere. H o w e v e r , unlike the striatal ME-i.r. release response, the Q U I N - i n j e c t i o n significantly reduced the ME-i.r. release in the ipsilateral, when c o m p a r e d to the contralateral globus pallidus (Fig. 2C). To d e t e r m i n e whether the M E - i . r . release response in the tissues o b t a i n e d from the Q U I N - i n j e e t e d animals refleeted a Ca2+-dependent process, experiments were conducted to examine the release in the absence of external Ca z+. In this regard, release experiments were p e r f o r m e d exactly as described previously, except that t h e striatal or the pallidal slices were incubated in K r e b s - H e n s e l e i t buffer from which Ca z+ had been omitted. The results of these experiments are shown in Fig. 2D. Omission of Ca 2+, while not influencing the spontaneous ME-i.r. release from the striatum or the globus pallidus of either hemisphere (data not shown), depressed the 30 m M K+-stimulated ME-i.r. release from all the tissue preparations by more than 98% (Fig. 2D). Thus, at seven days following an intrastriatal Q U I N - i n jection, the stimulated ME-i.r. release from the striatum and the globus pallidus of both hemispheres was found to be highly Ca2+-dependent.

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Fig. 2. The K+-evoked release of met-enkephalin-like immunoreactivity (ME-i.r.) from striatal or pallidal slices. A: representative ME-i.r. release profiles observed in the striatum (dotted line) and globus pallidus (solid line) obtained from untreated animals. Following a 40 min equilibration period in Krebs-Henseleit buffer, 5 × 15 min release fractions were collected. The release of ME-i.r. was stimulated by exposing the tissue slices to 30 mM K + during the third collection fraction. The mean K+-induced ME-i.r. overflows (i.e. the release in excess of the baseline release, expressed as a percentage of this release) were: striatum (944.1 - 225.9%, mean S.E.M., n = 4) and globus pallidusl (2052 -+ 410.6%, mean ± S.E.M., n = 4). Abscissa: time of incubation (minutes)• Ordinate: release of ME-i.r. expressed as pg/mg tissue/h.

B: release of ME-i.r. from striatal tissue obtained from animals at 7 days following an intrastriatal injection of saline (Sal) or quinolinate (QUIN, 72 nmol). Experimental conditions were as described above. The release of ME-i.r. was measured in both the injected (hatched bars) and the uninjected hemispheres (open bars). Ordinate: overflow of ME-i.r. expressed as a percentage of the baseline release. Each point is the mean - S.E.M. of 5-6 separate observations using 10-12 animals. *P < 0.05 when compared to the contralateral release response. C: release of ME-i.r. from pallidal tissue obtained from animals at 7 days following an intrastriatal injection of Sat or QUIN (72 nmol). Experimental conditions were as described in (A). The release of ME-i.r. was measured in both the ipsilateral (hatched bars) and the contralateral hemispheres (open bars). Ordinate: overflow of ME-i.r. expressed as a percentage of the baseline release. Each point is the mean -+ S.E.M. of 5-6 separate observations using 10-12 animals. *P < 0•05 when compared to the contralateral release response. D: effect of Ca 2+ omission on the release of ME-i.r. from striatal and pallidal tissue obtained from animals at 7 days post-QUIN-injection. Experimental conditions were as described in A, except that Ca 2+ was omitted from the incubation buffers for the duration of the experiment. The release of ME-i.r. was measured in both the ipsilateral (filled bars) and the contralateral (open bars) hemispheres. Abscissa: presence or absence of Ca 2÷. Ordinate: overflow of ME-i.r. expressed as a percentage of the baseline release. Each point is the mean +-- S.E.M. of 4-6 observations using 8-12 animals.

Time profile of QUIN action To d e t e r m i n e w h e t h e r the excitotoxin-induced changes in the ME-i.r. levels were t i m e - d e p e n d e n t , experiments were conducted in which rats were sacrificed at different times (2, 6, 12, 24, 48 h and 4, 7 and 14 days) following the intrastriatal injection of Q U I N (72 nmol). The resuits of these experiments are shown in Figs. 3 and 4. In the injected striatum, the ME-i.r. levels m e a s u r e d at 2 h post-infusion were r e d u c e d to a p p r o x i m a t e l y 65% of those d e t e r m i n e d in the saline-injected control animals (Fig. 3). A t 6, 12 and 24 h post-injection, the striatal M E - i . r levels rose steadily to a p p r o x i m a t e l y 85% of the control levels. A t 48 h and 4 days post-injection, the ME-i.r. content o f the injected striatum was c o m p a r a b l e to that d e t e r m i n e d in the saline-injected animals. The p e a k response to the Q U I N injection, reflecting an app r o x i m a t e 200% increase in the ME-i.r. content, oc-

curred at 7 days following the injection (Fig. 3). A t day 14 post-injection, the ME-i.r. content had r e t u r n e d to control levels. A similar time profile was o b s e r v e d in the uninjected striatum, however, in this striatum, the M E i.r. levels did not decline below those m e a s u r e d in the saline-injected control animals during the first 24 h period (Fig. 3). In the ipsilateral globus pallidus, the ME-i.r. content m e a s u r e d at 2 h post-infusion was similar to that determined in the saline-injected control animals (Fig. 4). Thus, the ipsilateral globus pallidus did not show the early decrease in the ME-i.r. levels a p p a r e n t in the injected striatum. H o w e v e r , at 6 and 12 h post-injection, the pallidal M E - i . r . t e n d e d to decrease slightly to 87.0 --- 14.0% (mean - S . E . M . , n = 5) and 80.0 --- 10.8% ( m e a n - S . E . M . , n = 5) of the control values, respectively. A t 24 and 48 h following the Q U I N injection, the

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Time (hours and days) Fig. 3. Time profile of the met-enkephaiin-like immunoreactivity (ME-i.r.) levels in the striatum obtained following the intrastriatal injection of quinolinate (QUIN, 72 nmol). Rats were unilaterally injected in the striatum and sacrificed at the designated times following the injection. Levels of ME-i.r. were determined in both the uninjected (open symbols) and the injected (filled symbols) hemispheres. Abscissa: time post-injection (in hours and days). Ordinate: level of ME-i.r. expressed as a percentage of the level of ME-i.r. in the saline-injected (control) animals. The striatai levels of ME-i.r. in response to an intrastriatal saline injection ranged from: uninjected striatum (4.6-6.6 ng/mg protein) and injected striatum (3.7-4.7 ng/mg protein), regardless of the time post-injection, Each point is the mean of 4-6 separate observations. Standard errors have been omitted for clarity, but were less than 20% of the mean value. *P < 0.05 when compared to the saline-injected (control) animals.

ME-i.r. levels were slightly greater than those measured in the saline-injected animals. A t day 4 post-injection, the ME-i.r. levels in the ipsilateral globus pallidus were significantly elevated (156%) above those measured in the saline-injected animals, and reached a peak (255%) at 7 days post-injection (Fig. 4). As was seen in the striatal profile of QUIN-action, at day 14, the pallidal MEi.r. levels had returned to control values. A similar time profile was observed in the contralateral globus pallidus (Fig. 4). Thus, the QUIN-induced changes in the MEi.r. levels in both the striatum and the globus pallidus were time-dependent. DISCUSSION The present study shows that the unilateral microinjection of Q U I N into the striatum produces a bilateral and time-dependent elevation in the ME-i.r. levels in the striatum and the globus pallidus. This finding is consistent with the results of two other pharmacological studies 31"32. Previously, it was found that Q U I N produced a dose-related and antagonist-sensitive increase in the striatal and the pallidal ME-i.r. levels, suggesting that the

50-

Time (hours

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Fig. 4. Time profile of the met-enkephalin-like immunoreactivity (ME-i.r.) levels in the globus pallidus obtained following the intrastriatal injection of quinolinate (QUIN, 72 nmol). Rats were unilaterally injected in the striatum and sacrificed at the designated times following the injection. Levels of ME-i.r. were determined in both the contralateral (open symbols) and the ipsilateral (filled symbols) globus pallidus. Abscissa: time post-injection (in hours and days). Ordinate: level of ME-i.r. expressed as a percentage of the level of ME-i.r. in the saline-treated (control) animals. The pallidal levels of ME-i.r. in response to an intrastriatal saline injection ranged from: contralateral pallidum (12.6-19.7 ng/mg protein) and ipsilateral pallidum (13.8-19.9 ng/mg protein), regardless of the time post-injection. Each point is the mean of 4--6 separate observations. Standard errors have been omitted for clarity, but were less than 20% of the mean value. *P < 0.05 when compared to the saline-injected (control) animals.

excitotoxin mediated its effect via the activation of an E A A receptor 31. Moreover, in a separate study, it was shown that the QUIN-induced rise in the contralateral ME-i.r. levels was attenuated by the injection of kynurenic acid, a non-selective E A A receptor antagonist, into the striatum contralateral to the QUIN-injection, suggesting that the QUIN-elicited contralateral response may also be due, in part, to the activation of E A A receptors by an endogenously liberated E A A 32. Although it has previously been demonstrated that E A A receptors are involved in the excitotoxin-induced elevations in ME-i.r. 31"32, the cellular mechanisms contributing to this effect remain unknown. Previous work on the rat hippocampus has suggested that an enhanced enkephalin biosynthesis may contribute to the excitotoxin-action 18. H o n g et al. 16 reported that the administration of the protein synthesis inhibitor, cyeloheximide, 6 h after a single intrahippocampal injection of K A , attenuates the KA-induced increase in the hippocampai MEi.r. Subsequently, it was shown that this elevation was associated with a marked increase in the levels of proenkephalin m R N A ~s. However, in the present study Q U I N injection into the striatum, while elevating ME-i.r., did not produce an increase in the striatal proenkephalin

123 mRNA levels. Indeed, QUIN was found to deplete the concentrations of mRNA for proenkephalin and cyclophilin in both the injected and the contralateral striatum, suggesting that this excitotoxin produces a generalized depletion in the striatal mRNA levels. In the injected striatum, the depletion in cyclophilin mRNA may be due to a QUIN-induced neuronal cell loss that was evident in an histological assessment carded out in a previous study31. In that study, the intrastriatal administration of QUIN, at a dose employed in the present investigation, produced an extensive lesion in the injected but not the uninjected striatum 31. Thus, QUIN-induced tissue damage may account, in part, for the depletions in cyclophilin mRNA observed in the injected striatum. Surprisingly, a decrease in cyclophilin mRNA was also observed in the contralateral striatum in which no histological evidence of cell loss was apparent. One explanation for this paradoxical observation is that in the contralateral hemisphere, QUIN may cause a subtle dysfunction (such as an altered transcription, peptide translation or peptide disposition) of neurons that is not reflected in a conventional histological assessment 31. However, while the concentrations of proenkephalin and cyclophilin mRNA decreased in both the injected and the contralateral striatum, a significant reduction in proenkephalin mRNA relative to cyclophilin mRNA occurred only in the injected striatum. This result is consistent with the unilateral nature of the QUIN-induced histological damage reported previously31, and suggests that QUIN attenuates enkephalin biosynthesis primarily in the injected striatum. The relative decrease in the proenkephalin mRNA, more so than the reduced proenkephalin mRNA concentration, may reflect the loss of enkephalin-containing neurons in the injected striatum. It would appear that the dramatic elevations in the ME-i.r. are not the result of an increased gene transcription. However, the possibility that those enkephalinergic neurons not adversely affected by the excitotoxin may compensate for any dysfunctioning neurons in the contralateral hemisphere and the lost neurons in the ipsilateral hemisphere, by generating increased proenkephalin mRNA levels, cannot be excluded. Such increases in the proenkephafin mRNA at the individual cell level would be consistent with the immunohistochemical evidence presented in otlier studiesTM and would not be detected in assays of global proenkephaiin mRNA levels, Alternatively, the QUIN-induced elevations in ME-i.r. may occur via an increased rate of proenkephalin mRNA translation and/or an altered disposition (degradation or release) of the peptide. Indeed, the inhibition of enkephalin degradation by 'enkephalinase' inhibitors has been found to enhance the striatal ME-i.r. levels 36. Moreover, the intrastriatal injection of KA has been shown to decrease

the activity of 'enkephalinase' in the striatum 23. The possibility that the elevations in ME-i.r. resulted from an impaired peptide release was suggested by the finding that certain pharmacological treatments (e.g. opioid receptor agonists 14'15'35 and GABA-mimetic 11 drugs) appear to elevate brain enkephalin levels while impairing its release. In the present study, this possibility was examined by evaluating the K+-evoked ME-i.r. release from the QUIN-injected and contralateral hemispheres in brain slice experiments. These experiments showed that the QUIN-induced elevations in ME-i.r. were not due to an altered release of the peptide. This conclusion is based on the following observations: (1) The intrastriatal QUIN injection, when compared to a saline-injection, bilaterally increased the striatal ME-i.r. levels, but failed to impair the ME-i.r. release in either hemisphere. (2) Similarly, the QUIN-injection, when compared to a saline-injection, also bilaterally increased the pallidal ME-i.r. levels, but did not influence the ME-i.r. release in either hemisphere. (3) Although the QUIN-injection significantly reduced the ME-i.r. release in the ipsilateral, as compared to the contralateral globus pallidus, this singular finding does not convincingly argue that the QUIN-induced increases in ME-i.r. levels are due to an impaired peptide release. The finding that a 'normal' release response occurred in the excitotoxin-damaged striatum raised the question of whether this response corresponded to the Ca2+-dependent release seen in the normal tissue. The present experiments clearly demonstrated that the K+-stimulated ME-i.r. release from the striatum and globus pallidus of both hemispheres was indeed highly Ca2+-dependent, suggesting that it most likely originated from the enkephalinergic neurons. Interestingly, the finding that a 'normal' release response occurred despite a substantial reduction in the proenkephalin mRNA levels, suggests that the enkephalinergic neurons which survived the excitotoxin-induced damage may be compensating with an increased content and release of the peptide. Indeed, it has been shown in two immunohistochemical studies that the residual striatal enkephalinergic neurons in the KAinjected striatum contain an increased amount of enkephalin-immunoreactive material 3,34. The study of the cellular mechanisms involved in the excitotoxin-action is complicated by the finding that the excitotoxin-induced changes in ME-i.r. are highly timedependent: maximal elevations in the striatal and the pallidal ME-i.r. levels of both hemispheres occurred at 7 days following the QUIN injection. However, the time profiles observed in the injected and the contralateral striatum differed in one respect. At 2 h post-injection, the ME-i.r. level in the injected, but not the contralat-

124 eral striatum, was significantly reduced when compared to that measured in control animals. The early reduction in the ME-i.r. level, which resembles that seen in the hippocampus following an intrastriatal KA injection ts, may have been due to a depletion of the peptide from neuronal stores following the QUIN-induced excitation of the enkephalinergic neurons. Thus, the delayed increase in the ME-i.r. level seen at seven days may reflect the initiation of intracellular mechanisms triggered to compensate for the early decrease in the peptide level TM. Kanamatsu et al. is have suggested that the early decrease in the hippocampal ME-i.r. level seen following an intrastriatal KA injection induces a compensatory increase in peptide production such that, at later time points, the peptide level exceeds the control level. However, this explanation is apparently inconsistent with observations on the contralateral striatum, in which the delayed increase in the ME-i.r. level was not preceded by an early decrease. The mechanism(s) underlying the delayed increases in ME-i.r. are unknown, but may involve early changes in peptide biosynthesis, the consequences of which only become apparent at later time points. Although the early decreases in ME-i.r. induced by Q U I N in the injected striatum resemble those observed in the hippocampus after a unilateral KA injection 18, the time profile of the QUIN-action in the basal ganglia nuclei differs from that of the KA-action in the hippocampus. In the striatum and globus pallidus, the maximal increase in the ME-i.r. level occurred at 7 days post-injection, while in the hippocampus this occurred at 48 h post-injection. In the latter region this elevation was maintained for two weeks TM, while the striatal and pallidal ME-i.r. levels returned to control values at two weeks post-excitotoxin-injection. The differences in the QUIN- and KA-actions in the basal ganglia and hippocampus, respectively, may be due to differences in the inherent excitability of these brain regions 2° or to the modulation of enkephalinergic cell function via different E A A receptor mechanisms. Although the peak elevation in the striatal and the pallidal ME-i.r. levels occurred at 7 days post-injection, a comparison of the regional time profiles of QUIN-action revealed two differences. First, the changes in the striatal ME-i.r. content within the first 12 h post-injection differed markedly from the changes in the pallidal ME-i.r. during the same time period. While the ME-i.r. content of the QUIN-injected striatum rose steadily during this period, the pallidal levels of ME-i.r. tended to decrease slightly when compared to the ME-i.r. levels measured at the 2 h time point. Second, in the globus pallidus, but not in the striatum, a small but significant

elevation in ME-i.r. was observed at four days postQUIN-injection. Although the reason for these differences in the regional profiles of the QUIN-action is not known, they may arise from the different neuronal environments of the enkephalinergic elements in the striatum and the globus pallidus e6. Thus, the heterogeneous distribution of excitatory and inhibitory neurotransmitters in the striatum and the globus pallidus may be a contributing factor in the differential profile of Q U I N action at the striatal and the pallidal levels 13. In summary, the present pharmacological study shows that the intrastriatal microinjection of Q U I N produces a bilateral and time-dependent increase in the striatal and the pallidal ME-i.r. levels, a response which may reflect adaptation to neuronal injury and is possibly unique to the opioid-containing neurons. Indeed, while excitotoxin-injections have been demonstrated to elevate brain levels of met-enkephalin 16'18'31'32 and dynorphin A TM, no such increases have been reported to occur in other neuropeptide systems, including somatostatin, neuropeptide Y, substance P and vasopressin 1-3. Although the role of opioids in the excitotoxic phenomenon is not known, it has been suggested that these compounds, in view of their neuro-inhibitory nature, may act as endogenous anti-excitatory agents and thereby serve a potentially neuroprotective function in the brain 31. In conclusion, the QUIN-induced increases in the ME-i.r. are apparently not associated with an increase in the levels of proenkephalin m R N A or an impaired release of the peptide. However, the results of this study do not exclude the possibility that the increased levels of ME-i.r. are due to an enhanced rate of proenkephalin m R N A translation or a reduced rate of enkephalin degradation. Although the QUIN-induced increases in the striatal and pallidal ME-i.r. levels appear not to resemble the enkephalin deficit associated with a progressed stage of HC 12"24, they seemingly are consistent with a recent report of an increased number of striatal enkephalin-immunoreactive neurons and an increased content of enkephalin-immunoreactivity within striatal neurons in a human subject suffering from severe striatal atrophy without the expression of chorea 33. Thus, the excit0toxin-induced changes in ME-i.r., which occur in the presence of histologically identifiable lesions, may more closely model the changes in enkephalin observed in the presymptomatic stages of HC.

Acknowledgements. This work was supported by the Medical Research Council of Canada, NIDA (Grant DA02265), and NIMH (Grant MH 42251). We would like to thank Drs. Sabol (NIH) and Sutcliffe (Research Institute of Scripps Clinic) for the generous gifts of the proenkephalin and cyclophilin eDNA, respectively.

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