A role for the basal ganglia in nicotinic modulation of the blink reflex

Craig Evingcr1• 2 , Micheie A. Basso"', Karen A. Manning2, Patrick A. Sibony3,

John J . PeliegrinP, and Anja K.E. Horn !

I Departmcn! of Ncurobiology and Behavior, 3 Dcpartment of Ophthalmology, und 4 Department of Psychology, StatC Univcrsity of New York at Swoy Braok, Stony ßrook, NY 11794, USA z Dcpartmcm of Anatomy, MedicaJ School, Univcrsity or Wisconsin, Madison, WI 53706, USA

Rccei llcd Dccembcr 6, 1991 / Acccptcd lune 23, t992

Summary. In humans and rats we fauod thaI oieotioe transiently modifies thc blink reflex. For blinks clicitcd by stimulation of the supraorbital branch of the trigeminal nerve, nicotine decrcased thc magnitude of the orbicularis ocu li clcctromyogram (OOemg) and incrcased the lateney of only the long-Iateney (R2) eornponenL For blinks elieited by eleetrieal stimulation of the cornea, nieotine deereased the magnitude and inereased the lateney of the single eomponen l of OOemg response. Sinec nieotine modificd only one component of the supraorbitally eli­eited blink reflex, nieotine must act primarily on the eentral nervous system rather Ihan at the museIe. The effeets of nieotine eould be caused by direet action on lower brainstem inierneurons or indireetly by modulating deseending systems im pinging on blink interneurons. Since p reco ll icular decercbration eliminated nieotine's ef­feets on the blink reflex, nieotine must ael through descen­d ing systems. Threc lines of evidence suggest that nicotine affects Ihe blink reflex through the basal ganglia by eausi ng dopam ine release in the striatum. First, stimula­tion of the substant ia nigra mimicked the effects of nico­tine on the blink reflex. Seeond, haloperidol, a dopamine (D2 ) reeeptor antagonist, bloeked the elfeet of nicotine on the blink renex. Th ird, apomorphine, a 0 2 receptor agon ist, mimicked the elfeets of nicotine on Ihe blink reflex.

Key words: Blink reflex - Nicotine - Basal ganglia -Orbieularis oeu li - Rat - Human

Introduction

Cigare lle smok ing transiently modi fies the human blink reflex elieited by stimulation ofthe supraorbital branch of the trigeminal nerve (SO; Evingeret al. 1988). Immediately aft er a person smokes a eigarelle, the eleetromyogram (EMG) amplitude ofthe lid-closing muscle, the orbieula ris

CQrrespondenc(' /0: C. Evingcr

oeu]i deereases and the lateney of the late R2 EMG eomponent inereascs. In eont rast, the lateney of the early R 1 EMG compouent does not change, although thc EMG amplitude ofboth eomponen ls deereascs. The bl ink reflex returns to nonnal 3- 5 min later. Two lines of evidenee suggest thaI nlcotine is the agent in eigarette smoke responsible for modifying the blink reflex. First, the time course of the rise and fa ll of blood nieotine levels following cigarette smOking (Benowi lz et al. 1989; Russell el al. 1980, 1983) is simil ar to the time course of blink modifieations with smoking. Seeond, nieoti ne is known 10 cross the blood- brain barrier readily. The presenl paper eonfirms that nieotine inha led in eigaretle smoke modi fi es the blink reflex (Evinger et al. 1988) by demonst raling Ihat nieotine ehewing gurn induces the same modifieation of the blink reflex in humans as docs eiga rette smoking. Moreover, intravenous injeetion of nieotine in anestbc­tized rats modifies eorneally and air-puff-clicited blinks in a similar manner to cigarette smoking in humans.

Nieotine eould alter the blink reflex direetly, by bind­ing to nicotinie reccpWrs on neurons that are part of the poslerior brainslem blink eireui ts, or indireetly, by aetivat~ ing deseending pathways that modulate the blink reflex. We eliminated the possibi lity of direct nicotinie effects by dcmonstrating thaI nieotine no longer mod ifies reflex blinking in deeerebrate rats. The basal ganglia is an ideal eandidate to aeeount for an indireel elfeet of nicoti ne. First, basal ganglia disorders modify the blink refl ex. Patients with Huntington's chorea, in whieh stnatal dop~ amine levels are relatively high, exh ibi t a decrease in blink refl ex exeitability and a small inerease in blin k latency (Bollen ct al. 1986; Carenceni ct al. 1976; Esteban el a J. 1981; Ferguson et al. 1978). In contrast, patients wilh Park inson's disease, in whieh the striatal dopam ine levels are abnormally low, possess a hypcrexeitable blink reflex (Klawans and Goodwin 1969; Messina ct al. 1972; Pearce CI al. 1969; Ru shwonh 1962). Seeond, the basal ganglia are rieh in nieotinie reeeptors, partieularly on dopamine­eontaining presynaptie terminals in the striatum (Araujo et al. 1988; Bolam el al. 1991; Clarke 1989; Hunt and Schmidt 1978; Scmba and Fibiger 1989). Aetivation of

these mcotmlc receptors in Ihe slrialum and substanlia nignl causes a release of dopaminc (Clarkc et al. 1985; Giorguieff-Chessclet el al. 1979; Lich lensteiger ct a1. 1982; Mereu et al. 1987; Sak urai et al. 1982; Schultz and Zigmond 1989; Westfall el al. 1989). These observations suggest that nicotine eould modu1ate the blink reflex by causing a surge in dopamine release in (he striatum. The presen l rcsuhs conflrm this hypothesis, by dcmonstrating that a dopamine (D,;) rcceplor antagonist blocks the effect of nicotine on Ihe bl ink rcnex and that a D 2 reccptor agonist mimies nicotine's effects. Thus, nicotine offen; a tool fo r investigaling how the basal ganglia may modulate reflexes in normal and diseased states.

Materials Qnd melhods

Human experiment!!

We im'cstigaled the reHex blinks or one man berore and after chewing nicotinc gum. The subject was awarc or thc goals oflhe inves tigation. All procedures were carried out in accorrlance wi lh the SUNY Stony IJrook Human Subject Guirlclines.

Prep(ll'atioll

EYinger et al. ( 1991) haYe giyen a compiete deseription or the measuremcnt tKhniques for lid posi tion and o rbieularis oculi EMG (OOemg) actiYilY in humans. BrieHy, we used the magnetie scarch coil tcchnique 10 measure lid posi tion by taping a 4·mm·diamcter coil on the tower margin or the upper eye[id and then placing thc subject in a ma&netie field (C-N.C Enterprises). To calibrate the output or the coil, the subjcct fluted different points along the vertical meridian ..... hi le the investigator measured the angle of lid rotation with a protraetor. Two miniature si lver electrodes « 2 mm diameterJ Illped to the lateral and medial portions orthe upper eyelid monitored OOcmg, and a gold cup elcctrode (9 mm diameter, Grass) scrved as a grou nd. Neither the OOerng electrodes nor the coil impaired lid mOycmenl or caused diseomfort to the subject. To el ici t Teßex Minks, two additional Grass skin elcetrodes wcre taped over the SO, one on the brow and another I cm above the first. Prior to e!cetrode a pplieation, aH skin surfaees were c1eaned with aleoho[ and thc elC(;u odes eoated with a oonduetive paste.

Prior 10 che ..... ing nieotine gum (2 mg nicOline per tablet; Nicorette, Lakeside Pharmaceutieal), we recorded 15 reßex blinks in response tO elcctr ieal stimulation of the SO. The subjcct thcn chewed 10 mg o r nieotine gum for 3 min. This was followed by 20 more electriea l stimuli 10 the SO, idenlicaJ to those: dcli\'ercd prior 10 ehewing the nicotine in gum. Electrical stimuli were delivercd every 2O±3 s. Data were collected and stored on-line on a personal computer. Ud and OOemg wete sampled at 5000 H7,; the compuler r«:lified the ODemg rocord and integrate<! the musele aetivity. The reeords could be analyzed indiyidually or averaged (Eyinger el al. 1991).

Rat experiments

Relle~ blinks ..... ert also el ici ted in 11 male Sprague-Dawley rats weighing betw~n 200 and 700 g. All animal e~periments werc performed wi th the approval of and in accordancc wi th SUNY Siony Brook Institutional Animal eare and Usc Committce, sta te, and fedcral guidelines.

Alleslhetized ralS

We anes thetizcd rals wi th i.p. injcctions or urethane (15 g,lkg in phosphate buffer pH 7.4). The femoral vein was cannu[a tcd 10 allow injeclion of nieotine. Mter fixing the hcad in a siereotaxic apparatus, we implanted a single-stranded, Tellon-coated, stainless stecl wire [A-M Systems 79 10; bare diameler O.0762 mm diameter with Tellon ooatingO.1143 mm], bared for I mm at thc tip, into the upper eyelid and a second into the lower c)'elid or the rat to measure OOemg. Reflex blinks were elieited by elcctrica[ stimulation of thc eornea ..... ith two smalI , si lver balls o r with brief air puffs directed at thc eye.

In three rats, we leslcd the effcct or nieotine injcction alone on the blink re11ex. Prior tO Lv. injection of nieotine, 15 or 20 reHex blinks wcre elieited by elcctrical stimulation ofthe cornea or wi lh an air pulf prescnted eyery 20 ± 3 s. Wilh this time bclwten stimuli, blinks were oonsis tent and robust. The ral was injccted with nicotine, thc aßlOunlS yarying rrom 0.32 m&fkg to 2.5 mgfkg depending upon the experiment, and Ihen r~ived 20- 40 stimuli identieal 10 those presented prior to Ihe nieotine injection. Two hOU TS after eomplclion of the first nicotine injecl ion we repeated the procedure on eaeh rat , using the same dose of nieotine, wilh similar results. Atlhe end ofthc experiment, the rat was perfused wi th warm 6% dextran in phos­phate bulfer (pH 7.4) fol1owed by cold 4% paraformaldchyde.

1'0 show that rostral brain regions were nOI eritieal for the nieotine elleet on blinking, the nieotine effecl on blinking was demonstraled on IWO rats and then the ra ts werc decerebratcd precollieularly by aspira tion. Two hours later, thc effKt of nicotine on the blink reflex was rclested.

To examine whether ba$31 ganglia aetiyation eould mimie the ellcct of nieoline, a stimulat ing mieroelectrode was implanted stereo­taxically in the substantia nigra of two uretbane·anesthetized rats. Twenty trials of OOemg aetivi ty evoked by stimulation ofthe cornea were ~rformed. Data from 2() more tria ls using identieal oorneal stimuli were eoliKted, but the substantia nigra region rccei\'ed a IO-Hz, 4O-1-IA, 6O-I-IS stimulation for 7 s immediatcly prcceding, but not ovcrlapping. eaeh eorneal stimulus. Substantia nigra stimulation alone neycr produeed any OOemg activity. The response to 20 addi tional corneal stimuli without nigra[ stimulation were Ihen eoliKted to show that the unstimulated blink reflex remained unchanged. The location of the stimulating elecn odes ..... as veri fled histologieal1y (or ellch experiment.

'1'0 evaluate the importance of dopamine re lease in nieotine's modulation of thc blink reflex in tWO urc thanc·anesthetizcd rals, the effects of i.v. injcctions of nicotine werc demonstrated and thcn the ra ts received i.p. injc..'Ctions of haloperidol (0.15 m&fk.g) to block D) reecptors. The rats ..... ere then ehal1cnged with nicotine 1 h [ater, whcn the haloperidol elTK t was maximal, and 4 hiater. when the halo· peridol effeet was over.

Alerl ralS

We prepared two rals for chronic stimulation and rccording. We anesthetized the rats with ketaminc (90 mg/kg) and xylal.inc t 10 mgJkg). Using ascptic proccdurcs, we implanted a nerve eu lT (Tellon tubing with a pair of stainlcss stcc! wires) around the SO and OQcmg clectrodes into the orbieularis oculi muscle near the anterior and posterior aspect& of the upper eyelid. These wires were [ed sUbcutaneously to the female end or a conneetor embedded in a dental aerylie mound allached to the skulI. A silver wire placed on the skull proyidcd a ground. Four to six self-tapping, stainless sttel, screws (1-72, 3/ 16 in. (4.763 mm), Small Parts MX-I72- 3] were screwed in to the skul1 tO anehor a dental a<: rylic base. The screws were eO\'ered with dental aerylie, which sen-cd as a base fo r thc stim ulating and recording leads. The ra ts recO\'cred for 2 days or longer befoTe testing bega n.

A lightweight lead plugged into thc conneetor on the dental aeryEe mound. This conneetion allowcd stimulation through the

nerve euff and rcoording of the OOemg while [he rat movcd fredy around the eage. Blinks were elieiled every 20 ± 3 s. The rats showed no signs of discomfort during [his proeedure. To demonstrate that aetivation of dopamine reccptors eould mimie thc effeet of nieotine on thc blink reflex, rats reccived apomorphine hydrochloridc (4 mg/kg subcutaneously; Sigma, AA393) after 15 eontrol blinks werc recorded. Fifleen reflex blinks were then rceorded every JO min for 1 h.

Drugs

For the aneslhetized-rat experiments, we utitized nico tine [( - )­nicotine di-( + }-Iartate salt, Sigma. N-5260) mixed with saline 10 form conccntrations ofO.25- 2.5 mg/ce. Haloperidol (Research Bio­chemicals, H-loo) was mixed with 1% ascorbie acid to a concentra­!ion ofO.2 mg/cc. For thc alert-rat studies, apomorphine was mixed in saline 10 a concentration of 4 mg/cc. All drugs weTe preparcd on the day of the experiment.

Dara collection and analysis

For both the human and the rats, data weTe collecte<! and stored on­line on a personal computer. In the human study, lid and OOemg were sampled a t 5000 Hz. The computer rectified the OOemg record and integratcd the rnusde activity. Thc records cüuld be analYlcd individualty or averaged (Evingeret al. 1991 ~ In rats, we sampled thc OOemg a\ 3000 Hz, but did not measure lid position. OOemg records were rectified and intcgrated over periods specified by the imestigator for cach series of trials. The data was analY7.cd off-line for latcney and OOemg magnitude. For both species, OOemg responses were fittere<! (band pass - 3 d B points at 300 and 5000 Hz) and eolleeted with 12-bil uccuraey (Data Translation DT2801-A).

Results

One advantage offercd by the blink reflex is the extra­ordinary eonsisteney and reliability of the response both within and belween subjeets. For example, stimulating the SO in alert rats, prcpared for eh ronie stimulation and reeordi ng, c1ieits virtually identieal OOemg responses day after day (Fig. I). Thc fact that the standard crrors of thc mean are smaller than the symbols in Figs. J. 5, 7, 9, and 11 furthcr emphasizes the small within-subjeet v.ariabili ty. ßetween subjeets, variabi lity is also remarkably smal !. For example, the response la'teney of the SO-elicilcd blink among 83 human subjects had Icss than 10% variability (Kimura 1989). Simi1arl y. the entire range of lateneics for cornea lly clieited blinks was onl)' 11.8- 13.6 ms for all our anesthetized rat subjeets. Thus, thc consisteney and de­pcndability ofthc bli nk reflex allowed us to reveal changes reliably with sma ll numbers of subjects.

EjJecr o[ nicotine 011 the blink reflex

Chewtng lIicotine gum. Rapidly chewing 10 mg of nieotine in gu m decreased blink magnitude and incrcased blink latcncy for a normal human (Fig. 2). Just as occurs with cigarclle smoki ng (Evinger ct al. 1988), nieotine decreased the magni tude of all OOemg components (t = 4.97, df = 14, P<O.OOI) and the ensu ring blink, but inercased the lal­cney of the R2 eomponent of the OOemg (t = 3.09,

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.,~I

R2

Fig. I. Brinks eticited by stimulation of the supraorbital branch of the trigeminal nerv!: (SO) in an alert rat. Euch trace is the average<!, rccrified orbicularis ocu!i etectromyogram (OGemg) response to 15 identical SO stimuli. The response shown by the solid line was collected 1 wcek prior 10 the response shown by the dashed line. R I , short-latcnc)" response: R2, long-latency response; Stirn, stimulus artifact

Before ~

v"

R2 Rl

j

Stim

30deg 1800 deg/s

gOms

After Nicotine

OOemg

Before Nicotine

Fig.2. A singte retlex blink of a human subjccl elici led by stimu la­tion ofthe SO ner~e before (thick Unes) and after (rhin lines) chewing 10 mg of nicotine gum. OOemg, rectificd orbicularis ocu!i EMG aClivity: Pos, upper eyclid position: Stirn, stimutus artifact; vet, lid velocity

df= 14, P<O.OI) with oul altering Rl lateney. Unlike the transient effeel of eigarctte smoking on the blink reflex, howcver, nieotine gum influenced the blink throughout the test pcriod (Fig. 3). The longer lasting effect of nieotine gum probably resulted from thc slower kinetics of nicotine absorption with nicotine gum than with eigarette smoking (Fagerström 1989; Russe!! et al. 1983). It is unlikely that

the 10 mg dose of nicotine gum was responsible for the longer lasting effect bccause chewing 10 mg of nicotine gum yields blood levels of nicotine only slightly higher than those associated with cigarctte smoking (Fagerström 1989). Thus, in humans, nicotine appears to bc the agent in eigarclle smoke responsible for modifying the blink reflex.

I ntravellOIlS illjeetion of lIieotine. Intravenous injection of nicotine into uretbane-anesthctized rats dccreased the OOemg amplitude of corneally elicited blinks and in­creased OOemg latcncy (Fig. 4). 80th rats and humans

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Fig. 3. R2 amplitude (. ) and latcncy (0) relative to mean prenicotine values (dolfed line) with supraorbital nerve stimulation before and after chcwing 10 mg nicotine in gum for one human. Each point is the mean of three blinks

BefaTe Nicotine

After Nicatine

.Jot Stirn 20ms

Fig. 4.. Averaged (5 trials), rectified OOcmg responses elicited by corneal stimula tion (Stirn) berore and after intravenous nicotine injcCliOrl (1.3 mg/kg) in a urclhanc·aneSlhetizcd rat

exhibit on ly a single componenl of OOemg activity with blinks elicited by stimulation of the cornea (Figs. 4, 6; Accornero cl al. 1980; Bcradelli el al. 1985), in contrast to activity shown with SO-clicited blink s. In some rats, the OOemg response evoked by corneal stimulation appeared similar to the R 1 and R2 responses clicited by SO stimula­tion in the alert rat (e.g., Fig. 4). More often, however, rats exhibited a single burst of OOemg response with corneal stimulation (Fig. 6). The corneall y c1icited and SO-elicited blinks exhibited very different physiological propcrties. For example, it was impossible 10 elicit an R l and R2 response with SO stimula tion in urethane-anesthetizcd rats, even though corneally c1icited blinks were robust. Neverthelcss, the latency of thc R2 cornponent of SO­elici ted blinks (Fig. 12) and the latency of Ihe corneally elicited OOemg activity (Figs. 4, 6) were similar.

Nicotinc doses equal to or abovc 0.32 mgfkg altered both blink amplitude and latency (Fig. 5). Doses of nico­tine larger than 0.32 mgfkg failed to increase the magni­tude of the effect on the blink reflex. Thus, doses equal Lo or greater than 0.32 mgfkg elicited the maximal elrcet of nicotine in the anesthetized rat. The increase in latency and deerease in OOemg ampli tude we re statistically signi­fleant for aB animals (P<O.OOI for alll tests). At all thrce nicotine doses tested, the injection compleLely suppressed the blink reflcx for 1- 3 min and redueed the amplitude for another 3- 7 min. Blink latency tended 10 recover more quickly then blink amplitude. For example, the OOemg amplitude ofthe rat receivi ng 1.3 mgfkg nicotine returned to prenicotine levels approximately 8 min after the nico­line injection, but blink latency recovered in less than 5 min. This observation suggesls that nicotine difTer­entially afTects blink amplit ude and latency. Consistenl wilh this co nc\usion, nicotine doses of 0.25 rngfkg pra­duced a strang suppression ofbli nk amplitude, but li ttle or no increase in OOemg latency (Fig. 6).

Decerebration. Nieotine could act directly on the pontine­medullary neurons subserving thc blink reflex, or nicotine

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Fig. 5. OOemg amplitude (filled symbols) and laleney (open symbols) relative 10 mean prcnicoline values (dotred fine) in rats receiving 0.32 ( _ ,0 ), 1.3 (e. 0), or 2.5 ( . , 1::. ) mg/kg nicotine. Each point is the mean of Ihree blinks

Before Decerebration After Decrebration

Before Nicotine

i~\\ After Nicotine j

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Fig. 6. A vcraged (5 tria ls), rectified DOcmg fTom corneal stimulation (Slim) befOTC and after 0.25 mgJkg nicOline, showing Ihe efTCCI or deccrcb rat ion

could modify dcscending neurons that modulate the blin k reflex. Wc elim inatcd descend ing influenccs by tcsting the effect of nicotine on the blink reflex following preeollieular dccerebration. Deccrcbration abolished the actions of intravenously injectcd nicotinc without climinating the blink refl ex (Figs. 6, 7). Prior to decerebration, thc de­erease in ODemg amplitude following a nieotine injeetion was statistieally signi fica nt (I = 2.83, dJ = 14, P <0.025), but it was no t signifiea nt after dccerebration (I = 0.4, df= 14, P=n.s.). Thus, nicotine must modulate the blink reflex by acting primarilyon ei reuits rost ral to the decere­bralion. Since the strialum is rieh in nicotinic receptors and basal ganglia disorders modify the blink reflex, nieo­ti ne could modify blinking through the basal ganglia.

Effec/ oJ slIbsralltia "lyra slimll/mion on the blink reflex

In t wo urethanc-anesthetized rats, electrical stimu lation in the region of the substantia nigra mimieked the effect of nieoti nc injections on the blink reftex (Figs. 8, 9). Stimu­lati ng the cornea o nccevery 10 s produccd modest habi tu­ation o f the blink re ft ex. Prcceding corneal stimulation by 7 s of 10-Hz, 40-p A, 6O-ps stimulati on on thc dorsal edge of the substanl ia nigra reticulata, however, decreased OOemg amplitude, inereaS(..'(! the rate of habituation, and incrcased OOemg latcney (Figs. 8, 9) relative to co rnea] sti mu lation alone (Figs. 8, 9; OOcmg amplitude: I = 2.74, dJ= 13, P < 0.025; latency: t =5.34, dJ= 13. P <O.OO I). De­terioration in the condition of the animal or the rc­sponsiveness of trigeminal palhways did not cause this modification of thc blink refl ex . Forty minutes after com­bined nigral and corneal stimulation, eJcctrical stimul ation of the cornea alone elicited blink s of normal amplitude and latency relative to the blinks evoked prior 10 nigral stimula tion (Fig. 9). These data are consistent with the suggestion that nico tine aets through the basal ganglia to decreasc bli nk ampli tude and increase blink latcncy. 1fthis hypothesis is eorrect, blocking dopami ne receptors sho uld

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Fig. 7. ODemg amplitude relative 10 mean prenicoline values (do/ted line) befoTe aud after 0.25 mgJkg nicotine, befote ( ... ) and after ( (:.. ) decerebralion. Each poillt is Ihe mean of three blinks

Normal Stimulated

Slim Stirn

Fig. 8. Reetified OOemg of tria ls I, 4, a nd 8 elici led by corneal stim ulation (Slim~ J4i , normal. Righl , corneal stimulation pref.'eded by nigral stimulation. Dashl'd Iinl'S indicatc Slart ofOOcmg activi ty o n tria l I

prevenl nieot inc's modulat ion of thc blink reflex bccause nicotine causes the relcase of dopamine from prcsynaptic termina ls in the stria tum (Giorguieff-Chesselet CI al. 1979; Sakurai el al. 1982; Sehultz and Zigmond 1989).

Phurmucologicu/ mUl1iplllatioll oJ fhe dopamine recep/ors

EjJect oJlwloperidol Oll Ulles/he/lzed rars. We investiga led the role of 0 1 rcceptors in modulating the blink reflex by

examining the effeet of nieotine on the blink reflex before and after administration of halopcridol, a D1 reccptor antagonist (Figs. 10. 11). Before halopcridol administra­tion, nieotine injeetion eaused the expccted deerease in OOemg magnitude with both eorneal- (Fig. 11; t = 6.53, df = 11 , P <0.001) and air-puff-cl icited (Figs. 10. 11; l = 2.54, df= 11 , P < O.025) blink-s. fnjcction of halopcridol (0. 15 mgfkg i.p.), however, red uced or abolished nieotine's modification of the blink reflex (Figs. 10, 11 ; cornea: t

= 1.39, df= 14, P =n.s.; air puff: t = 1.5, df=14, P = n.s.). Thus, nieotine appears to modulate the blink reflex via dopamine rcceplors.

Givcn that blockade of O 2 rcceptors prevents nicotine's modification of the blink reflex, activation of 0 2 receptors should mimic nicOl ine's efleel on the blink reflex. We investigatcd the efleet of apomorphine, a O2 agonist, on ODemg activity elieited by SO stimulation in chronie-

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Fig.lJ. OOcmg amplitude (filled symbols) and latency (open symbols) relative to first normal rene)[ blink ( 4 , 1::. ). (. , 0,) blinks preceded by nigral stimulation; ( . , 0 ,) recovery. Each point is the mean of thrce blinks

Normal A

B Post Nicotine

D

65ms

Fig.IO. A- D Rectified OOemg of individual blinkselicitcd by an air­pulf st imulus berOTe (A, C) and afler (8 , D) 0.25 mg/kg Ricoline. A, 8 BefOTe haloperidoL C. DOm: houT after 0.15 mg/kg haloperidol

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Fig. 11. ODemg amplilude of air-puff (open symbols) and corneal (jilled symtwls)-elicited blinks relative to mean preniootine value (dotled line) before ( 4 , ö ) and 1 h after ( • • 0) 1.5 mgfkg haloperidol

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! Jü~ Stirn

Fig.12. Individual, rectified ODemg responses 10 supraorbilal nerve stim ulation in an alert rat befoTe (top Imce) and after (botlom Irace) 4 mg/k.g apomorphine

ally prepared, alert rats (Fig. 12). lnjeclion of 4 mg!kg apomorphine produced a decreasc in both R 1 and R2 amplitude of the SO-clicited evoked blink (t = 4.47, df :: 16, P<O.OOI), but primarily increascd R2 1atcncy (t =: 5.49, df = 11, P < O.OOl; Fig. 12). Since Ihis dose of apomorphine caused the rats to chcw incessantly, OOcmg reeords become noisy, and accu rate measurcments of lateney and amplitude were possible only on 10- 15 selec­ted records for each rat. Oflhe two rats tested, one sbowed no change in R 1 latency following apomorphine, while thc seeond exhibited an insignificant, I-ms increase in RI latcncy. In contrast, R2 latency increased by 7.2 ms in tbc firs t rat and 5,4 ms in the second. OOemg amplitude ofthe two blink components dccreased by 31'% in thc fi rst rat and 41 "/0 in thc second rat. The elfeel lasted for the enlire hou r foliowing the apomorphine injcction. This result is qualitativc1y idcntical to that obtained with nicotinechew­ing gum. Thus, activation of D2 reccptors mimics the effccts of nicotine on the blink reflex.

Discussion

Comparison oJ rat and human reflex blinks

The reflex blinks of ra ts and huma ns are very similar. In both specics, stimula tion orlhe SO elieits a pair of OOcmg responses, RI and R2 (Figs. I, 12; Kimura 1989; Shahan i and Young 1973, for review). R I, whieh shows liule in tersubjl."Ct variabi li ty, probably arises fro m a three· neuron arc. The R2 lateney varies widely among subjects and c!early a rises from an oligosynaptie eircuit (Ongerboer de Visscr 1983). T he rapid aetiva tion oflhe rat OOemg from SO stimu lat ion (4-7 ms) strongly suggests thai rat R I also a rises from no more than a trisynaptic ci rcu it. As in humans, the rat R2 lateney of 14- 20 ms is eonsistent with an oligosynaptic dreuit. In the two speeies, cornea] stimu lation usually elici ts a si ngle burst ofOOemg aClivity (Fig.4; Accornero et a l. 1980; Bcradelli el al. 1985). The observa tion th aI corneal blink s and the R2 response have a simi lar lateney led to the proposa l tha i R2 and cornea I blinks o riginale from Ihe same neural ei rcuit (e.g., Rushworth 1962; K ugcJberg 1952). Reeeni human sludics, however, demonstrated that the two types of blink cou ld not utilizc identieal d reuitry (Beradelli CI al. 1985; Cruccu et al. 1986). Similarly, the fact Ihat there was differentia l modification of Ihe corneal blink reßex and the R2 respo nse in two eases shows that the same neu ral drcuit a lso docs not subserve the two responses in the ra t. First. although si milar to R2, the corneal blink has a distinctly shorter lateney th an the R2 component (Figs. I, 4, 12). Second, in urethane·aneslhetized rats, the corneal reflex is brisk, but SO·evokcd blinks never have an R2 eomponenl. Finally, the regions of the spinal trigeminal nucleus f(,o.ceiving primary afferents from the cornea and the SO are d ifferent (Marfurt and Dei Toro 1987; Pcllegri ni and Evingcr 199 1). Ncverthelcss, the cornea! blink a nd the R2 response could share some interneurons, si nce nicotine affects the two blinks similarly in the rat.

Despite the comparablc neural eircuitry underlyi ng blink s in ralS and humans, two differcnces exist. First, the rat blink reflex is not eonsensual, while both the human corneal blink reflex and the human R2 response with SO slimu la tion a re bilateral. In alert rats, sti mulation of the SO appcars only to aetivate thc cOnl talatcral OOemg whcn the rat is startlcd. In uret hane·anesthctized rats, unil ateral corneal stimulation never elicits a eontraiateral reaction. Nevertheless, both rat aud human RI responses are normall y ipsilateral. Second, the rat R I componenl is la rger and has a lower threshold than thc R2 component. The o pposite is true for IlUmans (e.g., Sanes and Iso n 1979). This seeond dilfe rcnee may refl eet the evolutiona ry significance placed on the R land R2 responses in frontal­and lateral-eyed species.

Nicorine·indllced modificarion ofrhe blink reflex

Cigarelle smoki ng transiently decreases blink magn itude and increases R21atency (Evinger et al. 1988). While other elements of dgarette smoke cou ld po ten tially modify thc blink reflex, nicotine appears to bc Ihe responsible agenl.

Nicotine gum produccs Ihe sa me alterations in the blink reflex as wbacco smok ing bUI over a Ion ger lime course. This d ilferencc may follow from the prolonged release of nicoti ne with nieotine gu m and the slowncss of gastro· intestinal uptake of nicot ine compared wilh the rapid passage of nicotine int o the bl oodstream through the lung and nasal passages with smok ing (Fagerström 1989; Rough ion 1945; RusseI! et a l. 1980, 1983). Intraveno us inject ion of nicoti ne in rat s, however, produeed the sa me modifieations as eiga relle smoking and matehed the time cou rse engendered with smoking. T hus, nicotine musl be the element in eiga retle smoke rcsponsible fo r modifying the blink reflex as we il as eye movements (Slbony Cl al. 1990).

Nieotine eould act direetly on blink reflex neurons in the lower brainslem with nicolinie receptors, or nieotine cou ld activa te a descending system impinging on blink circuits 10 modify the blink reflex indireetly. The fael that nicoti ne only modifies the latency of one component ofthe blink reflex and tha t dccerebration climinates the elfects of nicotine demonstrates that nicoti ne's main act ions are ccntrn) rat her Ihan on the o rbicularis oculi muscle. Si nce precollicula r dccerebration eliminates the nicotine effoct on the blink reflex, niCOl ine is unlikely to be acting exclusivcly on blink reflex interneurons, all of which lie ca uda l to the preteet um (Ongerbocr de Visser 1983). The abi lity of a dopaminergie receptor antagonist, haloperidol, 10 block nieotine's cffect implies that nieotine aets indio rectly on thc blink refl ex by causing the release of dop· amine. In addition to block ing dopaminc reccptors, halo· peridol also exh ibits some anticholinergic properties. This characteri stic mighl explain some of haloperidol's elim ina· tion of nicotine's alfect on (he blink reflcx. Nevertheless, the fac t Ihat apomorphine, a D 2 agonist, mimicked nicoti ne's eIfecis supports the proposal that nieotine pre· dominantly exerls il s action on the bli nk reflex via the release of dopamine. These observations implicale the basal gangl ia in nicotine's effeets o n the blink reflex.

Dopa minergic neurons of the basal ganglia are rich in nicotin ie receptors. Dopaminergie neurons of the sub· sta ntia nigra compacta eontain nicotinic reccptors on their terminals in the striatum, as weil as on their somata (Abood et al. 198 1; Bolam el a l. 1991; Clarke 1989; Clarke and Pert 1985; Hunt and Schmidt 1978; Semba and Fibiger 1989). Intravenous and subcutaneous injcction of nicotine aetivatcs dopaminergie neurons in a dose·de· pendent fashion (Clarke et a l. 1985; Liehlensteiger cl al. 1982; Westfall et a l. 1989), with a maximum increase in firing frequency eaused by 0.5 mg/kg nicotine adm inis· tered intravellously (Mereu et al. 1987). Nicotine eauses thc release of dopaminc in the st riaturn (Giorguieff­Chesselet et al. 1979; Sakurai el al. 1982; Schultz and Zigmond 1989). Thus, in ou r experimcll ts, nieotine should have markedly inereascd the release of dopam ine in the striatum. Moreover, si nee dopami ne re«ptor blockade obstrucled nieotine's elfect on the blink reflex, the in· creased release of dopamine appears to be a erueial step in modifying Ihe blink reflex.

Clinical st udies demonstrate tha i abnormally low levels of dopamine in Ihe sl riatum disrupt normal blink reflexes. Advanced stages of Parkinson's disease result in

an increascd exeitabili ty of both the R l and the R2 components of the blink reflex (Klawans and Goodwin 1969; Messi na el al. 1972; Pea rce et al. 1969; Rushworth 1962). Red uction in dopamine levels through t reatment with ualopcridol or reserpi ne also inerease the excitability of thc blink reflex (ß oiardi cl a l. 1975; Ferguson et al. 1978; Raffacle et a l. 1988). Similarly, reduction of dop­amine in rats by injeetion of 6-hydroxydopamine elevates blink reflex exeitability (Basso et al. 1991). T hese observa­tions suggest that inercased \cvels of dopamine in the striatum, such as those induced by nicotine should decrease the exeitabili ty of the blink reflex.

The basal ganglia ean infl uenee interneurons involved in thc blink reflex by modifying descending cortieal or superior eolliculus project ions. Early studics ofthc effeet of Parkinson's disease on the blin k reflex suggestcd that cortieal involvemcnt accounted fo r the inereased exeit­ability (Klawans and Good win 1969; Pearce et al. 1969). Recent studies in primates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- ind uced Park inson's disease reveal an increased inhibition of thalamie neurons (e.g. DeLong 1990), whieh should result in redueed eorlieal output. Similarly, the superior eollieulus should receive increased inh ibitjon in Parkinson's disease. Consistent with Ihis resu!! , injection of the gamma-aminobutyric aeid agonist, museimol, in to the superior colliculus produces an in­erease in blink reflex excitability similar to that present in rats with Patkinsonian-Iike symptoms (Basso et al. 1991 ; Basso and Evinger 1992). Thererore, descending cort ical and coll ieular neurons must modulate a network that inhibilS blink reflex interneurons. In this scheme, inereas­ing striatal dopaminc levels with nicotine would inerease inhibit ion of blin k reflex interneurons and decrease the magnitude of the blin k reflex.

AcknoKiJedgemenls. We wou/d like 10 thank Donna Malysl for lechnical assistancc. This work was supporte<! by NEI grant EY0739 I (CE), a Parkinson Disease Foundation summer fellowship (MAB), and a NATO fellowship from the DAAD (AKEU).

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