Exp. Brain Res. 30, 187-202 (1977)

Experimental Brain Research 9

Springer-Verlag 1977

An Attempt to Assess the Effects of Monocular Deprivation and Strabismus on Synaptic Efficiency in the Kitten's Visual Cortex C. Blakemore 1 and P. ttillman 2 The PhysiologicalLaboratory, Cambridge CB2 3EG, England

Summary. The relative effectiveness of the two eyes in exciting cells in the visual cortex was assessed, using both natural stimulation and electrical stimulation of the optic discs. It is argued that supramaximal electrical stimulation of the optic nerve could possibly reveal 'subliminal' synaptic inputs even after monocular deprivation or artificial strabismus has caused a loss of natural input from that eye, if such 'silent' synaptic input still survives. However, in kittens monocularly deprived for various periods of time or made artificially strabismic, there was usually excellent agreement between the relative visual excitability in the two eyes and their relative electrical excitability. In one animal, monocularly deprived continuously until 23 weeks of age, we examined the effect of reversibly turning off signals from the normal eye by pressure blinding. There was no evidence of a very rapid return of sensitivity to either electrical or natural stimulation of the deprived eye. Key words: Monocular deprivation - Visual cortex - Strabismus - Synaptic efficiency

Introduction There is a great deal of electrophysiological evidence that the effectiveness of afferent stimulation in exciting sensory neurones in the central nervous system can be modified by sensory deprivation or partial de-afferentation. In the visual cortex of cat and monkey, neuronal modification by deprivation is mainly restricted to a sensitive period early in life: during this period even brief deprivation of one eye causes most cortical cells to lose their effective input from that eye and become monocularly driven (Wiesel and Hubel, 1963; Hubel and Wiesel, 1970; Baker et al., 1974; Peck and Blakemore, 1975; Olson and Freeman, 1975). Other procedures that interfere with the normal simultaneous 1 Royal SocietyLocke Research Fellow 2 On leave from The NeurobiotogyUnit, The Hebrew Universityof Jerusalem, Jerusalem, Israel

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use of the two eyes, such as surgically-induced strabismus or alternating occlusion of the eyes also cause a gross reduction in the proportion of binocularly-driven cortical neurones (Hubel and Wiesel, 1965; Blakemore, 1976). Despite these and many other demonstrations of synaptic plasticity, the mechanisms by which the input to sensory neurones can change are largely unknown. One obvious possibility is physical withdrawal or growth of afferent axons (Hubel et al., 1975) in response to changes in their impulse-traffic, or similar changes in the dendritic fields of the cells on which the axons terminate. Another possibility (which could in fact happen simultaneously with changes in axonal distribution), is the occurrence of a more subtle modification of the efficiency of existing synapses, such as in the number of quanta of transmitter released per impulse in the presynaptic ending, or in the density of receptor sites on the post synaptic membrane. Merrill and Wall (1972) have recently demonstrated that many cells in the dorsal horn of the cat's spinal cord can be monosynaptically excited by electrical stimulation of any of a large number of nearby dorsal roots, via small axons, running longitudinally in the cord (Wall and Werman, 1976), even though the input providing the entire classical receptive field, for natural tactile stimulation of the skin, seems to arrive through only one root. The effectiveness of these 'subliminal' afferent inputs is normally rather low, but removal of a cell's preferred input by dorsal root section can lead to an increase in the synaptic strength of the terminals of intact, long-ranging axons, whose influence can otherwise only be demonstrated by electrical stimulation (Basbaum and Wall, 1976). We have applied the strategy of Wall's experiments to the kitten's visual cortex, and have asked whether the synchrony of impulse volleys set up by electrical stimulation of the visual pathway might reveal the continuing presence of afferent input that has (in terms of natural stimulation) been silenced by visual deprivation.

Methods We used the same general methods of preparing and maintaining animals, recording single units and plotting their receptive fields described by Blakemore and Van Sluyters (1975). The cats were paralysed, and anaesthetized during recording by artificial ventilation with about 80% N20. All receptive fields lay within 10 deg of the area centralis. This report is based on a study of 172 cortical cells from five animals.

Electrical Stimulation With the cat deeply anaesthetized by intravenous Althesiu (Glaxo), the conjunctiva was dissected free, up to its attachement around the limbus, and was then folded back over a metal ring to which it was glued with IS-12 cyanoacrylate adhesive. The ring was attached to a rod clamped to the stereotaxic frame, to hold the eye stationary. A small metal plate was positioned in close contact with the sclera, directly behind the ring, on the upper-lateral part of the globe, and we used a ground-down hypodermic needle as a trephine, mounted in a dental drill, to perforate the sclera, visible through a small hole in the metal plate, at a level just posterior to the ciliary body. This

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method, which was suggested to us by Dr. J.G. Robson, proved a very successful means of gaining entry to the globe, causing virtually no bleeding into either the eye or the orbit. Through the hole We introduced a coaxial bipolar electrode (0.5 mm shaft diameter; Rhodes Medical Instruments) held in a ball-and-socket arrangement. Under ophthalmoscopic observation, we then advanced and angled the electrode until it touched very lightly on the centre of the optic disc. For electrical stimulation of the discs, we used single shocks of 50-200 ~xsee in duration, with the inner conductor of the bipolar electrode as the cathode. A pair of similar bipolar electrodes, about 3 mm in separation, was lowered to the optic chiasma for recording gross potentials after visual or electrical stimulation of the eyes, and for stimulating the visual pathway. Signals from any electrode could be averaged on a Biomac 1000 averaging computer.

Pressure-Blinding and Enucleation It has long been known that retinal ischaemia rapidly blocks vision (Bornschein, 1958), This can be achieved by simply raising intra-ocular pressure above arterial pressure by injection of fluid intra-ocularly (Levick and Williams, 1964). The only major problem encountered with this method of pressure blinding is that slight clouding of the cornea develops after about 20 cycles of pressure application. The same technique of drilling was used to make a small hole in the lateral side of the eye, at about the equator. A short blunt tube was then introduced into the posterior chamber and was sealed into the eye by means of cyanoacrylate adhesive applied to a flange about 3 mm behind the tip. The tube was connected to a reservoir of Ringer's solution, the pressure of which could be raised and measured through a mercury manometer. Ophthalmoscopic observation showed that the retinal vessels collapsed at about 150 mm Hg, and at slightly higher pressures the potential evoked at the chiasma by flashing a bright light into the eye was eradicated rapidly and completely. In one cat, one eye was enucleated under deep Althesin anaesthesia: the equator of the globe was exposed and the whole anterior half of the eye was removed by quickly cutting around the equator. The vitreous body and the entire retina were then aspirated by suction, and the globe was collapsed around a plug of Sterispon absorbable gelatine sponge. Enucleation by this method produces minimal and short-lived bleeding. A long-lasting local anaesthesia (Anucaine) was infiltrated into the tissues of the orbit.

Results E a r l y in t h e s e e x p e r i m e n t s it b e c a m e c l e a r t h a t t h e r e was n o g r o s s d i s a g r e e m e n t b e t w e e n t h e r e l a t i v e e f f e c t i v e n e s s o f t h e t w o e y e s in d r i v i n g c o r t i c a l cells f o r e l e c t r i c a l c o m p a r e d w i t h v i s u a l s t i m u l a t i o n : it w a s v e r y u n u s u a l to f i n d a cell t h a t c o u l d b e e x c i t e d b y e l e c t r i c a l l y s t i m u l a t i n g t h e o p t i c disc o f a n e y e in w h i c h it did n o t o b v i o u s l y r e s p o n d t o n a t u r a l s t i m u l a t i o n . W e t h e r e f o r e d e c i d e d to m a k e a detailed study of the relative influence of the two eyes - the ocular dominance of t h e n e u r o n e s - u s i n g b o t h n a t u r a l a n d e l e c t r i c a l e x c i t a t i o n ; this r e q u i r e d t h e e s t a b l i s h m e n t o f s o m e s i m p l e q u a n t i t a t i v e m e t h o d o f classifying 'electrical ocular dominance', c o m p a r a b l e to H u b e l a n d W i e s e l ' s ( 1 9 6 2 ) s c h e m e f o r r a t i n g t h e r e l a t i v e i n f l u e n c e o f t h e t w o e y e s f o r visual s t i m u l a t i o n .

Electrical Ocular Dominance and General Observations on Electrically-Evoked Responses T h e o c u l a r d o m i n a n c e o f c o r t i c a l n e u r o n e s f o r n a t u r a l s t i m u l a t i o n is s i m p l y j u d g e d b y c o m p a r i n g t h e r e s p o n s e s w h e n e a c h e y e is s t i m u l a t e d in t u r n . W e

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devised a scheme closely similar to this but using electrical excitation. After arranging the concentric stimulating electrodes on the optic discs of the two eyes and adjusting the strength of the shocks (usually about 5 v) to give just supra-maximal excitation of each entire optic nerve (judged from the evoked potential recorded at the optic chiasma), we tested the response of every cell encountered to stimulation of each optic disc separately, and to simultaneous stimulation of the two. In general, as reported by previous workers (e.g. Hoffmann and Stone, 1971; Stone and Dreher, 1973; Singer et al., 1975) we found that many visual cortical neurones, especially simple cells, could not be made to fire impulses by electrical stimulation of the optic discs or optic chiasma. In our sample, only about half of all cells could be electrically driven, though all of them responded to visual stimulation of at least one eye. At first this finding seems to contradict the whole logic of this experiment - that electrical stimulation should be more effective than natural in revealing weak inputs. But since all cortical cells show EPSPs after chiasmal stimulation (Toyama et al., 1974), the low general effectiveness of electrical stimulation is usually attributed, not to the weakness of excitation~ produced in the cortex, but to the fact that at both the lateral genieulate nucleus and the cortex itself, inhibition is set up, by activity in the rapidly conducting 'Y' axons, which hyperpolarizes neurones and hence tends to prevent them from responding, especially if their major excitatory input comes through the more slowly conducting 'X' system (Singer and Bedworth, 1973; Stone and Dreher, 1973; Singer et al., 1975). Since all cells are probably influenced by electrical stimulation, and the failure to fire is mainly a consequence of the unusual timing of excitation and inhibition, it seems justifiable at least to compare the relative effects of stimulating the two eyes, e!ther by electrical or by natural means, amongst those cells that respond to both forms of stimulation, just as Ito et al. (1977) have very recently done in normal cats.

Patterns of Discharge: Short- and Long-Latency Responses Amongst those cells that did respond to shocks, the majority of which were complex units, the exact pattern of the discharge was extremely varied. The commonest form was a single, short-latency action potential (< 10 msec latency), which in many cases was probably due to direct monosynaptic excitation of cortical cells by geniculate axons (Watanabe et al., 1966; Hoffmann and Stone, 1971; Toyama et al., 1974; Singer et al., 1975), followed by a silent period of very variable length, and then, quite often, a ragged burst of spikes not well-locked in time to the stimulus. Occasionally the first spike was absent; in other cases there was a multiple early burst with short intervals between the spikes (Hoffmann and Stone, 1971; Stone and Dreher, 1973; Singer et al., 1975). The general form of excitation-inhibition-excitation is presumably attributable to early synaptic excitation, followed by intracortical inhibition, followed by 'rebound' from inhibition combined with late or residual excitation (Watanabe et al., 1966; Toyama et al., 1974; Singer et al., 1975).

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It is i m p o r t a n t to e m p h a s i z e t h a t t h e r e is little k n o w l e d g e a b o u t w h i c h components of the complicated electrical response play a major part in natural r e s p o n s e s ( C r e u t z f e l d t a n d Ito, 1 9 6 8 ; I n n o c e n t i a n d F i o r e , 1 9 7 4 ; C r e u t z f e l d t et al., 1 9 7 4 ) . I n a n y case, we w e r e c o n c e r n e d o n l y with t h e r e l a t i v e s e n s i t i v i t y to i n p u t f r o m t h e t w o eyes, a n d so s i m p l y e s t i m a t e d t h e total n u m b e r of s p i k e s t h a t f o l l o w e d e a c h s h o c k o n a v e r a g e ( i n c l u d i n g b o t h e a r l y a n d late i m p u l s e s ) , a n d c o m p a r e d this b e t w e e n t h e t w o eyes. I n fact, t h e p a t t e r n o f t h e e l e c t r i c a l l y - e v o k e d d i s c h a r g e was u s u a l l y v e r y s i m i l a r for s t i m u l a t i o n of e i t h e r eye, c o n f i r m i n g t h e o b s e r v a t i o n s o f Ito et al. ( 1 9 7 7 ) . I n v i r t u a l l y e v e r y case t h e e s t i m a t e o f e l e c t r i c a l o c u l a r d o m i n a n c e w o u l d h a v e b e e n t h e s a m e if o n l y t h e s h o r t - l a t e n c y spikes, b e f o r e t h e i n h i b i t o r y p a u s e , h a d b e e n t a k e n i n t o a c c o u n t (see Fig. 3 for a single e x c e p t i o n ) . M a n y c o r t i c a l cells h a v e fairly l o w s p o n t a n e o u s a c t i v i t y ( < 10 s p i k e s / s e c ) o r n o n e at all. S i n c e t h e e n t i r e r e s p o n s e g e n e r a t e d b y a s h o c k to t h e o p t i c disc is u s u a l l y c o m p l e t e w i t h i n a b o u t 50 msec, s p o n t a n e o u s a c t i v i t y o f less t h a n a b o u t 20 s p i k e s / s e c d o e s n o t i n t e r f e r e at all i n the detection of very weak responses. Each optic disc was stimulitted separately, usually 50-100 times, with several seconds rest between each shock. Finally both optic discs were stimulated simultaneously for a similar number of trials, before the cell was assigned to one of the following electrical ocular dominance groups. Group 1. Cells electrically influenced only through the contralateral eye. 2. Directly excited only through the contralateral eye, but simultaneous stimulation shows clear facilitation, suggesting a subliminal input from the ipsilateral eye. 3. Driven by both eyes but more strongly from the contralateral. 4. Equally excited for either eye. 5. Driven by both eyes but more strongly by the ipsilateral. 6. Driven directly only by the ipsilateral eye, but with subliminal influence from the contralateral, causing facilitation during simultaneous stimulation. 7. Driven by the ipsilateral eye alone. This classification, it should be noted, is similar but not identical to the one introduced by Hubel and Wiesel (1962) for natural responses. The principal difference is in the definitions of groups 2 and 6: for Hubel and Wiesel's scheme these groups mainly describe cells with very weak, but supraliminal influence from one of the eyes. We chose to use the scheme described above for two reasons: a) The response to a single electric shock, in terms of the total number of impulses, was generally much weaker than that to visual stimulation with a moving, optimally-oriented line. It therefore proved difficult to rank the degree of electrical dominance by one eye or the other in those cells that were clearly binocularly activated but not driven equally by the two eyes. b) It seems likely that subliminal influences from one eye, which only express themselves in terms of a modulation of the response generated by the other eye, might be more easily demonstrated with electrical than with natural stimulation. This is because the spatial arrangement of the patterns in the two eyes is of crucial importance in determining whether or not facilitation will occur during natural binocular stimulation (Barlow et al., 1967; Pettigrew et al., 1968; Bishop et al., 1971). Figure 1 shows responses from a complex cell with electrical dominance group 2, recorded in the right hemisphere of an adult animal made artificially strabismic at 21 days of age (see Fig. 4). Each trace consists of 10 superimposed oscilloscope sweeps, triggered by shocks to the optic disc. The neurone responded, though weakly, in the left eye, giving short latency (about 3.5 msec) and longer latency (about 8.5 msec) spikes. The cell never responded to stimulation of the right optic disc, nor to natural stimulation through that eye. On the other hand there was very distinct facilitation during binocular electrical stimulation: both the early and the late responses were enhanced. However, we were unable to demonstrate any binocular summation for natural stimulation. Thus we classified this cell as 'visual group 1, electrical group 2' (but is must be said that more sophisticated techniques, not normally employed in deciding visual ocular dominance, might have revealed subtle interactions

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Fig. 1. A complex cell from a strabismic cat (see Fig. 4) was stimulated electrically through each eye separately, and then through both eyes together. Each oscilloscope trace shows ten superimposed responses to a supramaximal cathodal shock (100 Ixsecduration) applied to the optic disc, with about 5 sec intervals between successive stimuli. The stimulus artifact is visible at the start of each trace. This cell, recorded in the right hemisphere, had no natural visual responsiveness through the right eye nor any obvious binocular facilitation, and hence was classified as 'visual ocular dominance group 1'. Electrical stimulation of the right eye also never resulted in a spike from the cell; but both early and late responses were obviously enhanced when the two eyes were stimulated. The cell was accordingly classified as 'electrical ocular dominance group 2'. (Spontaneous activity for this cell was about 2/sec, so it is very unlikely that more than one of the displayed spikes were spontaneous.)

between the two eyes during natural binocular stimulation: Bishop et al., 1971). Sample results corresponding to electrical groups 1, 3 and 4 are shown in Figure 3. Despite the slight differences in definition between the ocular dominance classifications for natural and electrical responses, we think that they are adequately comparable to permit a rough comparison to be made between the two forms of response.

Monocular Deprivation In an initial e x p e r i m e n t , a k i t t e n ( K 3 0 2 ) was d e p r i v e d in t h e right eye f r o m age 32 days to 42 d a y s a n d t h e n r e c o r d i n g s w e r e t a k e n f r o m t h e right h e m i s p h e r e . N o visual r e c e p t i v e fields w e r e m a p p e d in this a n i m a l but, f r o m p r e v i o u s e v i d e n c e ( H u b e l a n d W i e s e l , 1970; O l s o n a n d F r e e m a n , 1975; M o v s h o n a n d D i i r s t e l e r , 1977), t h e c o r t e x s h o u l d h a v e b e e n t h o r o u g h l y m o n o c u l a r . I n d e e d , of t h e 15 cells t h a t c o u l d b e e l e c t r i c a l l y e x c i t e d ( o u t of a t o t a l o f 41 r e c o r d e d ) , o n l y 2 r e s p o n d e d to s t i m u l a t i o n of the d e p r i v e d , i p s i l a t e r a l right eye a n d e v e n t h o s e less s t r o n g l y t h a n for s t i m u l a t i o n of the n o r m a l eye. E n c o u r a g e d b y t h e s e p r e l i m i n a r y findings we d e c i d e d to s t u d y both visual a n d electrical excitability in all o t h e r e x p e r i m e n t s , so we t o o k p a r t i c u l a r c a r e w h e n i n t r o d u c i n g t h e s t i m u l a t i n g e l e c t r o d e u n d e r o p h t h a l m o s c o p i c view to b e sure t h a t it did n o t o b s c u r e t h e r e g i o n o f t h e a r e a centralis. A s e c o n d a n i m a l ( K 3 0 3 ) was d e p r i v e d in t h e right eye f r o m 32 days until t h e e x p e r i m e n t at 52 days. I n this kitten, b o t h visual a n d e l e c t r i c a l r e s p o n s e s w e r e s o u g h t for e v e r y cell. R e c o r d i n g was f r o m t h e left h e m i s p h e r e , i p s i l a t e r a l to t h e n o r m a l eye. O f t h e 18 cells r e c o r d e d , 9 w e r e electrically i n e x c i t a b l e ; all b u t o n e o f t h e s e w e r e visually e x c i t a b l e only t h r o u g h t h e n o r m a l eye. O f the r e m a i n i n g 9, 8 c o u l d b e d r i v e n o n l y t h r o u g h t h e n o r m a l eye, b o t h visually a n d electrically.

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Fig. 2. Comparison of visual and electrical dominance for an animal (K311) that was briefly deprived in the contralateral (right) eye from 46--49 days. On the left is a histological reconstruction of the electrode track from 40 ~tm frozen coronal sections stained with cresyl violet: the solid circles represent electrolytic lesions placed during the penetration, which ran obliquely down the medial bank of the postlateral gyrus of the left hemisphere. In the two schematic reconstructions of the penetration, shown on the right, each pair of dots at the same horizontal level represents the visual and electrical ocular dominance of a cell recorded at that depth. EI and unfilled dots = electrically inexcitable cells. The breaks in the ordinates indicate a small area in which no single units were isolated. Three particular cells, L16, 18 and 22, whose responses are illustrated in Figure 3, are indicated on the schematic reconstructions. The histograms below the two columns show the total samples

O f t h e t w o cells w h i c h w e r e v i s u a l l y e x c i t a b l e t h r o u g h t h e d e p r i v e d e y e (as w e l l as t h e n o r m a l e y e ) , o n e was e l e c t r i c a l l y i n e x c i t a b l e . T h e o t h e r was t h e o n l y cell e l e c t r i c a l l y e x c i t a b l e t h r o u g h t h e d e p r i v e d e y e (as w e l l as t h e n o r m a l e y e ) : in fact w e c l a s s i f i e d it as e l e c t r i c a l l y g r o u p 5 b u t v i s u a l l y g r o u p 6. G i v e n t h e difficulty of making close comparison between visual and electrical excitability, these results indicate general agreement between the patterns of visual and

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Fig. 3. Raster-dot-displays (Wall, 1959) showing the latency distribution of impulses after electrical stimulation of the optic disc for 3 complex cells from K311 (see Fig. 2) and one complex cell from K129 (Fig. 5). Each box shows the result of 16 sequential stimuli with a pause of about 5-10 sec between shocks. Every horizontal line represents the time immediately following the electric shock, with spikes shown as dots. In each case the spontaneous activity was tow enough to be confident that few if any of the impulses appearing within the raster were not evoked by the shock

electrical dominance, just as good as in completely normal animals (Ito et al., 1977). Certainly we saw no evidence of large numbers of cells having subliminal input from the deprived, visually ineffective eye, which could easily be revealed by electrical stimulation. We decided to try to increase the probability of observing electrical excitability lingering after visual excitability had faded, by experimenting on an animal (K311) deprived in the right eye from 4 6 - 4 9 days, close to the minimum time needed to produce clear electrophysiological changes in the cortex. Recording in this animal was also from the left hemisphere, contralateral to the deprived eye. The distribution of visual ocular dominance during the penetration, shown in Figure 2, was typical of animals briefly monocularly deprived ( M o v s h o n and Diirsteler, 1977): the majority of cells were dominated completely by the experienced eye, with tiny residual 'columnar' regions, 0.5-1 m m apart, where one or two neurones recorded still responded through the deprived eye. Often their receptive fields in the deprived eye were not orientation selective 9 A m o n g s t the 10 cells that were electrically excitable (out of a total of 27), the electrical ocular dominance, shown in Figure 2, matched the visual ocular dominance quite well. Figure 3 shows sample spike-latency raster displays (Wall, 1959) for the cells marked L16, L18 and L22 on Figure 2. There was fairly close similarity b e t w e e n visual and electrical ocular dominance, except for unit L18, a quite exceptional cell, for which no receptive field could be plotted at all in the deprived right eye

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(visual group 7), yet which responded more strongly in toto for electrical stimulation of the right eye than for the left (electrical group 3). Even in this unusual case of apparently gross disagreement between electrical and visual dominance it is interesting to note that the strong response to stimulation of the right optic disc consisted entirely of long-latency (> 10 msec) spikes. Only stimulation of the left eye (visually dominant) generated occasional shortlatency, probably monosynaptic impulses (see Fig. 3). Unit L22 was one of the rare cells that only responded with late spikes. It was visually and electrically completely dominated by the deprived right eye, lying within a small region that was influenced by that eye.

Artificial Strabismus Figure 4 shows the penetration reconstruction for an animal (K129) given a divergent strabismus at 21 days and recorded at 26 months. The visual dominance distribution shows that most of the cells were monocularly driven by one or the other eye and they were encountered in successive columnar clusters (Hubel and Wiesel, 1965). The distribution of electrical ocular dominance, though based on the smaller proportion of electrically excitable cells, was again similar in general form to the visual dominance pattern, with alternating regions of dominance by one eye then the other. Raster displays for the unit R15 (marked in Fig. 4) appear in Figure 3 and responses of unit R8 are shown in Figure 1.

Correlation Between Visual and Electrical Dominance Figure 5 summarizes the correlation of visual and electrical dominance for the three animals in which both forms of stimulation were used. Of 63 cells recorded, 34 were electrically inexcitable. Nineteen of the remaining 29 were excitable both electrically and visually only through one and the same eye. Of the 10 cells excitable through both eyes, either visually or electrically, 8 were relatively more influenced electrically by the eye that was visually nondominant, Whereas the reverse was true for only one celt.

Extracellular Field Potentials With appropriate filtering (bandpass 0.3-10 KHz) and averaging we were able to record local field potentials in the cortex through the microelectrode. We looked mainly at the potentials set up by stimulating the optic discs, and although we did not study them in detail, we did make two general observations that are quite relevant to the question behind this experiment: a) Wherever field potentials could be recorded for stimulation of either eye, they were usually larger in amplitude for the eye dominating single neurones in that region of the cortex, b) Since the amplitude and even the waveform of the field potential

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Fig. 4. A reconstruction of results, as in Figure 2, but for an adult cat (K129, age 26 months) made artificially exotropic at 21 days. Responses for individual units R8 and R15, marked on the schematic reconstruction, appear in Figures 1 and 3 respectively. As the histological reconstruction shows, the track passed into the white matter, at the depth of the upper electrolytic lesion, and re-emerged into the grey matter deep in the medial bank. No orientation-selective units were recorded in the white matter. The schematic reconstruction only shows the first section of the penetration, before entering the white matter. It should be noted that the track was angled anteriorly and the two electrolytic lesions did not actually lie in a single coronal plane. Thus, although the penetration appears, in the upper 2 mm, to have passed almost exactly perpendicular to the surface in the coronal plane, it ran quite obliquely forwards across the palisades of cortical cells: therefore the fairly rapid alternation in ocular dominance is not unexpected. Three more cells recorded at the end of the penetration, when the electrode re-entered grey matter, are not included in the analysis in this diagram

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could vary quite considerably with movements of the electrode of about 100 ~tm, we assume that the signals recorded were mainly being generated close to the electrode tip. Clear short-latency gross potentials could, however, be picked up in many areas of the cortex where the eye being stimulated was incapable of driving any cortical cells recorded, either through visual or electrical stimulation (see Fig. 6). This implies that afferent fibres carrying signals from a nondominant eye do at least transmit impulses to a level at which they can be picked up as field potentials in the cortex, confirming the conclusion of Wiesel and Hubel (1963), who were also able to recorded cortical potentials after visual stimulation of the deprived eye.

The Origin of the Suppression of Dominant-Eye Signals It has been suggested and evidence provided, that part of the loss of effectiveness of a deprived eye in monocularly deprived cats, is due to active suppression of its inputs by signals from the experienced eye (Kratz et al., 1976; Duffy et al., 1976). We recorded from an animal (K274), continuously monocularly deprived in the right eye from the time of natural lid-opening until the experiment at 23 weeks, and examined the effects of two different techniques for cutting off signals from the normal left eye - pressure blinding and enucleation (see Methods). The special virtue of pressure-blinding is its reversibility. Approximately 15 sec after pressure was applied, all visual responsiveness from that eye, at the chiasma and the cortex, disappeared. The pressure was held for no more than 2-3 rain, and normal visual responsiveness then returned 0.5-1 min after release. Twelve units were tested in the hemisphere contralateral to the deprived eye: all were visually responsive through the normal eye but not visually or electrically driven through the deprived eye. After 2-3 rain of pressure-blinding of the normal eye, not one cell developed a responsiveness, electrical or visual, to stimulation of the deprived eye. Although all these units were responsive only through the normal eye, a field potential could usually be seen following electrical stimulation of the deprived eye. As shown in Figure 6, this potential appeared to be quite unaffected by pressure-blinding of the normal eye. In view of these results we decided to look at the effects of more drastic suppression of the normal eye's signals, by enucleation of that eye, which is known to result in an increase in the responsiveness of some cortical cells to stimulation of the deprived eye (Kratz et al., 1976). The retina was removed by suction, leaving the eye-cup intact (see Methods) and no deterioration whatever of the animal's general condition was noted (judged by ECG, EEG and expired CO2). There was little change in cortical spontaneous activity, except for a slight general increase, especially 12-24 hours after enucleation. A total of 56 more units were examined, up to 42 hours after enucleation, at which point the animal's physiological condition was still excellent. None responded to electrical stimulation of the deprived eye, and only 2 units (3.6 %),

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0 1 2 3 4 5 m sec Fig. 6. Effects of pressure-blinding of the experienced eye, and then its enucleation, on cortical field potentials evoked by electrical stimulation of the deprived eye, in a cat (K274) that was monocularly deprived in the right eye from the time of natural lid opening until recording in the left hemisphere at 23 weeks. Each trace is the averaged response for 32 successive shocks to the optic disc of the deprived right eye, at about 5 sec intervals (positive upward): each one starts with the stimulus artifact (lasting about 1 msec), and runs for 5 msec. The vertical amplitude calibration applies to all records. In the upper half of the illustration, marked 'before enucleatiou', the field potential is shown for two sites, where units L10 and L l l were recorded. At each point a pair of traces appears, the first without pressure-blinding and the second taken when the normal, left eye was temporarily blinded. The single units did not respond to electrical or visual stimulation of the deprived eye. After enucleation, the field potential is shown for four recording sites, where L15, 16, 58 and 68 were studied. (The first two were recorded 8.5 and 9 hours after enucleation, the second two towards the end of the experiment, 35 and 41.5 hours after enucleation.) None of these cells responded visually or electrically to stimulation of the intact deprived eye. Although the field potential obviously varied a little from place to place, presumably because of movement of the electrode through the laminar and columnar structure of the cortex, there was no consistent rise (or fall) in the amplitude of its components after enucleation

found close together, about 19 hours after enucleation, responded weakly to natural stimulation of the deprived eye and their receptive fields were non oriented. A s s e e n i n F i g u r e 6, t h e f i e l d p o t e n t i a l s o b t a i n e d f r o m e l e c t r i c a l s t i m u l a t i o n of the deprived eye Showed no obvious change after enucleation of the normal

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eye. (These examples were taken at the recording sites of a number of quite typical visually-unresponsive cells.) The problem of sampling units in the cortex after removal of the dominant eye is obviously formidable (Kratz et al., 1976): however the density of units detected was not obviously less than that in experiments on normal animals. The slightly elevated spontaneous activity of cells made their detection easier, but we also advanced the electrode very slowly, constantly moving a pattern of lines of all orientations in front of the remaining eye, to optimize our chances of finding cells driven by that eye. Nevertheless it is important to note that it is extremely difficult to establish the true proportion of cells that do develop sensitivity to input from the deprived eye after enucleation of the normal.

Discussion

There can be no strong general conclusion from this study: certainly we have no evidence for the widespread existence of naturally 'silent' but electrically-excitable synaPtic input from the ineffective eye in the visual cortex of deprived cats. On the other hand, the failure to excite cells by electrical stimulation of ineffective afferent input does not necessarily imply that there has been literal withdrawal of those axon terminals from their original synaptic sites. It could be that the terminals are still present but are actively suppressed by some inhibitory mechanism that is not overcome by electrically-evoked input. Equally, it is possible that the modification that causes shifts in cortical ocular dominance takes place in the terminals themselves or in the receptive area of post-synaptic membranes, without any obvious change in the distribution of axons or dendrites. There is morphological evidence in both monkey (Hubel et al., 1975) and cat (Thorpe and Blakemore, 1975) that monocular deprivation does cause an actual re-deployment of afferent fibres, the deprived input losing some of its distribution within the cortex. Ocular dominance 'columns' for the normal eye expand and displace those for the deprived eye. But it is possible that, although such redistribution of afferent fibres is indeed a major factor contributing to modification of cortical binocularity, there could still be more subtle effects, dependent on modulation of synaptic efficiency or active inhibition, perhaps especially in the border regions between expanding and contracting afferent axonal distributions. There is definite evidence that the loss of input from a deprived eye is partly due to active suppression of its signals by those from the normal eye. Kratz et al. (1976) have found that removal of the normal eye in monocularly deprived cats can lead to a return of sensitivity to input from the deprived eye in a fairly large fraction of cortical cells. We did not observe this phenomenon with either pressure-blinding or enucleation in one animal despite its excellent physiological state over more than 2 days of recording. However we must emphasize that we do not believe that this is necessarily a contradiction to Kratz et al.'s findings. Many of their cats were younger than our single animal (23 weeks) or were left for some time after enucleation before recording. It is even conceivable

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that their results simply represent the increased recordability of a separate population of cortical units, difficult to resolve with our particular microelectrodes, which always retain input from the deprived eye but whose weak responses are hard to detect w h e n m a s k e d by s p o n t a n e o u s and visualy e v o k e d activity derived f r o m the n o r m a l eye w h e n the latter is intact. In any case we can conclude from these and other unpublished results on pressure-blinding that active and rapidly reversible suppression by the n o r m a l eye cannot be the sole m e c h a n i s m involved in the effects of p r o l o n g e d m o n o c u l a r deprivation. W e are currently pursuing this question in m o r e detail. Finally, D u f f y et al. (1976) have m a d e the fascinating observation that a b o u t half of all cortical cells in cats m o n o c u l a r l y deprived f r o m the fourth week, regain a receptive field in the deprived eye u p o n intravenous injection of the y - a m i n o butyric acid antagonist, bicuculline, which is k n o w n to increase the excitability of cortical cells (Pettigrew and Daniels, 1973; R o s e and B l a k e m o r e , 1974; Sillito, 1975a, b). This result, like Kratz et al.'s (1976) can also be interpreted in terms of active inhibition of the deprived eye's signals. But again it only applies to a fraction of cells; and the technique of intravenous injection does not allow one to conclude w h e t h e r the relevant inhibitory m e c h a n i s m is in the visual cortex or at some earlier site. O u r own observation, and those of Wiesel and H u b e l (1963), that stimulation of the deprived eye can set up gross field potentials in large areas of the cortex where it is quite incapable of raising cortical cells to their firing threshold, suggests that deprived afferent axons are not totally n o n - f u n c t i o n a l nor entirely absent f r o m the cortex. It would be valuable to continue this study using m e t h o d s of observing sub-threshold post-synaptic activity.

Acknowledgements. This work was supported by a grant (No. G972/463/B) to C.B. from the Medical Research Council, London. We thank Lyn Cummings, Barbara Rhodes, Ron Dowsing and Phil Taylor for technical help. P.H. is grateful to Professor R.D. Keynes for permission to work in the Physiological Laboratory and warmly thanks C.B. and Professor H.B. Barlow for their hospitality.

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Bornschein, H.: Spontan- und Belichtungsaktivit/it in Einzelfasern des N. opticus der Katze. I. Der Einfluf3 kurzdauernder retinaler Isch/imie. Z. Biol. 110, 210-222 (1958) / Creutzfeldt, O.D., Ito, M.: Functional synaptic organization of primary visual cortex neul~ones in the cat. Exp. Brain Res. 6, 324-352 (1968) Creutzfeldt, O.D., Kuhnt, U., Benevento, L. A.: An intracellular analysis of visual cortical neurones to moving stimuli: responses in a co-operative neuronal network. Exp. Brain Res. 21, 251-274 (1974) Duffy, F.H., Snodgrass, S.R., Burchfiel, J.L., Conway, J.L.: Bicuculline reversal of deprivation amblyopia in the cat. Nature (Lond.) 260, 256-257 (1976) Hoffmann, K.-P., Stone, J.: Conduction velocity of afferents to cat visual cortex: a correlation with cortical receptive field properties. Brain Res. 32, 460-466 (1971) Hubel, D. H., Wiesel, T. N.: Receptive fields, binocular interaction and functional architecture in the cat's visual cortex. J. Physiol. (Lond.) 160, 106-154 (1962) Hubel, D.H., Wiesel, T.N.: Binocular interaction in striate cortex of kittens reared with artificial squint. J. Neurophysiol. 28, 1041-1059 (1965) Hubel, D.H., Wiesel, T.N.: The period of susceptibility to the physiological effects of unilateral eye closure in kittens. J. Physiol. (Lond.) 206, 419-436 (1970) Hubel, D.H., Wiesel, T.N., LeVay, S.: Functional architecture of area 17 in normal and monocularly deprived macaque monkeys. Cold Spr. Harb. Symp. quant. Biol. 40, 581-589 (1975) Innocenti, G. M., Fiore, L.: Post-synaptic inhibitory components of the responses to moving stimuli in area 17. Brain Res. 80, 122-i26 (1974) Ito, M., Sanides, D., Creutzfeldt, O.D.: A study of binocular convergence in cat visual cortex neurons. Exp. Brain Res. 28, 21-35 (1977) Kratz, K.E., Spear, P.D., Smith, D.C.: Postcritical-period reversal of effects of monocular deprivation on striate cortex cells in the cat. J. Neurophysiol. 39, 501-511 (1976) Levick, W.R., Williams, W.O.: Maintained activity of lateral geniculate neurones in darkness. J. Physiol. (Lond.) 170, 582-597 (1964) Merrill, E.G., Wall, P.D.: Factors forming the edge of a receptive field: the presence of relatively ineffective afferent terminals. J. Physiol. (Lond.) 226, 825-846 (1972) Movshon, J. A., Dtirsteler, M. R.: The effects of brief periods of unilateral eye closure on the kitten's visual system. J. Neurophysiol. (1977) (in press) Olson, C. R., Freeman, R. D.: Progressive changes in kitten striate cortex during monocular vision. J. Neurophysiol. 38, 26-32 (1975) Peck, C.K., Blakemore, C.: Modification of single neurons in the kitten's visual cortex after brief periods of monocular visual experience. Exp. Brain Res. 22, 57-68 (1975) Pettigrew, J.D., Daniels, J.D.: Gamma-aminobutyric acid antagonism in visual cortex: different effects on simple, complex, and hypercomplex neurons. Science (N. Y.) 182, 81-83 (1973) Pettigrew, J. D., Nikara, T., Bishop, P. O.: Binocular interaction on single units in cat striate cortex: simultaneous stimulation by single moving slit with receptive fields in correspondence. Exp. Brain Res. 6, 391-410 (1968) Rose, D., Blakemore, C.: Effects of bicuculline on functions of inhibition in visual cortex. Nature (Lond.) 249, 375-377 (1974) Sillito, A.M.: The effectiveness of bicuculline as an antagonist of GABA and visually evoked inhibition in the cat's striate cortex. J. Physiol. (Lond.) 250, 287-304 (1975a) Sillito, A.M.: The contribution of inhibitory mechanisms to the receptive field properties of neurones in the striate cortex of the cat. J. Physiol. (Lond.) 250, 305-329 (1975b) Singer, W., Bedworth, N.: Inhibitory interactions between X and Y units in the cat lateral geniculate nucleus. Brain Res. 49, 291-307 (1973) Singer, W., Tretter, F., Cyuader, M.: Organization of cat striate cortex: a correlation of receptive field properties with afferent and efferent connections. J. Neurophysiol. 38, 1080-1098 (1975) Stone, J., Dreher, B.: Projection of X- and Y-cells of the cat's lateral geniculate nucleus to areas 17 and 18 of visual cortex. J. Neurophysiol. 36, 551-567 (1973) Thorpe, P.A., Blakemore, C.: Evidence for a loss of afferent axons in the visual cortex of monocularly deprived cats. Neurosci. Letters 1, 271-276 (1975) Toyama, K., Matsunami, K., Ohno, T., Tokashiki, S.: An intracellular study of neuronal organization in the visual cortex. Exp. Brain Res. 21, 45-66 (1974)

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Received March 14, 1977