[1] 11w Functional damage of dopamine nerve terminals following intrastriatal kainic acid injection

[1] 155w The release of [3Hldopamine ([3H]DA) previously taken up into rat striatal slices was studied one week after a monolateral intrastriatal injection of kainic acid (KA). Different releasing stimuli (electrical pulses, veratrine, high-K +) were applied. The electrically evoked release in the KA-lesioned striata was drastically reduced with respect to the un!esioned contralateral stri~ta, in contrast, KA had no effect on the release of [3H]DA evoked by veratrine or high-K*. In unlesioned striatal slices, depolarized with 15 mM KCI, apemorphine reduced and (-)sulpiride increased the release of [3H]DA. The effect of apomorphine ~ as antagonized by (-)sulpiride indicating the presence of an autoreceptor system similar to that seen in unlesioned striata stimulated electrically. However, the effects of apomorphine and of (-)sulpiride were dramatically reduced in K+-dcpolarized slices prepared from KA-lesioned striata. The results suggest that the axon terminals in KA-treated areas remain intact in several of their properties but may be damaged in some critical processes,

[1] 37w Nerve endings are endowed with autoreceptors through which the neurotransmitter can control its own release or synthesis 4,~7. In the central nervous system presynaptic autoreceptors can be studied either by using brain slices or isolatea nerve terminals.

[2] 70w So far only one exception seems to exist in which release-regulating autoreceptors can be easily investigated in slices 7,m,19 but not in synaptosomes t6. For some unknown reasons, the dopamine (DA) system in the corpus striatum differs from other major transmitter systems (cholinergic, noradrenergic, serotonergic and GABAergic) in that classical synaptosome techniques have so far produced inconsistent results even with the most potent DA agonists (see 16 and unpublished data).

[3] 40w We thought a possible reason for this discrepancy might be that the autoregulatory mechanism of DA release involves some intrastriatal neuronal circuits which could be seen only in intact tissues (i.e. in a slice) but not in isolated nerve endings.

[4] 79w In order to test this hypothesis, intrastriatal injections of kainic acid (KA) were performed in the rat on the widely accepted assumption that such a procedure destroys neurons whose cell bodies lie in the striatum whereas axon terminals of extrinsic origin remain intact 5,6,12-14. During this study experimental evidence was obtained that not only emphasizes the peculiarity of the DA autoreceptor system but also casts strong doubts on the functional intactness of DA axon terminals in KA-treated corpus striatum.

[1] 38w The parameters of high-affinity choline uptake were determined in synaptosomes prepared from the KA-iesioned side and from the unlesioned contralateral side, respectively, 7 days after the lesion. The results (Table 1) indicate that the Vma x of [3H]choline

[2] 103w TABLE I High-affinity [~ H/choline and [3 H] DA uptake parameters in le. sioned and unlesioned contralmeral striata Uptake experiments were performed 7 days after lesioning. K m values are expressed as ~M. V~ as pmol of [3H]choline or [3H]DA taken up per mg of synaptosomal protein in 2 rain. Means + S.E.M. are reported. n Lesioned Unlesioned 4 [3H]DA uptake K. [SH]choline uptake K~ Vm~ 0.32 + 0.04 0.29 + 0.03 272.1 + 21.1 268.2 ± 19.0 2.3 -0.6 2.4 ± 0.5 66.2 + 1 !.2" 403.6 ± 56.5 "P < 0.001 when compared to the Vm. z of the contralateral side.

[3] 13w uptake was strongly decreased after KA treatment whereas the K m remained unchanged.

[4] 28w Similar studies on [3H]DA uptake clearly show that neither the Vma ~ nor the Km of the uptake process were modified by the intrastriatal KA injection (Table I).

[5] 39w The DA content also showed no significant effect of the KA lesion, being (in ng/g fresh tissue) 42.0 + 3.5 (n = 5) and 39.7 + 3.0 (n = 5) for the KA-icsioned and the contralateral unlesioned striatum, respectively.

[6] 38w Electrical stimulation. As can be seen from Fig. 1, the electrical overflow of radioactivity from unlesioned slices prelabeled with [3H]DA increased when either the stimulation period (at constant frequency) or the frequency (at constant stimulation period) was increased.

[7] 58w Interestingly, the electrically evoked release of tritium from the KA-lesioned slices was drastically re-The refractoriness to electrical stimulation of the KA-treated striatum was not irreversible, however; in fact no statistically significant difference could be found when slices from KA-lesioned and contralateral intact striatum were stimulated (3 Hz; 24 mA; 2 ms; 2 rain) 40 days after the lesion.

[8] 80w Depolarization with high-K +. Fig. 2 (left) illustrates the effect of increasing the concentration of KCI in the superfusion medium on the release of [3H]DA previously taken up by striatal slices. The overflow of radioactivity was incre&~ed by high-K + in a concentration-dependent manner. At the two concentrations of KCI employed (15 and 20 raM) the effect produced in the slices of KA-lesioned striatum did not differ significantly from that obtained in the slices prepared from the contralateral unlesioned striatum.

[9] 108w than 70% at 3 Hz and 2 min of stimulation and it was somewhat less at 5 min or at 5 Hz (Fig. 1). The KA treatment did not cause any change in the basal efflux which amounted to 0.219 + 0.004% (n = 24) and to 0.216 _+ 0.006% (n = 22) per min in the KA-lesioned and contralateral striata, respectively. Depolarization with veratrine. Veratrine stimulated the release of tritium radioactivity from rat striatal slices pre-incubated with [3H]DA in a concentration-dependent manner. The overflow provoked by 5/aM of the alkaloid was more thaw doubled when veratrine was present in the superfusion medium at 10/aM (Fig. 2, right).

[10] 27w Fig. 2 also shows that when tritium overflow in KA-lesioned slices was compared to that in slices from the unlesioned striatum, no statistical difference could be observed.

[11] 69w In striatal slices taken from unlesioned striata apomorphine decreased the electrically evoked release of [3H]DA. The effect of apomorphine was antagonized by (-)sulpiride (Fig. 3, left). These results are in keeping with those previoasly reported by various groups 7'1°'19. Because the electrically evoked release of [3H]DA was drastically reduced after KA treatment we found it difficult to investigate the possible effects of apomorphine on this very small [3H]DA release.

[12] 64w However, we could study the effects of apomorphine and suipiride on the release of [3H]DA in slices depolarized with 15 mM KCi. As shown in Fig. 3 (right), the effects of apomorphine alone or with sulpiride in the unlesioned striatum were almost superimposable to those seen in the same tissue exposed to electrical stimulation. Moreover, (-)sulpiride added before $2 increased the K+-evoked [3H]DA release.

[13] 47w Interestingly, the effects of the DA drugs were much less pronounced (actually the effect of 0.1/aM apomorphine disappeared) after KA lesion. Thus, in KA-lesioned striatum, although the K+-evoked release of [3H]DA seemed to be unaffected (Fig. 2), the effect of DA drugs in such release was impaired.

[1] 84w Seven days after the injection of KA the rats were sacrificed by decapitation. The brains were quickly removed and the lesioned corpus striatum along with the intact contralateral one were identified, rapidly dissected and kept in ice-cold saline. The tissues were sonicated in 0.1 M perchloric acid and the homogehates centrifuged at 20,000 rpm for 10 rain. Portions of the supernatants were processed for the determination of the endogenous DA content using a HPLC coupled with a coulochemical detector, according to Achilli et al.l.

[2] 128w Synaptosomes (P2 fraction) have been prepared from lesioned and contralateral striata of rats sacririced 7 days after surgery, essentially accorclmg to Gray and Whittaker 9. Briefly, the tissues were homogenized in 40 -ols of 0.32 M sucrose buffered p.: pH 7.4 with phosphate. The homogenates were centrifuged (5 rain, 1000 g) to remo~,~ nuclei and debris and synaptosomes were isolated from the supernatants by centrifugation at 12,000 g for 20 min. The synaptosomal pellet was then resuspended in a physiological medium with the following composition (raM): NaC1 125, KCi 3, CaCI 2 1.2, MgSO4 1.2, NaH2PO~ , 1, NaHCOs 22, glucose 10 (aeration with 95% 0 2 and 5% CO 2 at 37 ~) pH 7.2-7.4. Protein was measured by a modification of ~he method of LOWly i$.

[3] 109w Uptake of [3H]DA (in concentrations varying from 0.01 to 0.32/AM) or [3H]choline (0.16--5.62/~M) was studied in aliquots (about 0.15 mg protein) of both KA-treated and unlesioned contralateral striatal P2 suspensions. Synaptosomes were we-incubated 5 min at 37 °C; the radioactive substrates were then added and incubation was continued for 2 rain. The tisst,es were collected by vacuum filtration on GF/B ~, hatman filters which were washed rapidly with 3 x 4 nd of medium and counted for radioactivity. Blanks ~vcre determined at 0 °C. Results were analyzed by tl~e method of Scatchard and K m and Vm~ estimated by means of unweighed linear regression analysis of 1he transformed data.

[4] 50w Tritium release was studied using striatal slices prepared from KA-lesioned or contralateral corpora striata of rats treated 7 or 40 days before the experiments. Slices (0.4 mm thick) were prepared using a Mellwain Tissue Chopper, the blade advancing per. pendicularly from the rostrai to the caudal side of the striatum.

[5] 32w The slices were incubated with 0.05/IM [3H]DA and 1/IM [14C]choline for 15 rain at 37 °C, in 5 ml of a medium having the same composition as that used in uptake experiments.

[6] 155w After rinsing with standard medium, one slice was transferred to each of 12 parallel superfuskm chambers provided with platinum dectrodes t9 and superfused for 185 min at a rate of I ml/min. After a washing period of 45 min, fractions of different length were collected as following: 15, 30, 15, 20, 15, 30, 15 rain. Two periods of electrical stimulation were appried after 60 (St) and 140 (S,_) rain of superfusion. Pulse width was 2 ms and current strength 2~ mA. Frequency varied from 3 to 5 Hz and the duration of the stimulus from 2 to 5 rain (see Results). In ot,'ler experiments two 4-rain periods of high-K ÷ (15 m 20 ~nM) or veratrine (5 or 10 ~M) depolarization were applied similarly to electrical stimulation. After superfusioa, each slice was solubilized in 0.5 ml of soluene 350 (Packard Instruments). The solubilized tissues and 5-ml aliquots of the superfusate samples were counted for radioactivity.

[7] 98w As an interna! control of the lesion the dices from the KA-treated and from the contralategal striata were alway; double-labeled also with [t4C}cfioline (1 ~M). The ~4C-radioactivity found in the dices obtained from the unlesioned striata amounted to almost 2.5% of the tritium dtte to the different specific activity of the two isotopes. Ratios of the ~'~E/tritium contents were calculated for both lesioned and unle-sioned tissues in each experiment and only the slices of the KA-treated striatum in which this ratio was 40% or less with respect to the contralateral slice average ratio have been considered appropriately lesioned.

[8] 33w Apomorphine and (-)sulpiride were added to the superfusion medium 30 rain before $2. In some experiments (-)sulpiride was used throughout the superfusion. Controls without drug addition before $2 were always run in parallel.

[9] 28w The efflux of radioactivity collected in each fraction was calculated as a percentage of the total radioactivity present in the tissue at the onset of the fraction considered.

[10] 46w The stimulation-evoked overflow was calculated as the difference between the efflux in the 30-min fractions, at the beginning of which stimuli were applied, and the basal outflow calcvlated by summing the tritium released in the two 15-min fractions immediately preceding and following the respective 30min period.

[11] 15w In order to quantify effects of drugs on the stimulation-evoked overflow, ratios were calculated be-

[12] 181w 3Hz-2m~ 7 DAYS AFTER LESI~.*; 40 D~.YS AFTER LESION Fig. 1. Effect of intrastriatal kalnic acid on the efflux of radioactivity induced by electrical stimuli from striatal slices prelabeled with [3H]dopamine. Slices were prepared either from the right striatum (white bars), injected with 1.5/Jg kainic acid 7 or 40 days before sacrifice, or from the left untreated striatum (hatched bars). Release was evoked by applying an electrical stimulus having a pulse width of 2 ms and a current strength of 24 mA for 2 or 5 rain. The frequencies used were 3 or 5 Hz. The overflows reported in the figure are referred to S t . For other technical details see Materials and Methods. The data represent means :t: S.E.M. of 5-7 experiments in duplicate. * P < 0.005 and "" p < 0.001 when compared to the ~ve controh using a two-tailed Student's t-teU. tween the overflow (as percent of tissue tritium) evoked by S 2 and the overflow evoked by St in the drug-treated chambers and comparing the~e values with the ratios calculated under the respective control conditions.

[13] 10w Differences between two means were analyzed by two-tailed St;'dent's t-test.

[14] 64w [3H]DA (snec. ,~ct. 45 Ci/mmol), [3H]choline (spec. act. 75.4 Ci/mmol) and [14C]choline (spec. act. 50 mCi/mmol) were obtained from Amersham Radiochemical Centre (Buckinghamshire, U.K.); kainie acid (lot no. 124F-0179) and veratrine from Sigma Chemical Co. (St. Louis, MO, U.S.A.) and ketamine (Ketalar) from Parke Davis (Milano, Italy). Apomorpine hydrochloride and (-)sulpiride were a generous gift from Sandoz (Basel, Switzerland) and Ravizza (Milano, Italy), respectively.

[15] 26w Apomorphine was dissolved in HPLC-grade water containing 2 mg EDTA and 40 mg ascorbic acid x 100 ml; (-)su~piride was dissolved in 1 mM acetic acid.

[1] 50w Male Sprague Dawley rats (250-300 g) were anaesthetized with ketamine (150 mg/kg) and fixed onto a stereotaxic apparatus for small animals. KA (0.75 #1 of a 2 ~g/~i saline solution buffered at pH Correspondence: M. Raiteri, lstituto di Farmacoiogia e Farmacognosia, Universit~ di Genova, Viale Cembrano 4, 16148 Genova, Italy.

[2] 103w 7.4) was injected in the right corpus stria,.um by a Hamilton syringe at a constant rate in ? min. The coordinates for striatal injection were A 8.2, L 2.6, V 0.5, according to K6nig and Klippe I1' The cannula was left in place for an additional 3 rain to allow diffusion, then carefully removed and the scalp apposed with sutures. This treatment produced convulsions in almost all the animals as they recovered from anaesthesia. Although the phenomenon was of moderate intensity and lasted less than 30 rain, the animals that did not show this type of behaviour were rejected for reasons of homogeneity.

[3] 111w reported that intrastriatal KA injections produced, after about one week, neurochemical changes reminiscent of those observed in Huntington's chorea. Thus choline uptake, choline acetyltransferase activity and acetylcholine content in corpus striatum were reduced by about 70%. Similar effects were observed in the corresponding parameters of the GABA sys-tem. In contrast, no decrease was found in DA content and DA uptake. These data led the authors to suggest that neurons whose cell bodies lie in or near the injection site are destroyed by KA, while fibers of passage or axons terminating in the area affected by KA remain undamaged. In the last decade several investigators have based their work on this assumption.

[4] 100w However, the results of the present study suggest that the neuroche,nical changes previously observed (decreas~ of choline and GABA uptake, but not of DA uptake: decrease of acetylcholine and GABA content but not of DA content; decrease of the K +evoked release of acetylcholine and GABA but not of that of DA) 5"6' sA2"22 may not be sufficient to state that DA nerve endings in KA-lesioned striatum have not been damaged. In fact, the major property of an axon terminal, i.e. that of conducting nerve impulses, appears to be drastically impaired in the nigro-striatal pathway after intrastriatal injection of KA.

[5] 287w The mechanism of this neurotoxic action of KA at the level of striatal DA axon terminals is not clear. The transmitter content of these terminals is normal. Their capacity to take up [3H]DA also seems maintained, suggesting that the Na+-K + pump/ATPase system has not been damaged. Accordingly, DA terminals in the KA-treated striatum appear to maintain their polarization as they can '-z normally depolarized with high-K + and veratrine. The secretion-coupling mechanism also seems to be largely unaffected if we assume that the release evoked by veratrine and high-K + mimics the exocytotic release. Moreover, the release of [3H]DA evoked by veratrine which is known to depolarize excitable membranes by opening Na + channels did not differ between lesioned and unlesioned striata, suggesting that KA should not affect the veratrine-sensitive Na + channels, at least in the DA terminal boutons of the nigro-striatal pathway. The absence of KA effects on both the veratrine and K+-evoked release does not suppo~ the idea that the voltage-dependent Ca 2+ channels are involved in the action of KA on DA terminals. Finally, no structural damages have been reported to occur on the basis of histofluorescent, immunocytochemical and ele~ron microscopic studies (see for references 5) yet, some subtle but important damage must be caused by KA which apparently prevents the traffic of nerve impulses in the DA terminal axons of the ie-sioned striatum. How this can occur is difficult to say at present. As one speculation, the lesion could be localized just I~fore the bouton, in the terminal part of the axon which could be crucial for allowing electrically evoked release from a slice to occur. The wellknown refractoriness of synaptosomes to electrical stimuli would be in keeping with this view.

[6] 87w In any case, as shown in Fig. 1, the lack of response of KA-treated slices to electrical stimuli is not irreversible. I, fact, the electrically evoked [3H]DA release comes back to normal after 40 days, curiously when the damage of intrastriatal neuronal bodies has been reported to reach its maximum 21, Spontaneous recovery of functions altered following various types of lesions of central pathways is not uncommon (see for instance3"2°). Moreover, transient changes of some biochemical processes have been observed also in kainate-lesioned animals (see for instanccm).

[7] 94w Another property of DA nerve terminals which has never been considered previously in KA-lesioned striata is the autoreceptor-mediated negative feedback mechanism regulating DA release 7,ma9. The resuits of Fig. 3 clearly show that KA treatment impairs the ability of apomorphine to decrease the depolarization-evoked release of [3H]DA. Also the enhancing effect of the autoreceptor antagonist (-)sulpiride (which prevents the inhibitory action of the released DA on autoreceptors) was drastically reduced. The data indicate that the DA autoreceptor system in the KA-lesioned striatal slices is much less sensitive to activation than in the normal slices.

[8] 109w As mentioned in the introduction, although autoreceptors for several transmitters can be easily studied in synaptosomes 4.17 this has not been possible in the case of DA, if one excludes the recent report by Bowyer and Weiner 2 in which DA autoreceptors could be seen in striatal synaptosomes by using a special technical approach. Therefore to conclude that the DA autoreceptor system in the striatum is endowed with some peculiarities seems justified. However, whether the KA-induced damage observed in the present work affects the autoreceptor process in the DA nerve terminal or involves an impairment of some neuronal circuits present in the slice is difficult to say at present.

[9] 66w In conclusion, intrastriatal injections of KA destroy neuronal bodies. The frequent statement that nigro-striatal DA axon terminals are spared is based on the fact that several of their porperties remain indeed unchanged. However, as shown in the present work, other properties of major importance, such as the ability to release DA upon electrical stimulation and the autoreceptor-mediated inhibition of DA release are dramatically impaired by KA.