70

EJP 5’0x1

Drugs wcrc infused into the suhstantia dopamine striatum.

(DA) This

antagonists

nigra of the rat brain via a microdialysis

and 3,4-dihydroxyphcnylacctic approach allowed

acid was rccordcd from

the evaluation

in conscious animals.

Intranigral

of the sensitivity

administration

prohc. and the cxtraccllular

a second dialysis

of nigral

.lutorcccptors

of the specific III,

prohc implanted to various

agonists. quinpirolc

D,

concentration

and D,

(LY

of

in the ipsilatcral agonists and

1715.55: I. 10 and SO

~mol/l)

and (-1-N-0437 (IO ~mol/l), dccrcascd the output of striatal dopaminc, whcrcas the specific D, antagonist, ( - )-sulpiridc (IO and SO ~mol/l), stimulated slightly the rclcasc of dopamine from the ipsilatcrai corpus striatum. lntranigral administration on the

of the D, agonist. SKF

rclcasc

autorcccptors

of striatal

controlling

3X303 (I00

dopaminc. impulse

The

~mol/l).

results

and the D, antagonist.

indicate

flow in the nigra contrihutc

systemic dose of D2 agonists. The stimulating

cffcct of D,

that

to this cffcct. Advantngcs

23390 (100 ~mol/l). autorcccptors

equally to thl, dccrcasc of striatal

DA

was without

on ncrvc

et’fcct

terminals

and

rclcasc caused by a high

antagonists on the rclcasc of DA is mainly caused by relcasc-control-

ling autorcccptors localized on ncrvc terminals. The au&ccptors less (ahoul ZOR)

SCH

relcasc-controlling

in the nigra controlling

impulse flow contrihutcd

and disadvantages of the USC’of ;I microdialysis

somewhat

prohc to dclivcr drugs to rcstrictcd

brain arcas arc discussed.

Corpus striatum;

Suhstantia

nigra: Dopaminc rclcasc; Microdialysis:

1. introduction It is generally accepted that dopaminc (DA) D2 autoreceptors modulate the impulse flow, synthesis and release of dopaminc

in the corpus striatum and related

mesolimbic structures. Characteristic elcctrophysiologicat and .brochemical changes that arc obscrvcd after stimulation explained

or blockade

of

by a compensatory

nism (Carlsson

and

3,

recepiors

are

often

negative feedback mccha-

Lindqvist,

1963; Bunncy

et al..

1973; Groves et al.. 1975; Kehr ct al.. 1972; lmperato and Di Chiara, 1985, 19881. Various

sites of action have been identified

mechanistic striatonigral towards Lindqvist,

for the

significance of this feedback t-e-ponsc. A loop, which is believed to be inhibitory

nigral

dopaminergic

cells

1963; Bunney et al., lY73,

(Carlsson appeared

and to be

Dopaminc

D, rcccptorh: DO~X+X

rcccptors

involved in certain clcctrophysiological changes that occur after administration of DA antagonists and agonists (Bunney et al., IYX7). However. such a neuronal loop is not ncccssarily involved in the typical biochemical changes - in DA synthesis and metabolism - that occur in the ncrvc tcrm:nal areas when DA agonists and antagonists arc applied (c.g. Kchr ct al.. 1972; Di Chiara et a!., 1077; W?‘asterink and De Vries. IYRY; Murrin and Roth, lYX7). These biochemical changes wcrc explained by the prcscncc of autoreceptors

modu-

lating synthesis and release and localized on nerve terminals. Another mechanism that participates in the fecdback response of dopamincrgic

neurons

involves the

autorcceptors controlling impulse flow and localized on dopaminergic cell bodies and/or dendrites in the substantia nigra (Bunney et al., 1973. lY87; Groves et al.. 1975). To our knowledge, the relative contribution of these autoreceptors

C’orrespondrncr to: B.H.C. Westrrink. University Centre for Pharmacy. Antonius Deusinglaan 1. 4711 AW Cironinwn. The Nc~hcr. lands. Tel. 31.50.h.73 3117,Iax 31.50.~13331 I. * Present ;Iddreomatodendritic autoreceptors in the striatum.

to the modulatrnn

of DA release

OD:

0.Z I mm 1 was prcpiircd

htxiium met haiyi sulfonatc

from

polyacrylonitriic/

copolymer

(AN

09. Hospai.

Bologna. Italy). The in vitro rccovcry of DA at 7.51 pI/min (room tcmpcraturc) was: 9.1 + 1.25 (N = 5) tiw rhc striatum

nigra

CilllllUiil

cannuia.

7X

ilIld

The

Icft SN tccjordinatcs:

f

l.Yi

(N = 4) for the

probe was implanted A/P

3.X. L/M

from the intcral~ral iinc. at an angle of II”) icft htriaturn (coordinates: A/P 0.7. L/M h.tl. from hrcgma

point

impiantcd

gcncrai

under

t-i00 mg/kp The

i-p.) illld

cspcrimcn!G

implantation as described

di:llpiS

‘-(N-prop!:I-N-‘-thicn!:l-

mcmhranc:

((

cth~lamino)-c-h~~r~~~~t~tralin

sired in our !nhoratory 1~1 (LY 17iSC.i) and SKF X3

-~

1-N-0137.

synthc-

xn ct al.. IUSC): quinpiroic (KBI. Natick. MA. USA).

brief.

The

chiorai

probes were

hydrate

ItKill

iidocainc

wcrc

carried

8.7.

and in the 22, V/D anesthesia

(hr/ 1 analgesia. out

13-48

h after

of the probes. Brain dialysis of dopamine

was pcrformcd The following drugs wcrc dissolved in the pcrfu3ion tiuidand inluscd inlo the wh~tantia nigra via ;I micrcb

and hrn).

into the

3.X. V/D

with ;I fully automated ciscwhcrc

two polyethylcnc

(Westcrink

on-line

et al..

tubes (inner

system

1987).

diameter

In

= 0.28

mm) wcrc connected to the outlets of the dialysis tube. One tube (45 cm Icngth) was connected to the perfusion pump. and the other (45 cm length) to the injection valve of the HPLC

apparatus. The connection with

the HPLC

introduced

cquipmcnt

a lag time of about

t - I-Sulpiridc (Ravizri. Milano. Italy) and SCi1 2_?390 Natick. MA. L&A) wcrc dissolved in 0.1 moi/i xctic acid. Solutions f 10 mmol/l) wx2rc then ncutrai-

20 min. for which the data are corrected. An electronic timer was used to hold the injection valve in the ‘load’

izcd with 0.1 moi/i perfusion Iluid.

~1) was filled with dialysate. The valve then switched

NaOH

and diluted

further

in the

position for IS min. during which the sample loop (40 automatically

to the injection

position

for 15 s. This

procedure was repeated every 15 min. which was the time nccdcd to record a complete chromatogram. The SN

and

the

striatum

wcrc

perfused

The cspcrimcnts \vcrc pcrformcd with a I-shaped cannuis (Santiago and Wcstcrink. IYJtl). The cxposcd tip of the dialysis mcmbranc was 2 mm for the SN and

solution at a flow rate of 7.5-3.0

4 mm for the striarum. The dialysis tube (ID:

CaCi,

% of controls 100

0.22 mm:

pl/min

with

a Ringer

(perfusor

VI,

B. Braun. Melsungen. F.R.G.). The composition of the Ringer solution was (in mM): NaCi 140.0: KCI 4.0: 1.2; MgCi,

1.0. When the experiment

was termi-

XI

natecl

rat was given an overdose of chloral hydrate.

the

and the brain was fixed with 4% paraformaldchydc intracardiac wcrc

perfusion.

made,

according

Coronal

and dialysis

age dialysate concentrations of the control and drugs treatment were compared by Kruskal-Wallis analysis of variance by ranks. Means wcrc compared using the

via

sections (40 pm thick)

probe

placement

localized

to the atlas of Paxinos and Watson

Wilcoxon

matched-pairs

signed-ranks

two-sided test.

(I!IX2).

The implanted probe was in close contact with the substantia nigra zona reticulata in all experiments now 3. Results

described.

2.3. Chemical Dopamine (DOPAC) chemical pump

assays (DA)

and 3,4-dihydroxyphcnylacctic

3. I. Effect of yuittpirole (LY 171.5551 and ( - )-N-0137, ittftrsed itlro the substatttia n&a. otl the dialysare cotttettt of DA arid DOPAC recorded from the ipsilateral corpus swiatum

acid

were quantitatcd by HPLC with clectrodetection. A Perkin-Elmer series 10 HPLC

was used in conjunction

working

electrode

Ag/AgCI

set at -780

reference

with

electrode)

lands). An Altech-RSL

a glassy carbon The specific DA D, agomst. quinpirolc

mV (with respect to an (ANTEC.

cartridge

umn filled with reverse-phase

(150

C,,

The NetherX

4.6 mm) col-

5 pm material

used. The mobile phase consisted of a mixture of 0.1 M

detection

limit of the assay was about IO-15

171555).

During these infusions, the release of DA decreased in the ipsilateral striatum (fig. 1). A slight decrease was

was

seen at a concentration of 1 pmol/l and an apparently maximal increase was seen at both 10 and 50 pmol/l.

sodium acetate adjusted to pH 4.1 with acetic acid, 1.X mM I-heptanesulfonic acid, 0.3 mM NA?EDTA, 120 ml methanol/l. The flow was 0.7 ml/min.

(LY

was infused for 60 min at three different concentrations (I, 10 and 50 ~mol/l) into the substantia nigra.

and The

In contrast,

DOPAC

levels responded

to the infusion

of the D, agonist with a moderate increase. Control values ( +S.E.M., n = IX). DA: 11.2 f 0.9 fmol/min

fmol per

sample.

and DOPAC: 0.84 + 11.05 pmol/min. Like that of quinpirole, infusion of the D, 2.4. Expression of results atld sraristics

agonist.

( - l-N-0437

(IO ~mol/l), into the substantia nigra caused a decrease in the release of striatal DA (fig. 21.

The average of the last four stable samples bcforc drug treatment was considered as the control and was defined as 100%. All values given arc cxprcsscd as

The decrease in DA was again accompanied by an incrcasc in the DOPAC content of the dialysates. Control values t +S.E.M., n = 71, DA: 11.7 i 0.9

percentages

fmol/min

of controls. Differences

bctwcen

the aver-

and DOPAC:

0.54 &- 0.02 pmol/min.

125 %

of

controls 100

50 -

25

-

0”“““““” 0

Fig. 2. Effect squares)

of intranigral

in the ipdatcral

infusion of

(-

corpus srriatum.

significantly different

60

30

I-N-0437

IO pmol/l

90

(black bar) on the dialpare

150

catput

180

210

and DWAC

(filled

(bars) value% (V = 7\ exprtwd 3s percentage5 of control\. &‘v’alhhf~dI:~.vd hy Wilcoxon IK\I). are indicated: * I’ T ii.il!i tbuskal-

Val~c>

The dala iirc mcam++S.E.M.

rrom the control vdue

120

of DA (open square\)

IIt.5 _+ 0.9 pmol/min.

fniol/min

and

DOPAC:

I .04 + 0.05

e spt‘cific D1 antagonist. ( - kulpiridc. w3s infor MI min into the substantia nigra. Two diffcrc’nt conccntr~tions (ItI and 50 ~mol/lI wre used. Ektraccllular kvcls of A. rccordcd from the ipsilatcrrrl corpus striatum. responded ivith a slight incrcasc t,thc mssimal

The D, agonist. SKF 383% was infused for 60 min at a concentration of 100 pmol,/l into the substantia nigra. The cxtracellular concentration of DA and DOPAC in the ipsilatcral striatum was unchanged during the infusion of the D, agonist (fig. 4). Control

I-0 ’ 5 of controls). Both conc~ntrati0ns tcstcd wc‘rt‘ equally cffcctivc (fig. 3). The DOPAC conwntrations in striatal dialysatcs wax unchanged.

rise bv;~sahvut

Control

values ( f S.E.M..

n = I I ). DA:

125 %

of

controls 109

0 0

30

60

90

120

150

180

210

mln r Fig. 1. Effect ::f intr wgra! warrs)

;.n.won F .’ of S5LF 3KJY2 IIN) pmol/l

(hlxk h;~r) on the dialpate output of DA (open squ;uvs) and DOPAC (filled i.1 the ipsilulrrd corpu* wiatum. T!x data arc mcun\f S.E.M. (bars) v;ducs IN = .1) rupresxd as prrcrntqys of the conwols.

X3

125

of controls 109 %

0 0

30

60

90

120

150

160

210

min ------3r Fig. 5. Effect of intranigral infusion of SCH t33YO tfH1~mol/l

thlack bar) on the &&sate output of DA (open squares) and DOPAC (filled squares) in the ipsilateral corpus strialum. The data are means + S.E.M. (bars) value%(N = 4) expressed ah percentages of the ccm~rols.

values (+S.E.N., n = 41, DA: 6.6 -of:1.2 fmol/min DOPAC: 0.83 + 0.07 pmol/min.

and

The D, antagonist, SCH 23390, was infused at a concentration of 100 pmol/l. No effect was noticed on the extracellu!ar levels of DA and DOPAC recorded

from the ipsilateral striatum (fig. 51. Control values (fS.E.M., n = 41, DA: 9.3 + 0.9 fmoi/min and DOPAC: 0.62 f 0.06 pmol/min,

4. Discussion In this study drugs were infused via a dialysis probe into the substantia nigra, and fIA was recorded with a second probe in the striatum, The use of a microdialysis probe is a novel method for delivering drugs to discrete brain areas. We now used this approach to investigate the properties of DA receptors controlling impulse flow and localized on cell bodies En the nigra. The doses of the intranigrally infused D, agonists and antagonists suggest that the maximal effects are in the order of 10 pmol/l. This is about one order of magnitude less effective than that found in experiments in which these compounds were applied directly to the striatum (lmperato and Di Chiara, 1988; Westerink and De Vries, 1989; Timmerman et al., 1989a), assuming a similar drug passage across the different dialysis membranes used in these studies. The relative insensitivity of the substantia nigra to D, compounds can be interpreted in two different ways. First, the D, autoreceptors in this area might be less sensitive to agonists or antagonists. However, there is littlc evidence from the literature to support such a conclusion. A more likely explanation for the higher concentrations of the D, compounds needed is the fact that they first must diffuse through the whole somatodendritjc

area before the full pharmacological effect becomes detectable in the ipsilateral striatum. This would mean that the potency of the drug is greater than the infused concentrations suggest. The diffusion characteristics and the metabolic fate of the infused cqmpounds will contribute considerably to the apparent potency. With the exclusion of the latter drawback, it is emphasized that the use of microdialysis probes to administer compounds to brain ares has several advantages. Infusion via the dialysis membrane means that mechanical contact with the tissue is minimized and that a regular concentration gradient of the drug is built up in the surrounding tissue which does not exceed that of tire infused concent~tion. The microdialysis probes cause less damage than do injection cannulas and pushpull perfusions (Quan and Blatteis, 19X9), although a recent report emphasizes that the damage due to a microdialysis probe is still considerable (Shiuaib et al., 1990). The present study showed that infusion of DA-rcceptor specific drugs into the substantia nigra resulted in characteristic changes in the release of DA in the ipsilateral striatum: D? agonists decreased the release of DA and the D, antagonist, sulpiride, stimulated the release of DA. That the observed changes are mediated by Dz receptors is supported by the finding that neither high doses of D, agonist, SKF 38393, nor the D, antagonist, SCH 23390, had any effect on the release of DA from the ipsilateral striatum. It is of interest to compare these results with those of earlier studies on the role of D, and D, autoreceptors. Some reports have krcated that systemically administered SCH 23390 increases the firing of nigral dopaminergic cells (Mereu et all., 1985), whereas the release of striatal DA is stimulated after systemic as well as after intrastriatal adm;nistration of low doses of SCH 23390 (Imperato et al., 1987; Imperato and Di Chiara, 19881.

siblc ssplnnation DA

is that the dccrc;tsc in the rc!ztsc

in the ipsil;ttcr;ll

of DA

by rclativ4y

striatum

stimulates

c>f

the synthesis

un~~ccupi~d syntl~~sis-ct~ntrolling

autcjrcccptors (Roth CI al., 1973). The cxtraccllular DOPAr Icvcls in thcsc cxpcriments apparently reflect chitngcs in DA

synthesis

Jcasc. This obscwiltion rigittion

rate rather

than in DA

is prcscntly under further

rc-

invcs-

in our Irtborittory.

In summary, the present dual-cannul:I approach makes it possibk to study the properties of DA impulse-flow rt‘xuks on

ncrw

controlling

autorcccptors

in the nigra. The

indicate that rcl~~s~-c~~ntr~~lling autoreceptors tcrminds and impulse-flow controlling au-

torcccpk~rs in the nigra contribute crcasc of stri:M

equally kr the de-

DA rclcasc cituscd by a high systemic

dose of D, agonists. The stimulating effect of D, itnt~lg~~nis(s on tht: r&case of DA is mainly caused by

rclcwxontmlling autorcccptors localized on nave t~rlnin~~ls~ the ~ut~)r~c~pt~~rsc~ntr~~lling impulse flow in the nigra conlributcd this 12fftct.

somewhat less (about 20%) to

M.S. waz the rccipiznt of a post-doctoral fetlowship from the hfiniaterio ric Education y C‘iencis. Spitin.

rcctly into the striatum. an increitw in Ihe relcasc of A to i\bout ISOr; of the controls WIS ohs~~d Ilmpcrato and Di Chiara. 19%. 19SX; Zctterstriim ct al., 19Sh: Wcstcrink and De Vrics. 19X9). The present study c~~~fir~is that D,

~nt~g~~nists arc ahlc to block

impulse flow modifying autorcccptors in the nigra, but the fin;ll cffcct on the rckast‘ of striatal DA is r&ativcly slight: 3 maximum of 205 of the total ncurolcptic-induced increase in CA rcleasc is apparently mediated bp autoreceptors in the nigrtt. Rcccnt observations in which the relcasc of DA was mcasurcd in the substa~?~~ nigra during trcrttmcnt with D, agonists and antagonists (Santiago and Wcstcrink, in press a). rcsuited in very similar percent changes. These data suggest that the increase in impulse flow activity recorded after systemic treatment with DA antagonists ~Agh~jiin~~n and Bunney, 1973; Bunncy et al., 1973, 1987) contributes only to a minor cxtcnt to fhc total ~~~r~~l~~tic-ind~c~d increase in the rclcusc of DA in the striatum. Finally, WC would like to c~~rnrn~nt on the drug-induced changes in extracellular concentration of DOPAC in the ipsilatcrzl striatum, It was somewhat unexpected that striatal DOPAC increased while the rekase of DA WAS decreased at the same time. WAX observed after infusion of TTX

effects

Similar

or ha-

clofen into the nigra Gantiago and Wcstcrink. in press b; ~np~h~~~b~d~~bs~~~ti~?ns in our l~b~~r~to~~. A pas-

References Aph;tjzrnian. G.K. and B.S. Bunney. 1973. Central dopaminergic neurons: rtrurophybiolcr~ic;lI identification and rcponscs to drugs. in: Frontiers in Catrchnlamine Research. eds. E. Usdin and S.H. Snyder f Prrgamnn rrcss. New York) p. hJ3. Bunncy. B.S.. J.R. Walters. R.H. Roth and C.K. Aghajanian. 1973. D~~p~lmin~r~ic neurons: effect of ~nt~p~ych~iticdrugs and amphc’taminr on single cell activity. J. Pharmacol Exp. Thrr. 1X5. %a. Bunney. B.S.. S.R. S~sack and N.L. Silva. 1’1x7. Midhruin dopaminrrgic systems: neurophysiological and elcctrc)phybiological pharmctcalogy. in: Psychopharmacolcqy: The Third Generation of Progress. cd. H.Y. Mcltzer (Raven Press. New York) p. II>. Carbon. A. and M. Lindqvist. 1963. Effect ai? chlorp:c>mazine OF haloprridul an f(~rrn~tit~n of ~-m~th~)~ytyrumin~ and normrtanrphrinr in mouse brain. Acfa Phnrmacol. 20. 1%). Di Chiars. G.. M.L. Porceddu. P.F. Spano and G.L. Gessa. lY77. ~~;tl~~p~ri~il~lincreases and ~pc~m(~rphin~ decreases striatal dopaminr metabolism after drstructitrn of striate1 dopaminr-srnsitive adenylatr cyclase hy kainic acid. Brain Res. 1.30. 374. Groves. P.M.. C.J. Wilson. S.J. Ytrung and G.V. R&cc, IYX Self inhihition hy dopaminergic nrurons. Science 100, 23. Impcrato. A. and G. Di Chiara. IWS. Dopaminr relrasr and metaltolism in awake rats after systemic nruroplcptics as studied hy tran+stri;lt;d dialysis. J. Ncurtxxi. S. 197. ImpcrJto. A. and G. Di Chiera. I~~%%Effects of i