PVisionRes.,Vol.37,No.11,pp. 1511–1524,1997

01997 ElsevierScienceLtd.AUrightsreservedS OPrintedin GreatBritain

0042-6989/97$17.00+ 0.00

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Plasticity Monocular H-OKN NMDA Pretectum Adult frog

I

Glutamate, when activating NMDA receptors, has beenstronglysuggestedto play a key role in visualplasticityinyoung animals. Involvement of the NMDA receptor inneocorticalplasticityhas been demonstratedby studiesofphotic responses in the visual system in mammals:application of the competitive NMDA antagonist 2-amino-5-phosphonovalerate(APV) prevented some cor-tical changesnormally inducedby monoculardeprivation(Bear et 1990; Rauschecker & Hahn, 1987); itdisrupted segregation of retinogeniculate afferents inferret lateral geniculate nucleus (Hahn et 1991) andformation of a neuronal map in rat superior colliculus(Simon 1992).APV also disruptsoculardominancecolumns in 3 eyed tadpoles (Cline et 1987). Theseblocking effects of NMDA antagonists on visualresponses appeared to be restricted to the critical periodduring the development of young animals affecting theadult visual system much less (Tsumoto et 1987).However, in adult animals such as Xenopus,NMDA canrestore the ability to realign the visual tectal map afteralteration of an eye position, even after the end of the

*Laboratoirede Neuroscience Comportementaleset Cognitive, URA1939CNRS, 12 rue Goethe, 67000 Strasbourg,France.

TTowhom all correspondence should be addressed [Fax +33-08 3584 42].

critical period (Scherer & Udin, 1989, 1991; Udin &Scherer, 1990).

In previous work, we have shown such a visualplasticityphenomenonin adult frog. In lower vertebrates,monocular horizontal optokinetic nystagmus (H-OKN)displays a directional asymmetry, the stimulation in thetemporal–nasal (T–N) direction being always moreefficient than the stimulation in the nasal–temporal (N–T) directionin evokingthe reflex.In the frog, the H-OKNN–T component is even absent. However prolonged (8days) monocular deprivation obtained by unilateraleyelid suture, abolished the monocular H-OKN asym-metry by provokingthe appearanceof a N–T component.The H-OKN slow phase velocity gain which was almostnil for a N–T stimulation 1 hr after eyelid suture, wassignificantly increased 8 days later, and no longerdisappeared. The T–N component was not significantlymodified, and no difference between the gain of bothcomponentswas observed (Yucel 1990). Chronicadministrationof NMDA antagonistsfor the duration ofdeprivation prevented the appearance of a symmetricalmonocularH-OKN in frogs: followingrepeated intraper-itoneal injections of either MK 801, CGS 19755, orintrapretectal microinjectionsof APV, the N–T compo-nent did not appear and the H-OKN remained asymme-trical (Jardon & Bonaventure, 1992a).

In the frog, the pretectum and the nucleus of the basal1511

1512 B. L. N. JARDON and N. BONAVENTURE

optic root (n BOR) were shown to be the mesencephalicstructures responsible for OKN (Lazar et 1983;Montgomery et 1982; Yucel et 1991). Thepretectum receives the visual information directly fromthe contralateralretina and mainly from the central retinafor the pretectal l M(Montgomery 1981, 1985). The pretectal regionis a very heterogeneousbrain region, both anatomicallyand physiologically.It is located at the mesodiencephalicjunction. The role of the l

and of the postero-lateral thalamic nuclei is wellestablished in the genesis of the H-OKN: a pretectallesion entails the abolition of this reflex in the frog(Montgomery 1982), and the salamander (Man-teuffel 1983). Physiological analysis of theresponses of neurons of this area has shown that thepretectal cells have a preferential direction of dischargeand are inhibited for stimulationin the oppositedirection(Katte & Hoffmann, 1980). Moreover, it was stronglysuggested that the retinal output to these mesencephalicnuclei was mainly glutamatergic (Nunez-Cardozo &Kamphuis, 1995;Hickmott & Constantine-Paton,1993).A high density of NMDA receptor binding sites ‘wasevidenced in the frog pretectum (McDonald 1989).

All these data strongly suggested the involvement ofNMDA receptor activation in building up the plasticityphenomenonobserved in the frog oculomotorsystem. Inthe present paper, we have reported direct proof of theimplication of NMDA in this compensatory process.Moreover, we have analyzed to what extent lightstimulation of the open eye was necessary to evoke thisplasticity phenomenon following an NMDA treatment.Indeed, it was previouslyshown (Yiicel 1990)that,when frogs were put into total darkness during the weekof unilateral eyelid suture, the monocular H-OKNremained asymmetrical, and the N–T component didnot appear.

However, before studyingthe eventual involvementofthe pretectal NMDA receptors in the plasticity phenom-enon we have evidenced, the acute effects of the drugupon the directionality of the monocular H-OKN wereanalysed. In the first part of this report, monocular H-OKN was recorded before and after administration ofNMDA or LY 285265, an NMDA agonist (Schoepp

1991) into the pretectum or by systemicroute. In thesecond part of this paper, the effects of successiveinjectionsof NMDA or of LY 285265 on monocularH-OKN were analyzed during the week of monoculardeprivation to see if the pretectal NMDA receptoractivation could shorten the period of the build-up ofthe plasticity phenomenon. In the third part, frogs weredaily treatedwith NMDAorLY285265 in totaldarknessduring the week of “monocular deprivation”, to see towhat extent the effects of NMDA on the build-up ofmonocular symmetricalH-OKN dependon light stimula-tion.

e using the magnetic field search coil technique,in head restrained animals. Recordings were carried outbefore and after administration of the drug eitherintraperitoneally, into the occluded non-recorded eye,*or directly into the pretectumcontralateralto the viewingeye. All animals underwent eyelid suture of the left eyeunder local anesthesia and were tested 1 hr later forcontrol.

S

The frogswere placed in an optokineticdrum (300 mmin dia and 450 mm in height) with alternating black andwhite vertical stripes, distributed equally on its innersurface (10 mm wide). The drum was rotated clockwiseand counter-clockwiseat constant speed by means of anelectroniccontrolsystem.The range of the constantdrumspeeds used was between 1 and 9 degJsec. Roomillumination was kept constant at 80 lx at the level ofthe frog’s eye.

Eye H-OKN was recorded using a magnetic coilsystem as described by Koch (1977). One pair of coils(200 mm dia) carrying a current with a frequency of50 kHz, generated a homogeneousmagnetic field. Thesecoilswere mountedon a staticplatform. The sensingcoil(1 mg, diameterof the copperwire 50 pm; inner diameterof the coil 2 mm, 70 turns, provided by Sokymat,Switzerland) was fixed on the eyeball in such a way tobe oriented perpendicularly to the inter-aural axis, andwas placed in the centre of the magnetic field. Thevoltage of the sensingcoil, proportionalto the sine of thehorizontalangulardisplacement,was amplified,rectified,filtered and displayed on a paper recorder (BBC).

The system was calibrated before each recording bychecking the linear relationshipbetween the sensor coilangular displacement and the voltage induced in thesensor coil. The slow eye speed was measured using thecumulativecurve of at least three successiveslow phasesat steady state, after elimination of the eye resetting fastphases. The slow phase velocity gain was defined as theratio between the slow phase speed and the drum speed.

For purpose of data analysis, a Wilcoxon signed ranktest was used.

In order to record monocular H-OKN, the lids of oneeye were sutured, while the sclera of the other wasexposed by removing the superior eyelid under localanesthesia (Cebesine, Chauvin Blache). The frog’s headwas immobilizedby means of a nut, fixedon the skullandattached to a bar placed in the drum.

The sensing coil was secured (under local anesthesia)on the sclera with a drop of glue just before theexperiment.The drugswere prepared as describedbelowand administeredeither intravitreally,intraperitoneallyor

T

MonocularH-OKN was recorded in adult frogs

*It was previouslyshown (Jardon & Bonaventure, 1992b)that drugs,when proved to cross the blood–brainbarrier are conveyedby thebloodstreamto central structureswhen injected into the closed eye,as after systemic injections.

NMDA AND PLASTICITYIN FROG MONOCULARH-OKN 1513

directly into the pretectum, contralateral to the viewingeye.

Recordingswere carried out before and after injectionof the drug either into the closed eye (30@ of solution),intraperitoneally (50 pl of solution) or directly into thepretectum (0.2@ of solution). This last group of frogsunderwent cannula implantation into the

M of the .pretectum.

Frogs were prepared the day before the experimentunder general anesthesia (MS 222 Sandoz). Experimentswere carried out in accordance with the EuropeanCommunities Council Directive of 24 November 1986(86/609EEC). A permanent stainlesssteel guide-cannula(o.d. 0.4 mm; id. 0.3 mm) was chronically implantedinto the pretectum using a stereotactic procedure. Thecoordinates, according to the atlas of Wada et (1980)were A/P, 0.9 mm; M/L, 0.5 mm; D/V, 0.4 mm; with ahead angle of 20 deg. The intersection of the anteriorborder of the left tectum and the sagittal midline wastaken as reference.

The guide cannula, as well as the nut previouslydescribed, were anchored to the skull using retainingscrews and dental acrylic cement. A stainlesssteel sterilemandrel (o.d. 0.27 mm) of the same length was insertedinto the guide cannula to avoid obstruction.

Unilateral microinjections were performed in awakeanimals. A stainless steel injection cannula (o.d.0.28 mm; id. 0.18 mm) was introduced into the guidecannula, so that it extended 0.1 mm beyond the tip of theguide cannula. The injection cannula was connected to1 pl Hamilton microsyringe via polyethylene tubingfilled with distilled water. It was filled with the drug byaspiration.A small air gap separated the two liquids.Thedrugs or the vehicle were injected in a volume of 0.2 pl

over 20 sec. Movement of the air gap down the tubingwas indicativeof successfuladministration.The injectioncannula was left in place for an additional 30 secfollowingdrug administration.The mandrelwas replacedinto the guide cannula. The theory that a pretectalinjection of saline has no effect upon the recorded OKNwas tested.

ascertain the injection site (Fig. 1), frogs weredeeply anesthetized in MS222 following 8 days of post-injectionsurvival time. After transcardiac.perfusionwith0.9% saline followed by 4% formalin, brains wereremoved and placed into a 470 formalin solution.Paraffin-embeddedbrains were cut in 20pm slices andprocessed with cresyl-violet stain. All the animals inwhich the cannula track could not be clearly localizedwere excluded from the study.

NMDA (Sigma Chemical Co) was injected directlyinto the pretectum,since it does not cross the blood–brainbarrier. LY 285265 (D,L-tetrazol-5YL)(glycine) (ElyLilly Company) (Schoepp a 1991) is an NMDAagonist which crosses the blood–brain barrier; it wasinjected into the occluded eye, intraperitoneally ordirectly into the pretectum. The subtype of NMDAreceptorsit bindswith is presentlyunknown(D. Schoepp,personal communication).

The drugs were respectively dissolved in phosphatebuffer saline (PBS) 0.02 M (pH = 7.4) or in distilledwater. The concentrationsused were determinedfrom theliterature and from pilot studies.

The concentration of NMDA injected into thepretectum was 2,9 ng (10-4 M), and 0.29 ng (10-5 M).The concentrations of LY 285 265 injected into the

FIGURE1cannula tra

Photomicrographshowinga coronalsection (20 #m) of the mesodiencephalicregionof the frogbrain. The~ckrepresents the site of injection and is located medially to the anterior part of the optic tectum in th,

L M

tip of thee nucleus

1514 B. L. N. JARDONand N. BONAVENTURE

(A)

‘-N U

1degls

N-T

b3 degls

‘-”JJ-AA6 degls

N-T

T-N

(B)

T-N

N-T

T–N

‘-T Ww

9 degls

N–T

FIGURE 2. Coil recordings of monocular eye OKN evoked by four different constant dmm speeds in a monocular viewingcondition,before (A) and half an hour(B) followingan injectioninto the closed eye of LY 285265 (0.21 #g). The speed andthedirection of the stimulus are indicated on the left of the recordings.Calibration:vertical bar, angular displacementof 10 deg;

horizontalbar: 10sec duration.

occluded eye were 21 pg (5 x 10–3M), 2.1 pg (5x10-4 M), 0.21 pg (5x 10-5 M), 21 ng (5x 10-6 M);intra eritoneally,

P3.5 pg (5x 10-4 M), 0.35 pg (5X

10- M), and into the pretectum 0.14 ng (5 x 10-6 M).

P

Recordings began half an hour after intravitreal orintraperitonealdrug administration;they began immedi-ately following intrapretectal drug injection in order to

disregardthe drug diffusioninto cortical structures.Somerecordingswere made later to test the reversibilityof thedrug effect.

AH animals underwent eyelid suture of the left eyeunder local anesthesia, and were tested 1hr later forcontrol.

Three seriesof experimentswere conducted:in the twofirst series, all experiments were done in normal day–night illumination.In the first series, studying the acute

NMDA AND PLASTICITYIN FROG MONOCULARH-OKN 1515

effects of NMDA, frogs were tested before and afteradministrationof variousconcentrationsof drug, injectedeither by systemic route (n = 44) or directly into thepretectum (n= 35).

In the second series of experiments, studying the roleplayed by NMDA in the plasticity phenomenon, frogswere divided into four groups:

1 or control group (n= 13) received no druginjection during the week of monocular deprivation.

2 received daily an injection of PBS(phosphate buffer saline, 0.05, pH=7.4) during 8 daysof monocular deprivation, either by intraperitonealroute (n = 7) or directly into the pretectum (n = 6).They were tested daily.

3 = frogs received an intraperitonealinjection of LY 285 265 (3.5 pg, n = 20 or 0.35 pg,n = 32) the first 2 days of the monocular deprivationweek. They were tested daily.

4 frogs received a unilateral microinjection ofeither NMDA (0.29 ng, n = 21) or LY 285 265(0.14 ng, n = 12) into the pretectum contralateral tothe open eye, the first 2 days of the monoculardeprivationweek. They were tested daily.

In the third series of experiments, frogs were put intotal darkness the week of “monocular deprivation”.They were divided into the same four groups:

1 or control group (n =6) received no drugduring the week of monocular deprivation.

2 = received an injection of PBS daily,either by intraperitoneal route (n = 6) or directly intothe pretectum (n = 10).

3 = received an intraperitonealinjectionof LY 285265 daily (0.35 pg, n = 15 or 3.5 pg, n = 8)during the 7 days of total darkness.

4 = received a unilateral microinjectionof NMDA daily (0.29 ng, n = 17) or LY 285 265(0.14 ng, n = 25; 1.4 ng, n = 18) into the pretectumcontralateral to the open eye, during the 7 days of totaldarkness. Cannula implantation was conducted 1 dayprior to placing the animals in total darkness.

Intraperitoneal and intrapretectalwere carried out in weak red light.

c

drug administration

The monocular H-OKN was recorded in each animal,before injection,at four differentdrum speedsand in bothdirectionsof stimulation.In these conditions,the viewingeye predominantlyfollowed the stripesmoving in the T–N direction (n = 79). When the stimulationwas appliedin the N–T direction, no eye movement was detected,irrespective of the drum speed tested [Fig. 2(A)].Injection of PBS did not change the OKN gain when

1.200

1.000

F

0.800

“~ 0.600

0.400I

0.200t

4(A) O T-N before injection

A N–T before injection

● T–N after injection

A N–T after injection

%$-..~

0 ~o 2 4 6 8 10 0/s

Drum speed

1.000

0.800 [

= 0.600.-$

0.400 I

0.200

}

Ii(B) 0 T–N before injection

A N–T before injection

\

● T-N after injectionAN–Tafter injection

1

0.000 ~o 2 4 6 8 10 0/s

Drum speed

1.000

0.800

F

0.600a.-

80.400

I

0.200 }

(

AO T–N before injection

A N–T before injection

● T–N after injection

A N–T after injection

1,

o.OOOo~o O,s

Drum speed

FIGURE 3. Means values of slow phase velocity gain of monoculareye OKNbefore (opensymbols)and 30 min after injectionsof LY 285265 (filled symbols) into the closed eye. (A) 21 #g, n = 9; (B) 2.1 pg,~ = 10; (C) ().21pg, n = 6. Tire vertical bars indicate the standard

deviation.

administered either intraperitoneally (n = 3), into theoccluded eye = 3), or into the pretectum (n = 3).Additionally, surgical cannula implanted into the pre-tectum did not modify the control OKN.

a

spontaneous eye movement was observed afteradministration of LY 285 265, irrespective of the con-centrationused (21 pg: n = 9; 2.1 pg: n = 10; 0.21 ,ug:n = 6; 21 ng: n = 2).

The H-OKN recording started 30min after druginjection pig. 2(B)]. For a T–N stimulation, no changewas noted in the monocular H-OKN when compared tothat recorded before injection for the three highest drumspeeds tested [Fig.3(A, B, C)]. The averagevelocitygain

1516 B. L. N. JARDON and N. BONAVENTURE

T d T ~

3degls

N-’r —- “-, m

‘-N A - J - - J‘-N6deg/s

N ‘ N ~

I

‘ -‘ v

9degls

N-T q ~-’ p-

11

(A) (B)

T-N ~ T

N N

T J

N N

‘-N J -

N-T

I__N-T

‘-N -’-”N N

L L

—W—Y——=——————W

FIGURE 4. Coil recordings of monocular eye OKN evoked by four different constant drum speeds in a monocular viewingcondition,before (A) and immediatelyfollowing(B) a unilateral microinjectionof either I: LY 285265 (0.14pg) or 11:NMDA

(0.29 ng) into the nLM. Calibration:vertical bar, angular displacementof 10deg; horizontal bar, 10sec duration.

did not change significantly from the control value.However, for the lowest drum speed (1 deg/see) thevelocity gain markedly and significantly increased(f’ < 0.005) for the two strongest drug concentrations[Fig. 3(A, B)]. In particular for the highest one, thevelocity gain even exceeded 1 [Fig. 3(A)].

For N–T stimulation, frogs displayed an eye H-OKNwith slow phases following the alternating blacldwhitevertical stripes, and resetting fast phases which were notobserved in control animals (Fig. 2). The averagevelocity gain which was null before injection increasedfor all drum speeds tested [Fig. 3(A, B, C)]. The H-OKNbecame symmetrical with identical T–N and N–Tcomponents. The difference between the velocity gainof H-OKN evoked by a T–N stimulationand that evokedby a N–T stimulation, which was significant beforeinjection was no longer significant after LY 285 265(P> 0.2) irrespective of the drum speed and the drugconcentrationused.Three hours after injectionof LY 285265, the N–T component was still present. This effectproved to be reversible, since 24 hr after injection,the N–T component has totally disappeared. The same resultswere obtained when LY 285 265 was injected byintraperitonealroute (n = 11).

H ii c

The effects of both drugs were identical: afteradministration of LY 285 265, 0.14 ng = 8) orNMDA, 0.29 or 2.9 ng = 13) frogs not only followedthe alternating black/white vertical stripes in the T–Ndirection, but also those moving in the N–T direction[Fig. 4(1and II)].

Slow phases and resetting fast phases were observedfor both directionsof stimulation.The H-OKN recordedin the T–N direction was not modified compared to thatrecorded before injection, even at the lowest drumspeeds,converselyto what was observedafter intravitrealadministrationof LY 265 285. The slow phase velocitygain of the T–N component was not significantlymodifiedcompared to that of the control.However, therewas a significantincrease (f’ < 0.005) in H-OKN gain forthe N–T stimulation at the four drum speeds tested forboth drugs used. The monocular H-OKN becamesymmetricalwith both components. [Fig. 5(A, B)].

At a lower concentration 5) NMDA did notprovoke an effect upon the monocular H-OKN, and the

NMDA AND PLASTICITYIN FROG MONOCULARH-OKN 1517

1.000 r (A) O T–N before injection

I A N–T before injection0.800 ~

● T–N after injection

~<

AN-T after injection0.600

G.-ao 0.400

J-W0.200

0.000 ~o

1.000

0.800

0.600a.--

0 0.400

0.200

In mm ➤

2 4 6 8 10 0/s

Drum speed

(B) o T–N before injection

A N-T before injection

I

. T–N after injection

A N-T after injection

. . . .0 2 4 6 8 10 0/s

Drum speed

FIGURE5. Meanvalues of slowphase velocity gain of monoculareyeOKN, before (open symbols) and 10min after unilateral microinjec-tions (filled symbols)of (A): LY 285265 (0.14 rig),n =8; (B) NMDA(0.29 rig),n = 13 into the nLM contralateral to the open recorded eye.

The vertical bars indicate the standard deviation.

N-T component did not appear. Frogs died at concentra-tions higher than those usually used.

M f im d di

1 or control group = 13): frogs received nodrug nor vehicle injection; 1hr after unilateral eyelidsuture, the frog’s open eye followedpredominantlythestripes moving in the T–N direction, and no eyemovement could be detected when the stimulus wasapplied in the N–T direction, as mentionedabove [Fig.6(A)].

After 8 days of monoculardeprivation,frogs displayeda monocular eye OKN when stimulated in the T–Ndirection,but also when stimulated in the N–T direction.The slow phase velocity gain which was almostnil for anN-T stimulationincreased significantlyand no differencebetween both T–N and N–T componentswas observetas has been previouslydemonstrated(Yiicel 1990).

2 = Following a week of monoculardeprivation with daily intraperitoneal 7) orintrapretectal = administration of PBS, frogsfollowedthe stripesmovingin theT–N as well as in theN–T direction.Thus, chronic injectionsof PBS did notprevent or improve the appearance of the N–T com-ponent during the 8 days of unilateral eye deprivation

[Fig. 6(B)]. The difference between the slow phasevelocitygain of the OKN evokedby a T–N stimulationand that evoked by a N–T stimulation was no longersignificant (P > 0.3) for all drum speeds used [Fig.7(A)].

3 On the first 2 days of visualdeprivation frogs received an intraperitoneal injectionof LY 285265 at a concentrationof 0.35 pg =or = They were tested daily during theexperimentalweek.

After the first injectionof LY 285265, frogs displayeda symmetricalmonocularOKN with almost identical T–N and N–T components as was seen previously. Thedisinhibitory effect upon the N–T component lasted atleast 4 hr; it was totally reversible: 24 hr after injectionthe N–T component had totally disappeared. At thismoment, 24 hr following the first injection, a secondinjection of LY 285 265 at the same concentration wascarried out and monocularOKN was again recorded.TheN–T componentreappearedwith the same characteristicsas those observed before, in particular, the velocity gainwas again identical to that measured from the T–Ncomponent. However, in contrast to what was observedafter the first injection, the N–T component no longerdisappeared (without complementary injection). Themonocular OKN recorded on the following days stilldisplayed an N–T component, identical to the T–Ncomponent, the H-OKN remaining symmetrical [Figs7(B) and 8(A)].

The same results were obtained with both concentra-tions of LY 285265.

Each of the first 2 days of visual deprivation,frogs received a unilateral drug administration ofNMDA (0.29 ng, n = 21) or LY 285 265 (0.14 ng,n = 12) into the pretectum contralateral to the openeye.

Frogs were tested immediately after administrationofthe drug: as seen above, they displayed a symmetricalOKNwith both identicalcomponents.However, the N–Tcomponent disappeared as soon as the dmg effectvanished. Following the second intrapretectal adminis-tration of the drug, however, the N–T componentreappeared with the same characteristics as those of theT–N component which remained identical to thatrecorded in the control before injection [Fig. 8(B)]. Themonocular H-OKN was symmetrical. In this case also,the H-OKN recorded following an N–T stimulation didnot disappearon the following days, without adding anysupplementarydrug injection.

Thus, followingtwo successiveinjectionsof NMDA orNMDA agonist (over a period of 24 hr), the plasticityphenomenonseen after a week of monocular deprivationin control animals is built up earlier, only 2 days aftereyelid suture.

We wanted to know the optimaldurationbetween bothinjections, in order to obtain a permanent N–T compo-nent when LY 285265 (0.35 pg) was intraperitoneallyinjected. The results are shown in Table 1.

1518 B. L. N. JARDON and N. BONAVENTURE

(A)

T-N

1 degls

N–T

T–N

3 degls

N-T

T-N

6 degls

N-T

T-N

9 degls

N-T

1

(B)

T-N

T-N

‘-TwT-N

N-T

T-N

N-T

FIGURE 6. Coil recordings of monocular H-OKN evoked by four different constant drum speeds (A) 1 hr after monoculareyelid suture; or (B) following8 days of monoculardeprivationwith daily intraperitonealinjectionof PBS.Calibration:vertical

bar, angular displacementof 10deg; horizontalbar, 10sec duration.

Thus, to obtain a durable and permanent N–Tcomponent, the delay between both injections could befrom 3 to approx. 48 hr.

M f im 1 w

In order to study the role of light stimulation in these

plasticity phenomena, the same experiments as thoseconductedin normal illuminationwere carried out in totaldarkness for 1 week.

1 or control group = Frogs received nodrug nor vehicle injection; after 8 days of monoculareyelid suture,frogsdisplayedan asymmetricalH-OKNwith a T–N component identical to that of controls.

NMDA AND PLASTICITYIN FROG MONOCULARH-OKN 1519

~ 0.0 ~o 3 6 9

DRUM SPEED (DEG/S)

~ 1.000

<0

[

(B) ● T–N> 0.800~

o N–T1-

u$ 0,(300 ~

o 2 4 6 8 10

DRUM SPEED (DEG/S)

FIGURE7. Mean values of slow phase velocity gain of monocularH-OKN (A) 1hr (circles) or 8 days (triangles) after unilateral eyelidsuture. (B) Three days after unilateral eyelid suture, the frogs havingundergone two daily intraperitoneal injections of LY 285 265(0.35pg). Filled symbols, slow phase gain for T-N stimulation;opensymbols, slow phase gain for N–T stimulation. The vertical bars

indicate the standard deviation.

The N–T component did not appear. This observationconfirms a previous one (Yucel et 1990).

Frogs received either an intraperitoneal(n= 6) or an intrapretectal (n= 10) administrationofPBS daily. Under these conditions, the N–T compo-nent did not appear, the H-OKN recorded following aT–N stimulation was identical to that recorded innormal conditions of illumination. The H-OKNremained asymmetrical [Fig. 1O(D)].

These frogs received an intraperitonealadministration of LY 285 265 (3.5 pg, n = 8;0.35 ~g, n = 15) daily during the 7 days of totaldarkness.They were tested in the optokineticdrum 10-15 min after being brought out from darkness. Theanimals displayed an H-OKN for a T–N stimulation,but the N–T stimulation was unable to provoke anynystagmicresponse.The animalswere then placed intoa day–night illumination for 24 hr, without receivingany other drug infusion. The H-OKN recorded at thismoment displayed a T–N component identical to thecontrol; an N–T component appeared with the samecharacteristics as those of the T–N component. Themonocular H-OKN became symmetrical [Fig. 9(A)]and remained symmetrical for many days later.

(Animals were not tested beyond 15 days.) Thevelocity gain was identical for both components [Fig.lIn some experiments (n= 8, LY 285 2650.35 pg)

animals were maintained for only 4 days in totaldarkness: results were analogous to those describedabove, when they remained for 7 days in total darkness,i.e., symmetrical H-OKN was recorded when animalswere placed for 24 hr in day–night illumination.

Animals received a unilateral microinjectionof either NMDA (0.29 ng, n = 17) or LY 285 265(1.4 ng, n = 18; 0.14 ng, n = 25) into the pretectumcontralateral to the open eye. This treatment wascarried out during 7 days in total darkness.Frogs werethen tested in the optokinetic drum 10-15 min afterbeing brought into the light. Animals immediatelydisplayed a symmetrical H-OKN with both identicalT–N and N–T componentsas soon as they were placedin the moving drum [Fig. 9(B)]. The differencebetween the slow phase velocity gain of the H-OKNevokedby a T–N stimulationand that evokedby an N–T stimulationwas no longer significant (P > 0.3) forall drum speeds [Fig. IO(A,B)]. The effects of bothdrugs were identical,but it should be noted that at thelowest drum speed (1 deg/see) the gain was increasedup to 1 for the T–N component following NMDAadministration.

The presence of the N–T component for at least 15days was checked (the test was not carried out beyondthis time). In some experiments = 12), frogs weremaintainedfor only 4 days in total darkness:resultswereanalogous to those described above, when animals weremaintained for 7 days in total darkness.

Thus, in total darkness, frogs received daily either:

(1) a unilateral injection of NMDA or NMDA agonistinto the pretectum contralateral to the open eye for4 or 7 days: as soon as the animalwas brought intolight, the recorded monocular H-OKN was im-mediately symmetricaland the N–T componentnolonger disappeared.

(2) or an intraperitoneal(symmetrical)injectionof LY285265 for 7 or 4 days: as soon as the animal wasbrought into the light, the monocular H-OKNproved to remain asymmetrical, only the T–Nstimulation was able to provoke the reflex.However, if the frog was placed for 1 day innormal day–night illumination, the N–T compo-nent appeared and no longer disappeared.

In monocular viewing conditions, frogs display anasymmetricalhorizontal OKN, reacting only to stimula-tion in the T–N direction and not to stimulation in theoppositedirection.However, our resultsdemonstratethatsystemic or intrapretectal administrationof respectivelyNMDA agonist(LY 285 265)or NMDA itself, causes the

1520 B. L. N. JARDON and N. BONAVENTURE

(A) (B)

T–N N–T T-N N–T

J—J—J—J~ -1 degls

. UCg,, “ “~v

6 degls “&

%--- ~9 degls

FIGURE8. Coil recordings of monocularH-OKNevokedby four different constant drum speeds, 3 days after monoculareyedeprivation, the frog having undergone two daily (A) intraperitoneal injections of LY 285265 (0.35 pg); (B) unilateralmicroinjectionsof LY 285265 (0.14 ng) into the pretectum contralateral to the viewingeye. Calibration:vertical bar, angular

displacementof 10 deg; horizontalbar 10sec duration.

appearance and the increase of a N–T component, thusabolishing the asymmetry of the reflex.

The systemic injection of LY 285265 does not enablelocalization of its site(s) of action. However, the similareffect of the drug, when injected either by general routeor directly into the pretectum, leads us to conclude thatNMDA receptors involved in this process must belocalized at least partially in the pretectum. The highdensity of NMDA binding sites found in this structure(McDonald a 1989) supports this conclusion.

We have previously shown that, in frogs, prolongedunilateral eyelid suture (8 days) abolishedmonocularH-OKN asymmetry, provoking the progressiveappearanceof an N–T component which does not exist in controlanimals. Chronic administration of NMDA antagonistsduring the week of monocular deprivationprevented theabolition of monocular H-OKN asymmetry: the N–T

componentdid not appear, remaining almost nil (Jardon& Bonaventure, 1992a). Conversely, the results of thepresent paper indicate that the administrationof NMDA,or of a potent agonist, such as LY 285265, on the first 2days of monocular deprivation, reduces the delay of theappearance of an N–T component and at the same timeabolishesthe monocular H-OKN asymmetry. One of themost interesting points is that the monocular H-OKNremains symmetrical the following days, without addi-tional drug administration. Thus, NMDA receptoractivationseems to reduce the duration of the compensa-tory phenomenon development seen in control animals,during the week of monocular deprivation. Therefore,glutamate, when acting through NMDA receptors,appears to be involved in the mechanism underlyingsuch a plasticityphenomenonobserved in adult animals.This has already been shown in another model of visual

TABLE 1.

Delay between both injections Number of frogs Durationof the observationsof a stable N–T component

3 hr 26 17 days at least’24 hr 17 9 days at least*48 hr 5 11 days at least5 days 5 N-T appeared and was maintained only the day after each injection, after that it vanished

*For technical reasons, it was not possible to test the frog beyond this period. Delay between both intraperitoneal injections of Ly 285265(0,35 Kg)necessary to obtain a stable N-T component.

NMDA AND PLASTICITYIN FROG MONOCULARH-OKN 1521

(A) (B)

T–N N-T T-N N–T

M

9,%,s * 1

FIGURE 9. Coil recordings of monocular eye H-OKN evoked by four different constant drum speeds, the frog havingunderrzoneseven dailv successive (A) intraperitoneal injections of LY 285265 (0.35 @ in total darkness, recordings beingtaken~4 hr after the a~imalswere againpla~edin normaiday-night illumination;(B) un~ateral microinjectionsof LY>85 263(0.14 ng) into the pretectum contralateral to theopen eye in total darkness,recordingsbeing achievedimmediately(10-15 rein)after the animalswere againplaced in normalday-night illumination.Calibration:vertical bar, angulardisplacementof 10 deg;

horizontalbar, 10sec duration.

plasticity in frogs (Udin & Scherer, 1990).However,whyare two successive drug administrations necessary toprovoke the stability of this compensatory process?According to Skangiel-Kramska(1988), the activationofthe NMDA receptors must reach a critical threshold tobring about these plasticity phenomena.

Experiments carried out in total darkness have shownthat the N–T component of the monocular H-OKN doesnot appear in control animals. However, if, in theseconditions of light deprivation, frogs received aninjection of either NMDA or an NMDA agonist daily,the results differ according to the mode of drugadministration: when the drug is “asymmetrically”injected, i.e., into the pretectum contralateralto the openeye, frogs display an N–T component in the monocularH-OKN triggered by the open eye as soon as they arebrought into the right.When the drug is “symmetrically”injectedby intraperitonealroute, the H-OKN triggeredbythe open eye does not display an N–T component,remaining asymmetrical when frogs are brought out ofdarkness. But if the open eye is then normally light-stimulated for some hours, the N–T component appearsand will no longer disappear for at least 15 days. Theseexperimentsindicatethat unilateralpretectal injectionsof

NMDA are sufficientto obtain a permanent symmetricalmonocular H-OKN, light stimulation being unnecessaryto build up the plasticity phenomenon. On contrast, thesymmetrical injectionof the drug in total darkness is notable to provoke alone, the appearance of the N–Tcomponent. A unilateral light stimulation is required inaddition, for a short period, to provoke a symmetricalmonocular H-CIKN.

Thus, to build up the plasticity phenomenon we haveseen, a unilateralNMDA pretectal activation is required.It is obtained by direct unilateral administration ofNMDA or an NMDA agonist into the pretectum; it canalso be obtained by monocular illumination during 8days. However, in this case, a previoussymmetricaldrugadministration,even in total darkness reduced the periodof the build-up of the symmetrical monocular OKN.Therefore, maintaining adult animals in total darknesswith NMDA administration,seems to reduce the periodof build-up of this p~asticityphenomenon.It has alreadybeen shown (Fox et 1991;Williams et 1996) thatdark rearing prolongs plasticity and delays the loss ofNMDA receptorfunctionin the visual system,but only inyoung animals.

Now, the problemremains to determinewhat mechan-

1522 B. L. N. JARDON and N. BONAVENTURE

1.000

[

(A) (B)o T–N 1.400

I(

o T–ri0.800 ● N-T 1.200 ● N–T

<

1.000~ 0.600

.-CU ● .: 0.800

“ 0.400 I(),000 ~ 0 ~

o 2 4 6 8 10 0/s o 2 4 6 8 10 0/s

Drum speed Drum speed

1.000

[

( c1,000

[-

(D)0 T–N o T–N

0.800 ● N–T 0.800● N-T

~ 0.600

:\

0.600 –G.- .-

* a

o 0.400 ~ 0.400 —

0.200 ~’ 0.200 –

I●

0.000 I ( 10.000- ● I

● Io 2 4 6 8 10 0/s o 2 4 6 8 10 0/s

Drum speed Drum speed

FIGURE 10. Mean values of slow phase velocity gain of monocular eye H-OKN following 7 days of total darkness in fourdifferent experimental conditions, frogs having undergoneseven daily unilateral microinjectionsof drugs into the pretectumcontralateral to the open eye. (A) LY 285 265 (0.14rig); (B) NMDA (0.29rig); or (D) PBS. Frogs have undergone 7 dailyintraperitoneal administration of (C) LY 285 265 (0.35pg). Recordings were achieved immediately (10-15 rein) after the

animals were again placed in normal day–nightillumination(A, B, D) or 24 hr later (C).

ism underlies the effect of NMDA receptor activation.Itseems that the compensatory process observed inmonocular H-OKN occurs too quickly to be related toanatomicalchanges.Nevertheless,this hypothesiscannotbe totally dismissed:indeed, it was observedthat chronictreatmentswith low levels of NMDA entail fine synapticstructural alterations and local synaptic rearrangementswithin the retino-tectalneuropilein developingfrog (Yen

1993).The activation of NMDA receptors was found to

initiate long-term potentiation (LTP) of synaptic trans-mission (Collingridge& Bliss, 1987); it is also involvedin the plasticity of visual responses (Bear et 1990;Iwakiri & Komatsu, 1993; Udin & Scherer, 1990; Clineet 1987; Udin 1992). The question remainswhether the long-term adaptative visual modificationsobserved in monocular H-OKN characteristics arecontrolled by a mechanism analogousto that underlyingLTP. Recent studieslend supportto a role for Ca2+influxthroughNMDA receptors in synapticplasticity(Perkel

1993): as for LTP, the intracellular influx of Ca2+could serve as a second messenger to trigger enzymetranslocation or activation, protein-kinase C playing anessential role in this mechanism.This enzymaticcascadeonce induced, could provoke durable metabolic and/ormolecular modificationsin the cell.

It has previouslybeen shownthat pretectalGABAergicand cholinergic system are also involved in these

compensatory processes. (YucelJardon & Bonaventure, 1992b). The

1990, 1991;administration of

the GABAA agonist THIP transiently abolished themonocular H-OKN symmetry by suppressing the N–Tcomponent which appeared after a week of unilateralocclusion. In contrast, intrapretectal administration ofGABAAantagonists induced the appearance of an N–Tcomponentin the frog’s monocularH-OKN. In the sameway, the N–T componentwas also induced by adminis-tration of ACh muscarinic agonists, while atropine, anACh muscarinicantagonisttransientlysuppressedthe N–T component which appeared following prolongedunilateral visual deprivation. Thus, the plasticity phe-nomenonobserved in monocularfrog’s H-OKN seems torequire a reduction of GABAergic inhibition and aconcomitant activation of muscarinic cholinergic trans-mission occuring progressively in the pretectum. Inter-actions between GABAergic and cholinergicmechanisms were demonstrated at the pretectum level(Bonaventure& Jardon, 1992). But are these alterationsin GABAergic and cholinergic transmissions linked toNMDA receptor activation?

In other cortical structuresit was shown that carbacholcan potentate NMDA responses in the rat hippocampus(Harvey 1993) and muscarinic receptor activationcan induce a long-lasting enhancement of Purkinje cellresponses to glutamate (Andre 1993). Moreover,intact cholinergic input was necessary to provoke the

NMDA AND PLASTICITYIN FROG MONOCULARH-OKN 1523

shift of ocular dominance after monocular visualdeprivation during the critical period in kittens (Bear &Singer, 1986).

On the other hand, the induction of LTP in the visualcortex of the rat requires both activation of NMDAreceptors and the concomitant reduction of GABAinhibition (Artola & Singer, 1987); a GABAergicmechanism inhibits the formation of LTP in the ratsuperior colliculus (Hirai & Okada, 1993). These dataindicate that GABAergic mechanisms might control theNMDA-mediated processes. Moreover, it was recentlyshown that GABAergic receptor responses can besuppressed by NMDA application in hippocampalneurons isolated from the adult guinea-pig (Chen &Wong, 1995). On the other hand, it has been suggestedthat the directionality of monocular eye H-OKN wasdetermined by the directional selectivity of pretectalcells: many units recorded in the frog pretectum arestimulated in the T–N direction and inhibited bystimulation in the N–T direction (Katte & Hoffmann,1980;Cochran a 1984),the inhibitionof unit activityduring the N–T stimulation resulting in GABAergicactivation. By analogy with the results of Chen andWong, it is tempting to think that the pretectal NMDAreceptor activation could suppress the GABAergicinhibition at this level. Thus, it is suggested that NMDAreceptor activationrequiresACh stimulationand entailsareduction of GABAergic inhibition.

According to these data we propose the followinghypothesis: the chronic activation of pretectal NMDAreceptors causes modifications resulting in significantfunctional reorganization in monocular H-OKN. Thisactivation appears to be facilitated by ACh inputs actingon pretectal muscarinic receptors (Jardon a 1991),and seems simultaneously inhibit GABAergic terminalsfound in this structure (Yiicel et 1988).

R

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A are grateful to Ely Lilly (IndianapolisU.S.A.),and especially to Doctor D. Schoeppfor donating the LY 285265.