BrainResearch, 380(1986)309-316 309 Elsevier

BRE 11931

Intracerebral Quinolinic Acid Injection in the Rat: Effects on Dopaminergic Neurons

SILVIO MAZZARI t, CATERINA ALDINIO a, MAURO BECCARO 1, GINO TOFFANO t and ROBERT SCHWARCZ 2

1F1DIA Research Laboratories, Abano Terme (Italy) and 2Maryland Psychiatric Research Center, Baltimore, MD21228 (U.S.A.)

(Accepted December 31st, 1985)

Key words: quinolinic acid - - dopamine - - Huntington's disease - - basal ganglion

Intrastriatal infusion of the endogenous excitotoxin quinolinic acid (QUIN) leads to the degeneration of neuronal cell bodies around the injection site. Dopaminergic afferents not only survive the toxic insult but react by increasing their activity in the acute and suba- cute phases following the injection of QUIN. Measurements of the tissue concentrations of acidic dopamine metabolites, and determi- nations of L-DOPA accumulation after DOPA-decarboxylase inhibition, indicate an increased dopamine turnover within 90 min after the administration of 50 f~g QUIN. At the later timepoints examined (6 h, 4 and 11 days after QUIN), dopaminergic parameters are increased in the injected striatum only while no changes can be detected in the homolateral substantia nigra. Local norepinephrine lev- els are elevated 4 and 11 days after an intrastriatal QUIN injection but remain unchanged at distant sites or earlier postinjection peri- ods. The acute increase in nigrostriatal activity may be mediated by an excessively stimulated, yet functional striatonigral feedback loop whereas subsequent changes represent local reactions of dopaminergic nerve terminals secondary to neuronal degeneration in the striatum. In accordance with this interpretation, no monoaminergic changes can be observed in the hypothalamus 4 days following the local injection of 50 itg QUIN, a dose which does not cause neuronal necrosis in this brain area. These data are concordant with, and are discussed in the context of, a possible involvement of QUIN in the pathogenesis of Huntington's disease.

INTRODUCTION

Intrastriatal injections of the endogenous excito-

toxin quinolinic acid (QUIN) in the rat have been

shown to cause selective lesions of a circumscribed

area around the inject ion site and provide an animal

model for the human neurodegenerat ive disorder,

Hunt ington 's disease (HD) 3°. By inference, a

dysfunctional brain Q U I N system has been speculati-

vely linked to the pathogenesis of HD 26.

An examination of a possible etiological relation-

ship between a pathological overabundance of

QUIN and the precipitation of HD pathophysiology

also ought to include an assessment of nigrostriatal

dopamine (DA) neurons. While this pathway, in con-

trast to the majori ty of striatal cell bodies, clearly

does not degenerate in HD 37, a D A component of the

disease is indicated by both clinical and biochemical

observations. Moreover, exogenous excitotoxins

such as kainic acid have been shown to exert both

acute and chronic influences on nigrostriatal D A

neurons 2'3"1°'24'2s'36'38'41. In view of the presence of

the t ryptophan metaboli te Q U I N in the brain 4°, of

possible differences between the effects of kainate

and QUIN 12'26 and of the possible link between

QUIN and the pathogenesis of the human dis-

ease 26'3°, the present study was therefore designed to

examine the neurochemical effects of intrastriatal

QUIN injections on striatal and nigral D A para-

meters. In separate experiments, QUIN injections

were made, and monoaminergic systems subsequent-

ly assessed, in the arcuate nucleus of the hypothala-

mus, a brain area which shows particular resistance

to the neurotoxic actions of Q U I N 29.

A prel iminary account of this work has been pub-

lished 23.

MATERIALS AND METHODS

Male Sprague-Dawley rats (150-250 g) were used

in all experiments. Unilateral microinjections into

the striatum (coordinates according to the stereotax-

Correspondence: S. Mazzari, FIDIA Research Laboratories, Via Ponte della Fabbrica 3/A, 1-35031 Abano Terme, Italy.

0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

310

ic atlas of K6nig and Klippel2°: A 7.9, L 2.6, V 4.8 be- low the dura) and arcuate nucleus of the hypothala- mus (A 7.3, L 0.3, V 9.8) of anesthetized rats were made as described previously 29'3°. Solutions (pH 7.4) of QUIN (Sigma Chemical Co., St. Louis, MO) or

phosphate-buffered saline (PBS) were infused in a volume of 1 pl over a period of 2 rain.

At various timepoints after the operation (90 min, 6 h, 4 or 11 days), animals were killed by decapitation and the brain areas of interest rapidly dissected and frozen on dry ice. Samples were sonicated in 300-500 /~1 0.1 M HC104 and the homogenates centrifuged (28,000 g, 15 min). A portion of the supernatant was applied directly to a high performance liquid chro-

matography (HPLC) system and the levels of seroto- nin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and 3,4-dihydroxyphenyl- acetic acid (DOPAC) measured according to the method of Sperk 3s. A portion of the same superna- tant was used for the HPLC analysis of catechol- amines al. Protein was determined in the pellet ac- cording to the method of Lowry ~1 using bovine serum albumin as a standard.

DA-turnover was measured using m-hydroxyben- zylhydrazine dihydrochloride (NSD 1015; 100 mg/kg, i.p,) as an inhibitor of DOPA-decarboxyl- ase 6. Tissue levels of dihydroxyphenylalanine (DOPA) were measured on supernatant samples

r e ~g

Fig. 1. Photomicrographs depicting striatal neurons at various timepoints following an intrastriatal injection of 50 ¢tg QUIN. A, con trol striatum; B, 90 rain; C, 6 h; D, 4 days after the injection. Bar: 120/~m.

311

(prepared as above) according to the me thod of Shum et al. 32.

Light microscopic analysis of bra in tissue was per-

formed on 30 ~ m thionin-s ta ined cryostat sections

following perfusion of the rats with a solut ion con-

taining 2% pa ra fo rma ldehyde and 1% glutaralde-

hyde.

RESULTS

Morpho logy

The progress ion of neurona l degenera t ion with

t ime following the intrastr ia tal inject ion of 50 ktg

Q U I N is depic ted in Fig. 1. Only minor morphologi -

cal changes could be de tec ted 90 min following the in-

ject ion (Fig. 1B, cf. control s t r ia tum in A) . A t 6 h,

the beginning degenerat ive process was clearly evi-

dent at the light microscopic level. Disp lacement of

Nissl-substance and re la ted chromatolyt ic changes

resulting in great ly reduced staining of the degener-

ating neurons could be observed throughout the af-

fected area (Fig. 1C). On pos topera t ive day 4, nerve

cell degenera t ion was comple te , yielding the typical

appearance of a striatal exci totoxic lesion (Fig. 1D).

A virtually identical micrograph (not shown) was ob-

ta ined 11 days after Q U I N applicat ion. • "; '9 In confi rmat ion of a previous study" , no abnormal

appearance of the neuropi l was observed 4 days after

the in t rahypotha lamic inject ion of 50 g Q U I N (mi-

crograph not shown).

Behavior

No strict behaviora l assessment was pe r fo rmed in

the course of the present study. However , all animals

were observed for behaviora l response for 3 - 4 h af-

ter intrastr iatal Q U I N adminis t ra t ion. In accordance

15o

IOO

50

L

a k

. . . . .

90min 6hr 4d lid Fig. 2. DA levels in the homolateral striatum (hatched bars) and substantia nigra (open bars) at various timepoints follow- ing an intrastriatal injection of 50 pg QUIN. Data are ex- pressed as a percentage of respective PBS-injected controls and are the mean + S.E.M. of 9-10 animals at each timepoint. * P < 0.01 vs controls, Dunnett's test.

with a previous repor t 3°, rats injected with 20-100 ttg

Q U I N showed in termi t tent ton ic -c lon ic movements

of the contra la tera l forepaw, combined with occasio-

nal bar re l - ro ta t ion upon awakening from anesthesia•

Biochemis try

Intrastriatal injections. In initial exper iments , the

effects of an intrastr iatal inject ion of Q U I N on local

D A paramete r s were examined 4 days pos topera-

tively, a t imepoint at which all striatal cell bodies vul-

nerable to a given dose of the toxin had degenera ted

(as judged at the light microscopic level; see above).

In a dose-dependen t manner , the tissue content of

D A , D O P A C and H V A increased with increasing

doses of Q U I N , the metabol i te levels being partic-

ularly severely affected (505% of control for

D O P A C , 296% of control for H V A at 100 ~g Q U I N ,

Table I).

TABLE I

DA, DO PA C and HVA levels in the ipsilateral striatum 4 days after an intrastriatal Q UIN injection

Values are expressed as pmol/mg protein and are the mean + S.E.M. from 5-6 separate animals.

DA D OPA C HVA

Control (PBS) 553.6 + 25.4 58.6 + 5.8 41.6 + 3.5 QUIN 5#g 553.8 + 46.5 62.3 + 7.2 34.5 + 2.8 QUIN 20ktg 642.6 + 30.9 97.3 + 13.2 55.4 + 9.3 QUIN 50ttg 648.4 + 18.7" 157.6 + 10.8" 88.8 + 6.1" QUIN 100ttg 758.5 _ 62.6* 295.8 + 42.7* 123.0 + 18.1"

* P < 0.01 vs PBS-injected striata (Dunnett's test).

312

o

<~ "~ 1.50 > • "r ,.-

0

,oo

25C

200 2

.__~j I ....

,~ **

90min 6hr 4d lid

m

o o E

o

3 0 0

250

200

I O0

50

0 1 -1-

~-~, -r __. ,-~

,-q

90min 6hr 4d II d

Fig. 3. Striatal H V A and D O P A C levels at various timepoints following an intrastriatal injection of 50/~g QUIN. Data are ex- pressed in relation to respective PBS-injected controls and are the mean _+ S.E.M. of 9-10 animals at each timepoint. Hatched bars, injected side; open bars, contralateral side. ** P < 0.001 vs controls, Dunnet t ' s test.

The chronological development of dopaminergic changes following an intrastriatal injection of 50 ~tg QUIN is depicted in Figs. 2-4. In the striatum, signi- ficant increases in DA levels were detected at the 4- day timepoint only (Fig. 2). In contrast, the more pronounced elevations in the levels of DOPAC and HVA were observed as soon as 6 h after QUIN appli- cation (but not after 90 min) and remained constant for at least 11 days (Fig. 3A, B). No significant changes were noted in the contralateral striatum at any postoperative timepoint.

A qualitatively different picture was obtained in the substantia nigra. Significant increases in the lev- els of DOPAC and HVA were noted after 90 rain only and for both metabolites were detected in the contralateral substantia nigra as well (Fig. 4A, B). Beside a modest decrease in DA levels on the in- jected side after 6 h, no changes in nigral neurotrans- mitter content were noted following intrastriatal

QUIN injection (Fig. 2). Norepinephrine (NE) levels in the injected stria-

turn increased by 87 _+ 8% (n = 9; P < 0.01) and 43 + 13% (n = 10; P < 0.05), respectively, 4 and 11 days following an intrastriatal injection of 50 ktg QUIN (control levels 4 days after a PBS injection: 3.8 + 0.1 pmol/mg protein; n = 9). No such changes were noted at earlier timepoints, and in the contralateral striatum and the substantia nigra (data not shown). No changes in the striatal or nigral levels of 5-HT could be detected 4 days after an identical intrastria- tal QUIN injection (cf. also ref. 1).

DA-turnover in both the striatum and substantia nigra was assessed 90 rain and 4 days following an in- trastriatal injection of 50 #g QUIN. As compared to PBS-injected controls, D O P A accumulation after DOPA-decarboxylase inhibition was significantly in- creased in the injected and contralateral striatum and in the ipsilateral substantia nigra 90 rain after the op- eration (Table II). After 4 days, the only significant (P < 0.05) change detected was a 44% increase in the lesioned striatum (n = 5, data not shown).

Intrahypothalamic injections. No changes in hypo- thalamic DA, NE, 5-HT and 5-HIAA levels were ob- served 4 days after the local injection of 50 ktg QUIN (Table III). A slight decrease in DOPAC content was noted in the injected hypothalamus but is difficult to interpret (and may be of negligible biological signifi- cance) in view of the low concentration of the meta-

313

"FABLE If

DOPA accumulation in the striatum and substantia nigra 90 rain after the intrastriatal injection of 50 #g QUIN

Animals were sacrificed 30 rain after the i.p. administration of 100 mg/kg NSD 1015. Values (mean ± S.E.M.) are expressed as pmol/mg protein. The number of separate animals in each group is indicated in parentheses.

Injected side Contralateral side

Striatum PBS 33.0 + 1.4 (9) 35.6 ± 2.3 (7) QUIN 65.2 ± 3.7** (8) 46.3 + 1.6" (9)

Substantia nigra PBS 14.1 ± 0.9 (10) 21.5 ± 1.0 (10) QUIN 32.5 ± 2.5** (8) 23.7 ± 1.7 (9)

* P < 0.01 as compared to the homolateral side of PBS-in- jected control.

** P < 0.001 animals (Student's t-test).

bo l i te in this bra in reg ion (close to the de t ec t ion l imit

o f the m e t h o d ) . H V A levels were no t de t ec t ab l e in

the hypo tha l amus .

DISCUSSION

Fol lowing the first r epor t on se lec t ive n e u r o n a l de-

g e n e r a t i o n caused by the in t ras t r ia ta l in jec t ion of the

e x o g e n o u s exc i to tox in kainic acid s, w o r k in severa l

l abora to r i e s has shown tha t D A af ferents are no t

only spared (hence ' axon- spa r ing ' exc i to tox ic le-

sions) in the l e s ioned a rea bu t reac t to the loss of the i r

pos t synap t ic ta rge t neu rons by ini t ial ly inc reas ing

the i r activity. E v i d e n c e for this p h e n o m e n o n , which

has b e e n t en ta t ive ly i n t e r p r e t e d as a c o m p e n s a t o r y

m e c h a n i s m in r e sponse to the loss of s tr iatal dopa -

mine recep to r s , has c o m e p r imar i ly f r o m b iochemica l

A

zoo

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> IOO - - " ~',~

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I

90min 6hr 4d

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.-q

.-q

l i d

B 250

200 I

150 ~-**

o ~

.~ Joo - - ~ . . . . . . . . . . . . . .

5 o

90rnin 6 hr 4d II d

Fig. 4. Nigral HVA and DOPAC levels assessed at various timepoints following an intrastriatal injection of 50 ~g QUIN (n = 9-10). Data are expressed and presented as in Fig. 3. ** P < 0.001 vs controls, Dunnett's test.

TABLE III

Monoamine and metabolite levels in the arcuate nucleus 4 days after a local injection o f 50 #g QUIN

Results are expressed as pmol/mg protein and represent the mean ± S.E.M. of the number of animals indicated in parentheses. Ipsi and Contra represent injected and contralateral sides, respectively.

DA DOPA C NE 5-HT 5-H1AA

lpsi Contra Ipsi Contra Ipsi Contra lpsi Contra lpsi Contra

PBSinjected 31.9±2.3 32.6±2.4 8.5±0.4 5.8±0.9 163.2±2.9 159.8±5.2 67.7±1.6 67.8±2.1 (7) (7) (6) (7) (7) (7) (7) (7)

QUINinjected 32.4±4.0 34.5±3.4 4.8±0.8* 6.4±0.8 170.8±6.8 165.7±3.3 71.8±3.7 71.5±2.2 (8) (8) (s) (s) (8) (8) (8) (8)

33.9±2.0 33.5±1.4 (7) (7) 34.9±2.5 32.5±1.6 (8) (8)

* P < 0.01 vs the homolateral side of PBS-injected animals (Student's t-test).

314

and behavioral studies in which the activity of tyro- sine hydroxylase 8'28, DA turnover 2'9"28,36'41, turning

behavior 3s and the pharmacological effects of dopa- minergic drugs in lesioned striata 22'27 were exam-

ined. Electrophysiological data from kainate-le- sioned animals, though more complex in nature, also indicate that substantial changes take place in nigro- striatal DA neurons following chemical destruction of the striatum 3'1°. The high intensity of striatal cate- cholamine fluorescence 4'15 and tyrosine hydroxylase immunohistofluorescence 28 observed at the lesion site within 2 days after a striatal kainate injection fur- ther supports the notion that DA neurons are func- tionally affected by the lesion.

In contrast to the extensive and multifaceted inves- tigations of the various effects of striatal kainate le-

sions, there exists a remarkable paucity of studies on the acute sequelae of an intrastriatal excitotoxin in- jection. Ultrastructural analyses performed during the first few hours after the injection do not provide an answer regarding the timepoint of beginning func- tional disintegration of the striatonigrostriatal circuit in the course of the degenerative process 7. Clearly kainate and its congeners can powerfully, and vir- tually instantly, excite striatal neurons I7 and typical behavioral phenomena (contralateral posturing and turning, barrel rotation and periodic myoclonus) can be observed in the very early stages of the lesion s'9'3s.

Intrastriatal kainate injections also result (within mi- nutes) in an acute increase in the firing rate of nigro- striatal D A neurons 3. Significant increases in striatal DOPAC levels have been noted as soon as 1 h after a local kainate injection 3. Thus, it appears that DA neurons react very rapidly to the physical introduc- tion of an excitotoxin in their terminal field, probably independent of beginning degenerative events and at least in part mediated by feedback mechanisms with- in the basal ganglia. This notion is certainly also in ac- cord with several in vitro studies demonstrating the stimulation of striatal D A release by glutamate, kai- nate or Q U I N 13'18'24'25.

The present data, obtained after intrastriatal injec- tions of QUIN, by and large reveal the expected pic- ture with regard to both acute and subacute biochem- ical effects in the basal ganglia. In agreement with our previous report 3°, there was no evidence for a de- generation of dopaminergic neurons at any timepoint following an intrastriatal QUIN injection. As soon as

90 min after application of the amino acid, striatal and nigral DA turnover was significantly increased, suggesting an increase in the firing of dopaminergic neurons. The levels of both acidic DA metabolites, H V A and DOPAC, were concomitantly elevated in the substantia nigra. We cannot, at the present time, explain our failure to detect elevations of striatal DOPAC and H V A levels 90 min after the QUIN in- jection. However, there can be little doubt that an activation of dopaminergic neurons, which is also manifested by the acute behavioral syndrome (see above), occurs in the initial phases following QUIN injection. At 6 h, when degenerative events have clearly begun to cause chromatolytic changes in stria- tal neuronal cell bodies (cf. Fig. 1C), large increases in striatal HVA and DOPAC levels were observed. At this and the later postoperative timepoints ana- lyzed in the course of the present study, no biochemi- cal changes could be detected in the substantia nigra. Thus, it appears that excitotoxin-induced striatal de- generation does not interfere with dopaminergic function in the substantia nigra (e.g. dendritic DA re- lease) hours and days following an acute increase in nigral dopaminergic activity. Apparent functional discrepancies between the region of DA cell bodies and their terminal field have been previously demon- strated using various experimental paradigms and the possible implications for basal ganglia physiology and pathology have been discussed 14.

The increased striatal DA turnover and metabolite concentrations 4 and 11 days after induction of the le- sion are in line with similar observations made in kai- nate-lesioned striata (see above) and in all likelihood reflect the response of D A terminals to the disruption of local neuronal feedback circuits. Similar mecha- nisms may be involved in the serotonergic reaction to the QUIN-induced degeneration of their target neu- rons 1. In contrast, long-term sequelae of striatal QUIN lesions on the two monoaminergic afferent systems are qualitatively different from each other (manuscript in preparation).

The substantial increase in the levels of NE in the striatum is clearly related to QUIN-induced striatal nerve cell loss. A similar finding has been reported to occur in kainate-lesioned striatum where the change in the levels of this catecholamine - - which is present in the striatum in very small amounts - - could not be linked to a change in NE turnover 34. While the mech-

315

anism as well as the possible functional significance

of this observat ion remains elusive at present , the sit-

uation is clearly different from the h ippocampa l for-

mat ion, where local inject ion of Q U I N results in a ra-

pid and t ransient dramat ic decrease in NE levels 39.

We have shown previously the failure of intrahy-

pothalamical ly appl ied Q U I N to produce lesions of

local neurons 29. These inject ions were therefore used

in the present study to examine our thesis that post-

synaptic nerve cell loss is the tr igger for the increased

local dopaminergic activity observed in animals with

subacute (6 h to 11 days after Q U I N ) striatal lesions.

While differences in the synaptic circuitry be tween the s t r ia tum 16 and the hypotha lamus 33 may compli-

cate the in terpre ta t ion of our present results, the

data are certainly in agreement with this not ion: no

effect of Q U I N on hypotha lamic fibers containing

D A , 5-HT or NE could be observed (cf. Table III) .

The da ta obta ined in the course of the present

study are in accordance with a hypothet ical l ink be-

tween (an overabundance of) Q U I N and HD, En-

hanced dopaminergic activity is thought to contr ibute

prominent ly to the prec ip i ta t ion of choreat ic move-

ments during the early and middle stages of the dis-

ease process 19, which are mimicked (neuropathologi -

cally speaking) by the acute and subacute stages after

intrastr iatal Q U I N inject ions. In contrast , a marked

reduct ion in hyperkinet ic symptoms, together with a

Parkinsonian-l ike appearance of the pat ient becomes

more prominent in terminal H D 31. Accord ing to the-

oret ical considerat ions, this phase should correspond to a hypodopaminerg ic state 5'19'31. Indeed , striatal

D A turnover and the levels of striatal D A metabo-

lites are significantly reduced as compared to con-

trols at long-term t imepoints following a striatal

Q U I N lesion (manuscr ip t in prepara t ion) . Thus, a re-

markab le correla t ion exists be tween clinical and ani-

mal da ta in spite of the fact that in H D , in contrast to

the animal model , the degenera t ive process takes its

course over a pe r iod of several years. A careful in-

vestigation of the Q U I N system in H D will be neces-

sary in order to decide if this brain consti tuent may

indeed be causally l inked to the pathogenesis of the

human disease.

ACKNOWLEDGEMENTS

This work was suppor ted by U S P H S Grants NS-

16102 and NS-20509 and grants from the Amer ican

Parkinson Disease Associa t ion and the Lena Marcus

fund (to R.S.) . The authors thank Mrs. Joan Rober ts

for her excellent secretar ial assistance.

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