Experimental Brain Research
Exp. Brain Res. 34, 351-363 (1979)
9
Springer-Verlag 1979
Modification of Orientation Sensitivity of Cat Visual Cortex Neurons by Removal of GABA-Mediated Inhibition T. Tsumoto 1, W. Eckart, and O.D. Creutzfeldt Department of Neurobiology,Max-Planck-Institutefor BiophysicalChemistry, Postfach 968, D - 3400 G6ttingen-Nikolausberg,Federal Republic of Germany
Summary.
The effects of an inhibitor of G A B A synthesis, 3-mercaptopropionic acid (MP), and of the G A B A antagonist bicuculline (B1C), on the direction and orientation sensitivity of visual cortical neurons were investigated using a computer-controlled stimulus presentation system. Intravenous administration of MP, which was usually more effective than if administered microelectrophoretically, induced a slight, but significant reduction in these properties of about half of the neurons tested. The effect of electrophoretic BIC was in the same direction but clearer than that of MP. In 71% of the simple cells, direction sensitivity was virtually lost during administration of BIC while orientation sensitivity was never completely eliminated in any neuron tested. Simultaneous administration of both drugs (MP systemically, BIC electrophoretically) caused more complete modification of the sensitivities than single administration of each. In four out of thirteen neurons tested, orientation sensitivity was completely abolished. The excitatory receptive fields slightly increased in size and became virtually round. The response magnitude to the optimal stimulus was increased by each drug alone and by both. The present results further support the hypothesis that intracortical inhibition plays a major if not an exclusive role for the orientation and direction sensitivity of cortical cells.
Key words: Visual cortex - Orientation sensitivity - GABA-inhibition Bicuculline - 3-Mercaptopropionic acid. Visual cortical (VC) neurons are preferentially or selectively responsive to particular visual stimulus parameters, such as to the orientation of a light slit or dark bar of a specific orientation moving in one direction (Hubel and Wiesel, 1962). Several lines of evidence suggest that the orientation and direction 1 Present address: Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa, 920, Japan Offprint requests to: Prof. O.D. Creutzfeldt, M. D. (address see above)
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sensitivity may be partially due to intracortical inhibitory mechanisms (Creutzfeldt and Ito, 1968; Benevento et al., 1972; Blakemore and Tobin, 1972; Bishop et al., 1973; Creutzfeldt et al., 1974). If this is so, pharmacological blockade of intracortical inhibition should be able to abolish or attenuate those sensitivities of VC neurons. There is abundant evidence suggesting that gamma-aminobutyric acid (GABA) may be an inhibitory transmitter in VC (Mitchell and Srinivasan, 1969; Iversen et al., 1971) and that its action is selectively antagonized by the alkaloid bicuculline at postsynaptic sites (Curtis et al., 1970, 1971; Curtis and Johnston, 1974; Sillito, 1975a). Using intravenous bicuculline, Pettigrew and Daniels (1973) reported that VC neurons of the complex type lost some of their sensitivities but simple cells did not. In both types of cells, Rose and Blakemore (1974) found that topical application of the same drug reduced such sensitivities and increased the size of their excitatory receptive fields (RF). More recently, Sillito (1975a, b, t977) reported that in simple cells the direction sensitivity was significantly reduced or eliminated but the orientation sensitivity was only slightly reduced, whereas in complex cells the latter was virtually lost. Although the results of these three groups are not fully consistent with each other, each supports only partially the hypothesis described above. This may be due to an incomplete block of inhibition, because the GABA-induced or electrically elicited inhibition of some cortical neurons was not blocked by bicuculline (Curtis and Felix, 1971; Sillito, 1975a). In the present experiments an inhibitor of GABA-synthesis, 3-mercaptopropionic acid (MP), was used in an attempt to abolish GABAinhibition. MP was reported to produce convulsions in mice (Sprince et al., 1969), and to markedly reduce the concentration of G A B A in the cerebral or cerebellar cortex (Rodriguez de Lores Arnaiz et al., 1972, 1973; Karlsson et al., 1974). This effect of MP is suggested to be due to inhibition of glutamate decarboxylase which is directly involved in G A B A synthesis (Lamar, 1970; Rodriguez de Lores Arnaiz et al., 1972, 1973; Karlsson et al., 1974; Wu, 1975). In a third step of our experiments, MP and bicuculline, which may thus act separately on pre- and postsynaptic sites, were administered simultaneously.
Methods Preparation The experiments were done on fourteen adult cats. Under sodium pentobarbitone (Nembutal) anaesthesia (i.p., 30-40 mg/kg), the trachea and femoral vein were cannulated for artificial ventilation and infusion, respectively. The animals were immobilized by an i.v. infusion of gallamine triethiodide (9-12 mg/kg/h). Since the experiments mostly took up to 48 h, 20-30 mg Nembutal was added every 6-8 h. Further details of the preparation and care of the animals throughout the experiments are given elsewhere (Tsumoto et al., 1978).
Administration of Drugs Four-barrel micropipettes, consisting of two double-chambered pipettes (theta-pipettes) were used for the extracellular recording of neuron activity and the electrophoretic administration of drugs.
GABA-Inhibition in Visual Cortex
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One channel of a recording theta-pipette was filled with a solution of 2-M NaC1 and the other with 1-M NaC1 solution saturated with Fastgreen dye for histological identification of the recording site. A second theta-pipette was attached to the recording pipette which protruded 20-30 ~tm beyond the orifice of the former pipettes (Hess and Murata, 1974; Tsumoto et al., 1978). In most of the experiments, both channels of the second pipettes contained a solution of bicuculline (5 mM in 165 mM-NaC1, adjusted to pH 3 with HC1) or a solution of MP (2 mM in 165 mM-NaC1, adjusted to pH 6 with NaOH). In some experiments, one channel was filled with the bicuculline and the other with the MP solution. The ejecting currents for bicuculline and MP were +20 to + 150 and -50 to -200 nA, respectively. To prevent leakage of the drugs from the micropipette tips, the retaining currents o f - 2 0 and + 30 nA were passed through the pipettes with bicuculline and MP, respectively. In most of the experiments, MP was administered intravenously via the cannulated femoral vein with an infusion pump. For this, a solution of 2% MP in 165 mM NaC1 was infused at a rate of t5-20 mg/kg/h. The EEG was recorded continuously from the sensorimotor cortex ipsilateral to the recording site. When convulsive signs were observed in the EEG, administration of MP was stopped.
Recordings and Visual Stimulation Penetrations were made through a closed chamber overlying area 17 of the cortex. Amplification and display techniques for unit activity were conventional. After a unit was isolated, its excitatory RF was plotted with hand-held black or bright targets. For quantitative analysis of orientation and direction sensitivities, a moving light slit or dark bar was projected from the rear onto a screen located 1 m in front of the cat. Movement, orientation, and direction of the bar were computer controlled by moving a mirror system in the path of the projection beam. Orientation and movement direction were changed after each sweep and the response stored. Pefistimulis time histograms (PSTHs) for the forward and backward movements at each orientation were constructed after completion of the desired number of stimulus cycles. The rotation started from the optimal orientation in a clockwise fashion at steps of 30 or 45 ~ so that forward and backward movements at different orientations were tested in each unit. In the following section (results) the different orientations are represented as the optimal, the clockwise (+) or anticlockwise (-) deviations at 30 or 45 ~ intervals and at the orientation 90 ~ from the optimal. PSTHs at each orientation were constructed from 3-8 sweeps with a bin width of 5-40 ms. Starting and returning points of the stimulus at each orientation were arranged so that the stimulus always crossed the center of the excitatory RF of the recorded cell. After a complete set of PSTHs had been calculated, the computer could display all the PSTHs simultaneously, or an enlarged PSTH at a given orientation alone. Also, it could display a polar plot or orientation tuning curve based on the amplitude of the averaged responses. Usually, the stimulus was 1.0-1.5 log units above or below the background illumination, which was kept in the scotopic-mesopic range (about 10 -a ed/m2). Only one eye was stimulated. In order to assess drug effects, the same stimuli were given and PSTHs were constructed before, during and after administration of the drugs.
Results T h e p r e s e n t r e p o r t is b a s e d o n 6 0 n e u r o n s r e c o r d e d f r o m a r e a 17 o f t h e c o r t e x . T w e n t y - f i v e o f t h e m w e r e s t u d i e d w i t h M P , 2 0 w i t h b i c u c u l l i n e a n d 15 w i t h b o t h drugs.
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Fig. 1. Effect of MP on orientation and direction sensitivity of a complex cell. Direction of motion and orientation of the slit (0.5 ~ • 6 ~ are indicated by diagrams above each PSTH in A. Relative values of the orientation to the optimal are given in the left of A. Stimulus speed was about 6~ Four sweeps for each PSTH, bin width 7 ms. Vertical calibrations at the foot of A indicate 16 spikes/bin. A control PSTH-set made before MP administration. B PSTH-set obtained during intravenous infusion of MP. Records were taken 28-32 rain after starting the infusion. C control PSTH-set made 62-66 rain after stopping the infusion
of control PSTHs. About 10 min after i.p. injection, the E E G showed very large slow waves and sharp seizure potentials, eventually leading into status epilepticus. Simultaneously, the cells discharged in high frequency clusters, independent of visual stimuli and then became silent, probably due to depolarization block. After some 10 rnin, the E E G recovered to the original state, but the unit was usually lost. When a smaller dose was given, the EEG, cell activities and responses were not significantly altered. These observations indicate that the tolerance range for the action of MP is very narrow, and that intraperitoneal administration was not suitable for our experiments. As described later, electrophoretic MP exerted weaker effects on the stimulus selectivity of VC neurons than intravenous MP. In most experiments, therefore, MP was administered intravenously (see methods). An example of
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GABA-Inhibition in Visual Cortex Table 1. Orientation sensitivity Drug
Type of units
No. of units
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3 (33%) 0 6 (60 %) 0 2 (33%) 0 11 (44%)
8 7 6 21
5 (63%) 0 3 (43 %) 0 2 (33%) 0 10 (48%)
8 7 5 20
2 (25%) 5 (71%) 2 (40%)
7 6 3 16
1 (14%) 5 (71%) 1 (17 %) 2 (33 %) 1 (33%) 0 10 (63%)
7 6 2 15
4 (57%) 2 (29%) 3 (50%) 2 (33%) 1 (50%) 0 12 (80%)
6 4 2 12
1 (17%) 5 (83%) 1 (25%) 2 (50%) 1 (50%) 1 (50%) 11 (92%)
abolished a
0 0 0 9 (45%)
abolished b
a Reduction and abolishment of orientation sensitivity during application of the drugs: responses (spike numbers) to motion in the preferred direction of the slit oriented 90~to the optimal increased to 1049% or to > 50% of those to the optimal stimulus, respectively b Direction sensitivity: the responses to non-preferred and preferred motion direction of the optimally oriented slit were compared
the effect o f i.v. M P on the responses of a V C n e u r o n is shown in Fig. 1. This n e u r o n was a " c o m p l e x " cell according to the criteria o f H u b e I and Wiesel (1962) and had a large excitatory R F (Creutzfeldt et al., 1974). It r e s p o n d e d maximally to a slit m o v i n g f r o m the left to the right at the optimal orientation ( - 3 0 ~ to the vertical) and had a wide r e s p o n s e field (top o f A). Significant responses were also elicited by the m o v i n g slit if the orientation deviated + 3 0 ~ f r o m the optimal. Thus, the " o r i e n t a t i o n t u n i n g " of this unit was broad, as is usual for c o m p l e x cells. Thirty minutes after starting the c o n t i n u o u s i.v. infusion o f MP, the responses to the preferred m o t i o n of the slit at optimal and + 3 0 ~ orientations were m a r k e d l y increased (B). Slight responses w e r e also elicited by all o t h e r orientations. Thus, M P p r o d u c e d a slight, but significant r e d u c t i o n of the direction and orientation sensitivity of this unit. A b o u t 60 rain after stopping the M P infusion, its responsiveness r e c o v e r e d to the control level (C). Such a reduction o f orientation sensitivity by M P was seen in 11 o f 25 units tested, and o f direction sensitivity in ten of t w e n t y - o n e direction-sensitive units (Table 1). A cell with R F o f unequivocal " h y p e r c o m p l e x " type ( H u b e l and Wiesel, 1965) was not recorded, and our n u m b e r s are t o o small to suggest differential effects on c o m p l e x and simple cells. In a few experiments, M P was administered electrophoretically. Fifteen to 30 min after starting the ejection of MP, an increase in the s p o n t a n e o u s discharge and optimal response and a very slight r e d u c t i o n of those sensitivities were o b s e r v e d in some V C neurons.
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Fig. 2. Effect of bicuculline on orientation and direction sensitivity of a complex cell. Dashed line in each PSTH shows the turning point of forward and backward movement of a slit (0.5 ~ x 6~ Relative values of orientation of the slit to the optimal are indicated to the left of each PSTH. Stimulus speed was about 5~ Four sweeps for each PSTH, bin width 30 ms. The lowest records of A - C are PSTHs of the spontaneous discharge constructed under conditions identical for PSTH compilation in the absence of visual stimulation. Vertical calibration at the foot of A indicates 16 spikes/bin. A control set of PSTHs made before bicuculline administration. B set of time histograms obtained during electrophoretic administration of bicuculline (+ 60 nA). Records were taken 10-14 min after starting the administration. C control set of PSTHs made 20-24 rain after stopping the administration
Effects of Bicuculline In most V C neurons, electrophoretic bicuculline caused a clearer modification o f their specificities than MP. The strongest bicuculline effects observed in our experiments are shown in the example of Fig. 2. This unit was a complex cell, and responded best to forward m o v e m e n t s of the slit oriented 40 ~ to the vertical (top of A). The number of spikes elicited by the backward m o v e m e n t of the slit
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GABA-Inhibitionin VisualCortex
357
was about 40% of that of the optimal response. During administration of bicuculline (+60 nA), responses to the non-preferred motion of the optimally oriented slit increased to the same magnitude as those to the preferred motion (top of B). Thus, the direction sensitivity of this unit was completely eliminated by bicuculline. This confirms previous reports (Pettigrew and Daniels, 1973; Rose and Blakemore, 1974; Sillito, 1975b, 1977; Ostendorf and Hess, 1975; Hess et al., 1976). On the other hand, the orientation sensitivity was not completely abolished, although during bicuculline administration, clear responses could be elicited by slits oriented at + 45 ~ and 90 ~ to the optimal (B). The number of spikes elicited by the slit at 90 ~ to the optimal was 20 % of that of the maximal response. In five of seven complex cells the orientation tuning of visual responses was significantly broadened by bicuculline (Table 1), but the responses to the stimulus at 90 ~ to the optimal never exceeded 50% of that to the optimal. The number of simple cells, in which the orientation sensitivity was modified by bicuculline, was relatively small (2 out of 8 cells) and in no case was it abolished (Table 1) (see also Sillito, 1975b; Ostendorf and Hess, 1975; Hess et al., 1976).
Simultaneous Administration of MP and Bicuculline Since the two drugs appear to act separetely on pre- and post-synaptic sites, it was expected that a combination of both might have a stronger effect on orientation and direction sensitivity due to a more complete block of intracortical inhibition than by either drug alone. This turned out to be the case. An example of this is shown in Fig. 3. This neuron was classified as simple. It showed clearly direction-selective responses to motion of the slit at the optimal orientation (A). Orientation tuning of its responses was very sharp, as seen in most simple cells. During the electrophoretic administration of bicuculline (B) the response to the preferred direction was increased and strong responses appeared also in the non-preferred direction (68% of the amplitude to the optimal direction). On the other hand, motion of the slit oriented 90 ~ to the optimal elicited no responses even during bicuculline administration. After the responsiveness had recovered to the original control level (C), MP was infused intravenously. About 20 rain after beginning the infusion at the rate of 20 mg/kg/h (D), responses to slit motion in the non-preferred direction at the optimal orientation increased to 48 % of those to the opposite motion, but also in this condition, no responses were elicited by the moving slit oriented 90 ~ to the optimal. Bicuculline was then ejected electrophoretically while continuing the intravenous infusion of MP (E). Responses to both directions of motion of the optimally oriented slit increased to 310% of the control response, and also the orientation sensitivity was almost completely abolished. Response magnitudes to forward and backward motion of the slit oriented 90 ~ to the optimal were 84 and 66% of those of the optimal response, respectively. Spontaneous discharges were only slightly increased to a level of 1.1 spikes/sec (see Fig. 4A). Responsiveness of this unit had recovered to the control level about 40 min after ceasing the administration of the two drugs (F). The effects
358
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GABA-Inhibition in Visual Cortex
359
of the simultaneous administration of both drugs were not simply summation of the effects of each alone, since each drug had no effects on responses to the slit oriented 90 ~ to the optimal. These combined drug effects on visual responses of two simple cells are graphically illustrated in Fig. 4. The curves show the magnitude of response of the cells to the eight standard testing stimuli (see methods). In addition to the loss of the direction and orientation sensitivities, the responses to the optimal stimulus were also increased by the drugs, indicating that the control responses to the optimal stimuli without drugs were not "maximal". This excludes the possibility that the loss of orientation and direction sensitivities might be due to a saturation of responses to the optimal stimulus. In two out of seven simple and two out of six complex cells tested with the two drugs, the combined administration strengthened the effect of each so as to abolish the orientation sensitivity (Table 1). In the cells which lost their orientation sensitivity during simultaneous administration of both drugs, it was seen that their excitatory response fields only very slightly increased in size (see Fig. 3E) and appeared to be virtually round.
Discussion The present results have demonstrated that simultaneous administration of 3-mercaptopropionic acid (MP) and bicuculline could lead to a remarkable reduction or even complete loss of orientation and direction dependent response specificities of VC neurons, while each drug alone had less clear effects. This suggests that electrophoretic bicuculline may not completely block G A B A - m e d i a t e d inhibition of VC neurons. In fact, previous reports indicate that in some cortical neurons the inhibition, which was induced by electrophoretic G A B A or elicited by electrical stimulation, was not totally blocked by bicuculline (Curtis and Felix, 1971; Sillito, 1975a), probably because the alkaloid did not gain access to all of the receptors activated by G A B A . MP, on the other hand, had only weak effects on the direction and orientation sensitivities of VC neurons if infused intravenously or administered electrophoretically. This may be explained by too low a dose of MP; as long as it
Fig. 3. A-F. Effect of simultaneous administration of MP and bicuculline on orientation and direction sensitivity of a simple cell. Only responses to a moving slit (0.5~ • 5~ at the optimal orientation and 90~to it are shown in this figure. Dashed line in each PSTH shows the turning point of the stimulus. Stimulus speed was about 4~ Four sweeps for each PSTH, bin width 40 ms. Vertical calibration in C indicates 16 spikes/bin. A control PSTHs made before bicuculline administration. B PSTHs obtained during electrophoretic administration of bicuculline (+60 nA). Records were taken 12-17 min after starting the administration. C control PSTHs made 15-20 min after stopping the bicuculline administration. D PSTHs obtained during intravenous infusion of MP. Records were taken 20-25 rain after starting the infusion. E PSTHs obtained during simultaneous administration of both drugs. Records were taken 5-10 rain after re-starting bicuculline (+ 60 nA) in addition to continuous MP infusion, which runs already for 32 min. F control PSTHs made 40-45 rain after stopping the administration of both drugs
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Modification of orientation and direction sensitivity of simple cells by the two drugs. Curves show response magnitude for each cell to a moving slit at testing orientations as indicated on the abscissa. Backward m o v e m e n t of the slit oriented at the optimal, at + 9 0 ~ and at _+45~ was represented as + 1 8 0 ~, - 9 0 ~ and _+135 ~, respectively. N u m b e r of spikes of spontaneous activity obtained with the same m e t h o d s as in Fig. 2 are shown on the right. Ordinates indicate total n u m b e r of spikes in a PSTH. A the same unit as Fig. 3. C o n t i n u o u s line with open circles shows control responses of the cell prior to administration of bicuculline. The other three lines with the symbols as indicated in the top right are obtained from full sets of P S T H s corresponding to Fig. 3B, D, and E. B another simple cell. Symbols are the same as in A. In this cell single application of MP was not made. Results of bicuculline were obtained from a PSTH-set m a d e 1 2 - 1 6 min after starting the electrophoretic ejection ( + 8 0 nA). Those of MP + Bic. were obtained 2 0 - 2 4 min after simultaneous application of M P (i.v., at a rate of 15 m g / k g / h and bicuculline electrophoretically + 80 hA)
GABA-Inhibitionin VisualCortex
361
was subthreshold for causing convulsive signs in the EEG, it was probably insufficient to completely inhibit the GABA-synthesizing enzymes. Alternatively, GABA released from presynaptic sites might be taken up by the nerve terminals for re-use, so that gabergic synapses could function for some time without new synthesis of GABA (Iversen, 1971; Storm-Mathisen et al., 1975). These insufficient pre- and postsynaptic effects of the two drugs given alone, may at least partly explain why the combined use of both was more effective than single administration. The fact that electrophoretic MP induced similar, albeit slighter, effects on activities of VC neurons to those by intravenous MP suggests that the latter effects may be at least in part due to a loss of inhibition at the cortical level, The effects of electrophoretic bicuculline on simple and complex cells is to some extent differential (see Table 1): Reduction or abolishment of the direction sensitivity was seen in 85 % of the simple cells, whereas reduction of the orientation sensitivity in 71% of the complex cells. This tendency appears to be consistent with Sillito's results (1975b, 1977) that the direction sensitivity was lost in simple cells, while the orientation sensitivity was abolished in complex cells. On the other hand, we never observed a complete loss of the orientation sensitivity in complex or simple cells under electrophoretic bicuculline administration. This is in partial disagreement with Sillito's findings but confirms the observations of Ostendorf and Hess (1975) and Hess et al. (1976). Also, the present results that effects of simultaneous administration of MP and bicuculline was not significantly different in simple and complex cells may not support the dichotomic conclusion that the direction sensitivity of simple and orientation sensitivity of complex cells may be differentially abolished by intracortical disinhibition. Two reasons might account for the different results obtained in the two laboratories (Hess, personal communication): Firstly, in this laboratory assembled electrodes were used (see methods) in contrast to multibarrelled pipettes because of their better recording properties. The different electrode distances might to some extent affect the distribution of drugs in relation to the recorded nerve cell and thus lead to different results. The second and more likely possibility is the influence of anaesthetics. While Sillito used nitrous oxide in his experiments, we worked on Nembutal anaesthetized animals. As Nembutal reduces central excitability and possibly enhances GABA-mediated inhibition, it might also reduce the effectiveness of electrophoretically applied BIC. The present findings that orientation and direction sensitivity of VC neurons could be nearly completely abolished by the two simultaneously administered drugs which interfer with gabergic inhibition supports the suggestion that these properties depend on intracm-tical inhibition (Creutzfeldt and Ito, 1968; Benevento et al., 1972; Creutzfeldt et al., 1974). The fact that the response to the optimal stimulus was increased by disinhibition as well, is also consistent with the model based on intracellular recordings from VC neurons (Creutzfeldt et al., 1974). Furthermore, responsive areas were virtually round during blockade of gabergic inhibition, as also suggested by intracellular recordings (Creutzfeldt et al., 1974) as well as by the response width in non-optimal
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o r i e n t a t i o n s d u r i n g g l u t a m a t e e x c i t a t i o n ( H e s s a n d M u r a t a , 1974). It is t h e r e f o r e r e a s o n a b l e to c o n c l u d e that i n t r a c o r t i c a l i n h i b i t i o n m a k e s visual cortical n e u r o n s m o r e o r less selectively r e s p o n s i v e to m o v i n g line stimuli o f c e r t a i n o r i e n t a t i o n a n d d i r e c t i o n . It r e m a i n s still a r i d d l e , h o w e v e r , w h y t h e i n h i b i t o r y m e c h a n i s m r e s p o n s i b l e for o r i e n t a t i o n sensitivity a p p e a r s to b e m o r e r e s i s t a n t to r e m o v a l o f g a b e r g i c i n h i b i t i o n t h a n t h a t for d i r e c t i o n sensitivity. T h e s t r o n g e r r e s i s t a n c e o f o r i e n t a t i o n sensitivity to d i s i n h i b i t i o n m a y i n d i c a t e t h a t t h e i n p u t to c o r t i c a l n e u r o n s a l r e a d y has an o r i e n t a t i o n a l bias as s u g g e s t e d by r e c o r d i n g s in t h e r e t i n a ( H a m m o n d , 1 9 7 4 ) a n d t h e l a t e r a l g e n i c u l a t e b o d y ( C r e u t z f e l d t a n d N o t h d u r f l , 1978). T h e fact t h a t s o m e n e u r o n s a r e n o t a f f e c t e d at all b y e i t h e r drug, suggests t h a t at least s o m e o f t h e m m a y b e s e c o n d o r d e r c o r t i c a l n e u r o n s , w h i c h a r e d r i v e n by o r i e n t a t i o n / d i r e c t i o n sensitive first o r d e r neurons.
Acknowledgements. W. Eckart, who developed the computer programs and collaborated in the design and performance of the experiments, died in September 1977, 6 months after completion of the experimental work. We gratefully acknowledge his ingenious and inventive contributions. We also thank Drs. R. Hess and B.B. Lee for helpful advice during the experiments and preparation of the manuscript.
References Benevento, L.A., Creutzfeldt, O.D., Kuhnt, U.: Significance of intracortical inhibition in the visual cortex. Nature 238, 124-126 (1972) Bishop, P.O., Coombs, C.S., Henry, G.H.: Receptive fields of simple cells in the cat striate cortex. J. Physiol. 231, 31-60 (1973) Blakemore, C., Tobin, E.A.: Lateral inhibition between orientation detectors in the cat's visual cortex. Exp. Brain Res. 15, 439-440 (1972) Creutzfeldt, O.D., Ito, M.: Functional synaptic organization of primary visual cortex neurons 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) Creutzfeldt, O.D., Nothdurft, H.C.: Representation of complex visual stimuli in the brain. Naturwissenschaften 6, 307-318 (1978) Curtis, D.R., Duggan, A.W., Felix, D., Johnston, G.A.R.: GABA, bicuculline and central inhibition. Nature 220, 1222-1224 (1970) Curtis, D.R., Duggan, A.W., Felix, D., Johnsto n, G.A.R., McLennan, H.: Antagonism between bicuculline and GABA in the cat brain. Brain Res. 33, 57-73 (1971) Curtis, D.R., Felix, D.: The effect of bicueulline upon synaptic inhibition in the cerebral and cerebellar cortices of the cat. Brain Res. 34, 301-321 (1971) Curtis, D.R., Johnston, G.A.R.: Amino-acid transmitters in the mammalian central nervous system. Ergebn. Physiol. 69, 98-188 (1974) Hammond, P.: Cat retinal ganglion cells: size and shape of receptive field centres. J. Physiol. (Lond.) 242, 99-118 (1974) Hess, R., Murata, K.: Effects of glutamate and GABA on specific response properties of neurones in the visual cortex. Exp. Brain Res. 21, 285-297 (1974) Hess, R., Ostendorf, D., Sanides, D., Negishi, K., Creutzfeldt, O.D.: Neural connections in the visual cortex as revealed by intracortical chemical stimulation. In: Afferent and Intrinsic Organization of Laminated Structures in the Brain, O. Creutzfeldt (Ed.), Exp. Brain Res. Suppl. 1, 384-388 (1976)
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Hubel, D.H., Wiesel, T.N.: Receptive fields, binocular interaction and functional alcchitecture in the cat's visual cortex. J. Physiol. (Lond.) 169, 106-154 (1962) Hubel, D.H., Wiesel, T.N.: Receptive fields and functional architecture in two non-striate visual areas (18 and 19) of the cat. J. Neurophysiol. 28, 229-289 (1965) Iversen, L.L.: Role of transmitter uptake mechanisms in synaptic neurotransmission. Br. J. Pharmacol. 41, 571-591 (1971) Iversen, L.L., Mitchell, J.F., Srinivasan, V.: The release of y-aminobutyric acid during inhibition in the cat visual cortex. J. Physiol. (Lond.) 212, 519-534 (1971) Karlsson, A., Fonnum, F., Malthe-S~renssen, D., Storm-Mathisen, J.: Effect of the convulsive agent 3-mercaptopropionic acid on the levels of GABA, other amino acids and glutamate decarboxylase in different regions of the rat brain. Biochem. Pharmacol. 23, 3033-3061 (1974) Lamar, C., Jr.: Mercaptopropionic acid: A convulsant that inhibits glutamate decarboxylase. J. Neurochem. 17, 165-170 (1970) Mitchell, J.F., Srinivasan, V.: Release of SH-y-aminobutyric acid from the brain during synaptic inhibition. Nature 224, 663-666 (1969) Ostendorf, D., Hess, R.: Antagonism between GABA and bicuculline in visual cortical neurones. Is GABA mediating synaptic inhibition? Eur. J. Physiol. 335, (Suppl.) R 98 (1975) Pettigrew, J.D., Daniels, J.D.: GABA antagonism in visual cortex: different effects on simple, complex, and hypercomplex neurons. Science 182, 81-83 (1973) Rodriguez de Lores Arnaiz, G., Alberici de Canal, M., DeRobertis, E.: Alteration of GABA system and Purkinje cells in rat cerebellum by the convulsant 3-mercaptopropionic acid. J. Neurochem. 19, 1379-1385 (1972) Rodriguez de Lores Arnaiz, G., Alberici de Canal, M., Robiolo, B., Mistrnrigo de Pacheco, M.: The effect of the convulsant 3-mercaptopropionic acid on enzymes of the y-aminobutyrate system in the rat cerebral cortex. J. Neurochem. 21, 615-623 (1973) Rose, D., Blakemore, C.: Effects of bicuculline on functions of inhibition in visual cortex. Nature 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) Sillito, A.M.: Inhibitory processes underlying the directional specificity of simple, complex and hypercomplex cells in the cat's visual cortex, J. Physiol. (Lond.) 271, 699-720 (1977) Sillito, A.M.: The use of iontophoretically applied bicuculline to investigate inhibitory mechanisms in the visual cortex. In: O.D. Creutzfeldt (Ed.). Afferent and intrinsic organization of laminated structures in the brain. Exp. Brain Res. Suppl. 1, 389-393 (1977) Sprince, H., Parker, C.M., Josphs, J. A., Jr., Magazino, J.: Convulsant activity of homocysteine and other short-chain mercapto acids. Ann. N.Y. Acad. Sci. 166, 323-325 (1969) Storm-Mathisen, J., Fonnum, F., Malthe-Sorenssen, D.: GABA uptake in nerve terminal. In: G A B A in Nervous System Function, E. Roberts, T.N. Chase, D. Tower (Eds.), pp. 387-394. New York: Raven Press 1975 Tsumoto, T., Creutzfeldt, O.D., Legendy, C.R.: Functional organization of the corticofugal system from visual cortex to lateral geniculate nucleus in the cat (with an appendix on geniculo-cortical monosynaptic connections). Exp. Brain Res. 32, 345-364 (1978) Tsumoto, T.: Inhibitory and excitatory binocular convergence to visual cortical neurons of the cat. Brain Res. (in press) Wu, J.-Y.: Purification, characterization, and kinetic studies of GAD and GABA-T from mouse brain. In: GABA in Nervous System Function, E. Roberts, T.N. Chase, D.B. Tower (Eds.), pp. 7-55. New York: Raven Press 1975 Received June 22, 1978
Exp. Brain Res. 34, 351-363 (1979)
9
Springer-Verlag 1979
Modification of Orientation Sensitivity of Cat Visual Cortex Neurons by Removal of GABA-Mediated Inhibition T. Tsumoto 1, W. Eckart, and O.D. Creutzfeldt Department of Neurobiology,Max-Planck-Institutefor BiophysicalChemistry, Postfach 968, D - 3400 G6ttingen-Nikolausberg,Federal Republic of Germany
Summary.
The effects of an inhibitor of G A B A synthesis, 3-mercaptopropionic acid (MP), and of the G A B A antagonist bicuculline (B1C), on the direction and orientation sensitivity of visual cortical neurons were investigated using a computer-controlled stimulus presentation system. Intravenous administration of MP, which was usually more effective than if administered microelectrophoretically, induced a slight, but significant reduction in these properties of about half of the neurons tested. The effect of electrophoretic BIC was in the same direction but clearer than that of MP. In 71% of the simple cells, direction sensitivity was virtually lost during administration of BIC while orientation sensitivity was never completely eliminated in any neuron tested. Simultaneous administration of both drugs (MP systemically, BIC electrophoretically) caused more complete modification of the sensitivities than single administration of each. In four out of thirteen neurons tested, orientation sensitivity was completely abolished. The excitatory receptive fields slightly increased in size and became virtually round. The response magnitude to the optimal stimulus was increased by each drug alone and by both. The present results further support the hypothesis that intracortical inhibition plays a major if not an exclusive role for the orientation and direction sensitivity of cortical cells.
Key words: Visual cortex - Orientation sensitivity - GABA-inhibition Bicuculline - 3-Mercaptopropionic acid. Visual cortical (VC) neurons are preferentially or selectively responsive to particular visual stimulus parameters, such as to the orientation of a light slit or dark bar of a specific orientation moving in one direction (Hubel and Wiesel, 1962). Several lines of evidence suggest that the orientation and direction 1 Present address: Department of Physiology, Faculty of Medicine, Kanazawa University, Kanazawa, 920, Japan Offprint requests to: Prof. O.D. Creutzfeldt, M. D. (address see above)
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sensitivity may be partially due to intracortical inhibitory mechanisms (Creutzfeldt and Ito, 1968; Benevento et al., 1972; Blakemore and Tobin, 1972; Bishop et al., 1973; Creutzfeldt et al., 1974). If this is so, pharmacological blockade of intracortical inhibition should be able to abolish or attenuate those sensitivities of VC neurons. There is abundant evidence suggesting that gamma-aminobutyric acid (GABA) may be an inhibitory transmitter in VC (Mitchell and Srinivasan, 1969; Iversen et al., 1971) and that its action is selectively antagonized by the alkaloid bicuculline at postsynaptic sites (Curtis et al., 1970, 1971; Curtis and Johnston, 1974; Sillito, 1975a). Using intravenous bicuculline, Pettigrew and Daniels (1973) reported that VC neurons of the complex type lost some of their sensitivities but simple cells did not. In both types of cells, Rose and Blakemore (1974) found that topical application of the same drug reduced such sensitivities and increased the size of their excitatory receptive fields (RF). More recently, Sillito (1975a, b, t977) reported that in simple cells the direction sensitivity was significantly reduced or eliminated but the orientation sensitivity was only slightly reduced, whereas in complex cells the latter was virtually lost. Although the results of these three groups are not fully consistent with each other, each supports only partially the hypothesis described above. This may be due to an incomplete block of inhibition, because the GABA-induced or electrically elicited inhibition of some cortical neurons was not blocked by bicuculline (Curtis and Felix, 1971; Sillito, 1975a). In the present experiments an inhibitor of GABA-synthesis, 3-mercaptopropionic acid (MP), was used in an attempt to abolish GABAinhibition. MP was reported to produce convulsions in mice (Sprince et al., 1969), and to markedly reduce the concentration of G A B A in the cerebral or cerebellar cortex (Rodriguez de Lores Arnaiz et al., 1972, 1973; Karlsson et al., 1974). This effect of MP is suggested to be due to inhibition of glutamate decarboxylase which is directly involved in G A B A synthesis (Lamar, 1970; Rodriguez de Lores Arnaiz et al., 1972, 1973; Karlsson et al., 1974; Wu, 1975). In a third step of our experiments, MP and bicuculline, which may thus act separately on pre- and postsynaptic sites, were administered simultaneously.
Methods Preparation The experiments were done on fourteen adult cats. Under sodium pentobarbitone (Nembutal) anaesthesia (i.p., 30-40 mg/kg), the trachea and femoral vein were cannulated for artificial ventilation and infusion, respectively. The animals were immobilized by an i.v. infusion of gallamine triethiodide (9-12 mg/kg/h). Since the experiments mostly took up to 48 h, 20-30 mg Nembutal was added every 6-8 h. Further details of the preparation and care of the animals throughout the experiments are given elsewhere (Tsumoto et al., 1978).
Administration of Drugs Four-barrel micropipettes, consisting of two double-chambered pipettes (theta-pipettes) were used for the extracellular recording of neuron activity and the electrophoretic administration of drugs.
GABA-Inhibition in Visual Cortex
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One channel of a recording theta-pipette was filled with a solution of 2-M NaC1 and the other with 1-M NaC1 solution saturated with Fastgreen dye for histological identification of the recording site. A second theta-pipette was attached to the recording pipette which protruded 20-30 ~tm beyond the orifice of the former pipettes (Hess and Murata, 1974; Tsumoto et al., 1978). In most of the experiments, both channels of the second pipettes contained a solution of bicuculline (5 mM in 165 mM-NaC1, adjusted to pH 3 with HC1) or a solution of MP (2 mM in 165 mM-NaC1, adjusted to pH 6 with NaOH). In some experiments, one channel was filled with the bicuculline and the other with the MP solution. The ejecting currents for bicuculline and MP were +20 to + 150 and -50 to -200 nA, respectively. To prevent leakage of the drugs from the micropipette tips, the retaining currents o f - 2 0 and + 30 nA were passed through the pipettes with bicuculline and MP, respectively. In most of the experiments, MP was administered intravenously via the cannulated femoral vein with an infusion pump. For this, a solution of 2% MP in 165 mM NaC1 was infused at a rate of t5-20 mg/kg/h. The EEG was recorded continuously from the sensorimotor cortex ipsilateral to the recording site. When convulsive signs were observed in the EEG, administration of MP was stopped.
Recordings and Visual Stimulation Penetrations were made through a closed chamber overlying area 17 of the cortex. Amplification and display techniques for unit activity were conventional. After a unit was isolated, its excitatory RF was plotted with hand-held black or bright targets. For quantitative analysis of orientation and direction sensitivities, a moving light slit or dark bar was projected from the rear onto a screen located 1 m in front of the cat. Movement, orientation, and direction of the bar were computer controlled by moving a mirror system in the path of the projection beam. Orientation and movement direction were changed after each sweep and the response stored. Pefistimulis time histograms (PSTHs) for the forward and backward movements at each orientation were constructed after completion of the desired number of stimulus cycles. The rotation started from the optimal orientation in a clockwise fashion at steps of 30 or 45 ~ so that forward and backward movements at different orientations were tested in each unit. In the following section (results) the different orientations are represented as the optimal, the clockwise (+) or anticlockwise (-) deviations at 30 or 45 ~ intervals and at the orientation 90 ~ from the optimal. PSTHs at each orientation were constructed from 3-8 sweeps with a bin width of 5-40 ms. Starting and returning points of the stimulus at each orientation were arranged so that the stimulus always crossed the center of the excitatory RF of the recorded cell. After a complete set of PSTHs had been calculated, the computer could display all the PSTHs simultaneously, or an enlarged PSTH at a given orientation alone. Also, it could display a polar plot or orientation tuning curve based on the amplitude of the averaged responses. Usually, the stimulus was 1.0-1.5 log units above or below the background illumination, which was kept in the scotopic-mesopic range (about 10 -a ed/m2). Only one eye was stimulated. In order to assess drug effects, the same stimuli were given and PSTHs were constructed before, during and after administration of the drugs.
Results T h e p r e s e n t r e p o r t is b a s e d o n 6 0 n e u r o n s r e c o r d e d f r o m a r e a 17 o f t h e c o r t e x . T w e n t y - f i v e o f t h e m w e r e s t u d i e d w i t h M P , 2 0 w i t h b i c u c u l l i n e a n d 15 w i t h b o t h drugs.
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of control PSTHs. About 10 min after i.p. injection, the E E G showed very large slow waves and sharp seizure potentials, eventually leading into status epilepticus. Simultaneously, the cells discharged in high frequency clusters, independent of visual stimuli and then became silent, probably due to depolarization block. After some 10 rnin, the E E G recovered to the original state, but the unit was usually lost. When a smaller dose was given, the EEG, cell activities and responses were not significantly altered. These observations indicate that the tolerance range for the action of MP is very narrow, and that intraperitoneal administration was not suitable for our experiments. As described later, electrophoretic MP exerted weaker effects on the stimulus selectivity of VC neurons than intravenous MP. In most experiments, therefore, MP was administered intravenously (see methods). An example of
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GABA-Inhibition in Visual Cortex Table 1. Orientation sensitivity Drug
Type of units
No. of units
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a Reduction and abolishment of orientation sensitivity during application of the drugs: responses (spike numbers) to motion in the preferred direction of the slit oriented 90~to the optimal increased to 1049% or to > 50% of those to the optimal stimulus, respectively b Direction sensitivity: the responses to non-preferred and preferred motion direction of the optimally oriented slit were compared
the effect o f i.v. M P on the responses of a V C n e u r o n is shown in Fig. 1. This n e u r o n was a " c o m p l e x " cell according to the criteria o f H u b e I and Wiesel (1962) and had a large excitatory R F (Creutzfeldt et al., 1974). It r e s p o n d e d maximally to a slit m o v i n g f r o m the left to the right at the optimal orientation ( - 3 0 ~ to the vertical) and had a wide r e s p o n s e field (top o f A). Significant responses were also elicited by the m o v i n g slit if the orientation deviated + 3 0 ~ f r o m the optimal. Thus, the " o r i e n t a t i o n t u n i n g " of this unit was broad, as is usual for c o m p l e x cells. Thirty minutes after starting the c o n t i n u o u s i.v. infusion o f MP, the responses to the preferred m o t i o n of the slit at optimal and + 3 0 ~ orientations were m a r k e d l y increased (B). Slight responses w e r e also elicited by all o t h e r orientations. Thus, M P p r o d u c e d a slight, but significant r e d u c t i o n of the direction and orientation sensitivity of this unit. A b o u t 60 rain after stopping the M P infusion, its responsiveness r e c o v e r e d to the control level (C). Such a reduction o f orientation sensitivity by M P was seen in 11 o f 25 units tested, and o f direction sensitivity in ten of t w e n t y - o n e direction-sensitive units (Table 1). A cell with R F o f unequivocal " h y p e r c o m p l e x " type ( H u b e l and Wiesel, 1965) was not recorded, and our n u m b e r s are t o o small to suggest differential effects on c o m p l e x and simple cells. In a few experiments, M P was administered electrophoretically. Fifteen to 30 min after starting the ejection of MP, an increase in the s p o n t a n e o u s discharge and optimal response and a very slight r e d u c t i o n of those sensitivities were o b s e r v e d in some V C neurons.
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Effects of Bicuculline In most V C neurons, electrophoretic bicuculline caused a clearer modification o f their specificities than MP. The strongest bicuculline effects observed in our experiments are shown in the example of Fig. 2. This unit was a complex cell, and responded best to forward m o v e m e n t s of the slit oriented 40 ~ to the vertical (top of A). The number of spikes elicited by the backward m o v e m e n t of the slit
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GABA-Inhibitionin VisualCortex
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was about 40% of that of the optimal response. During administration of bicuculline (+60 nA), responses to the non-preferred motion of the optimally oriented slit increased to the same magnitude as those to the preferred motion (top of B). Thus, the direction sensitivity of this unit was completely eliminated by bicuculline. This confirms previous reports (Pettigrew and Daniels, 1973; Rose and Blakemore, 1974; Sillito, 1975b, 1977; Ostendorf and Hess, 1975; Hess et al., 1976). On the other hand, the orientation sensitivity was not completely abolished, although during bicuculline administration, clear responses could be elicited by slits oriented at + 45 ~ and 90 ~ to the optimal (B). The number of spikes elicited by the slit at 90 ~ to the optimal was 20 % of that of the maximal response. In five of seven complex cells the orientation tuning of visual responses was significantly broadened by bicuculline (Table 1), but the responses to the stimulus at 90 ~ to the optimal never exceeded 50% of that to the optimal. The number of simple cells, in which the orientation sensitivity was modified by bicuculline, was relatively small (2 out of 8 cells) and in no case was it abolished (Table 1) (see also Sillito, 1975b; Ostendorf and Hess, 1975; Hess et al., 1976).
Simultaneous Administration of MP and Bicuculline Since the two drugs appear to act separetely on pre- and post-synaptic sites, it was expected that a combination of both might have a stronger effect on orientation and direction sensitivity due to a more complete block of intracortical inhibition than by either drug alone. This turned out to be the case. An example of this is shown in Fig. 3. This neuron was classified as simple. It showed clearly direction-selective responses to motion of the slit at the optimal orientation (A). Orientation tuning of its responses was very sharp, as seen in most simple cells. During the electrophoretic administration of bicuculline (B) the response to the preferred direction was increased and strong responses appeared also in the non-preferred direction (68% of the amplitude to the optimal direction). On the other hand, motion of the slit oriented 90 ~ to the optimal elicited no responses even during bicuculline administration. After the responsiveness had recovered to the original control level (C), MP was infused intravenously. About 20 rain after beginning the infusion at the rate of 20 mg/kg/h (D), responses to slit motion in the non-preferred direction at the optimal orientation increased to 48 % of those to the opposite motion, but also in this condition, no responses were elicited by the moving slit oriented 90 ~ to the optimal. Bicuculline was then ejected electrophoretically while continuing the intravenous infusion of MP (E). Responses to both directions of motion of the optimally oriented slit increased to 310% of the control response, and also the orientation sensitivity was almost completely abolished. Response magnitudes to forward and backward motion of the slit oriented 90 ~ to the optimal were 84 and 66% of those of the optimal response, respectively. Spontaneous discharges were only slightly increased to a level of 1.1 spikes/sec (see Fig. 4A). Responsiveness of this unit had recovered to the control level about 40 min after ceasing the administration of the two drugs (F). The effects
358
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GABA-Inhibition in Visual Cortex
359
of the simultaneous administration of both drugs were not simply summation of the effects of each alone, since each drug had no effects on responses to the slit oriented 90 ~ to the optimal. These combined drug effects on visual responses of two simple cells are graphically illustrated in Fig. 4. The curves show the magnitude of response of the cells to the eight standard testing stimuli (see methods). In addition to the loss of the direction and orientation sensitivities, the responses to the optimal stimulus were also increased by the drugs, indicating that the control responses to the optimal stimuli without drugs were not "maximal". This excludes the possibility that the loss of orientation and direction sensitivities might be due to a saturation of responses to the optimal stimulus. In two out of seven simple and two out of six complex cells tested with the two drugs, the combined administration strengthened the effect of each so as to abolish the orientation sensitivity (Table 1). In the cells which lost their orientation sensitivity during simultaneous administration of both drugs, it was seen that their excitatory response fields only very slightly increased in size (see Fig. 3E) and appeared to be virtually round.
Discussion The present results have demonstrated that simultaneous administration of 3-mercaptopropionic acid (MP) and bicuculline could lead to a remarkable reduction or even complete loss of orientation and direction dependent response specificities of VC neurons, while each drug alone had less clear effects. This suggests that electrophoretic bicuculline may not completely block G A B A - m e d i a t e d inhibition of VC neurons. In fact, previous reports indicate that in some cortical neurons the inhibition, which was induced by electrophoretic G A B A or elicited by electrical stimulation, was not totally blocked by bicuculline (Curtis and Felix, 1971; Sillito, 1975a), probably because the alkaloid did not gain access to all of the receptors activated by G A B A . MP, on the other hand, had only weak effects on the direction and orientation sensitivities of VC neurons if infused intravenously or administered electrophoretically. This may be explained by too low a dose of MP; as long as it
Fig. 3. A-F. Effect of simultaneous administration of MP and bicuculline on orientation and direction sensitivity of a simple cell. Only responses to a moving slit (0.5~ • 5~ at the optimal orientation and 90~to it are shown in this figure. Dashed line in each PSTH shows the turning point of the stimulus. Stimulus speed was about 4~ Four sweeps for each PSTH, bin width 40 ms. Vertical calibration in C indicates 16 spikes/bin. A control PSTHs made before bicuculline administration. B PSTHs obtained during electrophoretic administration of bicuculline (+60 nA). Records were taken 12-17 min after starting the administration. C control PSTHs made 15-20 min after stopping the bicuculline administration. D PSTHs obtained during intravenous infusion of MP. Records were taken 20-25 rain after starting the infusion. E PSTHs obtained during simultaneous administration of both drugs. Records were taken 5-10 rain after re-starting bicuculline (+ 60 nA) in addition to continuous MP infusion, which runs already for 32 min. F control PSTHs made 40-45 rain after stopping the administration of both drugs
360
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Modification of orientation and direction sensitivity of simple cells by the two drugs. Curves show response magnitude for each cell to a moving slit at testing orientations as indicated on the abscissa. Backward m o v e m e n t of the slit oriented at the optimal, at + 9 0 ~ and at _+45~ was represented as + 1 8 0 ~, - 9 0 ~ and _+135 ~, respectively. N u m b e r of spikes of spontaneous activity obtained with the same m e t h o d s as in Fig. 2 are shown on the right. Ordinates indicate total n u m b e r of spikes in a PSTH. A the same unit as Fig. 3. C o n t i n u o u s line with open circles shows control responses of the cell prior to administration of bicuculline. The other three lines with the symbols as indicated in the top right are obtained from full sets of P S T H s corresponding to Fig. 3B, D, and E. B another simple cell. Symbols are the same as in A. In this cell single application of MP was not made. Results of bicuculline were obtained from a PSTH-set m a d e 1 2 - 1 6 min after starting the electrophoretic ejection ( + 8 0 nA). Those of MP + Bic. were obtained 2 0 - 2 4 min after simultaneous application of M P (i.v., at a rate of 15 m g / k g / h and bicuculline electrophoretically + 80 hA)
GABA-Inhibitionin VisualCortex
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was subthreshold for causing convulsive signs in the EEG, it was probably insufficient to completely inhibit the GABA-synthesizing enzymes. Alternatively, GABA released from presynaptic sites might be taken up by the nerve terminals for re-use, so that gabergic synapses could function for some time without new synthesis of GABA (Iversen, 1971; Storm-Mathisen et al., 1975). These insufficient pre- and postsynaptic effects of the two drugs given alone, may at least partly explain why the combined use of both was more effective than single administration. The fact that electrophoretic MP induced similar, albeit slighter, effects on activities of VC neurons to those by intravenous MP suggests that the latter effects may be at least in part due to a loss of inhibition at the cortical level, The effects of electrophoretic bicuculline on simple and complex cells is to some extent differential (see Table 1): Reduction or abolishment of the direction sensitivity was seen in 85 % of the simple cells, whereas reduction of the orientation sensitivity in 71% of the complex cells. This tendency appears to be consistent with Sillito's results (1975b, 1977) that the direction sensitivity was lost in simple cells, while the orientation sensitivity was abolished in complex cells. On the other hand, we never observed a complete loss of the orientation sensitivity in complex or simple cells under electrophoretic bicuculline administration. This is in partial disagreement with Sillito's findings but confirms the observations of Ostendorf and Hess (1975) and Hess et al. (1976). Also, the present results that effects of simultaneous administration of MP and bicuculline was not significantly different in simple and complex cells may not support the dichotomic conclusion that the direction sensitivity of simple and orientation sensitivity of complex cells may be differentially abolished by intracortical disinhibition. Two reasons might account for the different results obtained in the two laboratories (Hess, personal communication): Firstly, in this laboratory assembled electrodes were used (see methods) in contrast to multibarrelled pipettes because of their better recording properties. The different electrode distances might to some extent affect the distribution of drugs in relation to the recorded nerve cell and thus lead to different results. The second and more likely possibility is the influence of anaesthetics. While Sillito used nitrous oxide in his experiments, we worked on Nembutal anaesthetized animals. As Nembutal reduces central excitability and possibly enhances GABA-mediated inhibition, it might also reduce the effectiveness of electrophoretically applied BIC. The present findings that orientation and direction sensitivity of VC neurons could be nearly completely abolished by the two simultaneously administered drugs which interfer with gabergic inhibition supports the suggestion that these properties depend on intracm-tical inhibition (Creutzfeldt and Ito, 1968; Benevento et al., 1972; Creutzfeldt et al., 1974). The fact that the response to the optimal stimulus was increased by disinhibition as well, is also consistent with the model based on intracellular recordings from VC neurons (Creutzfeldt et al., 1974). Furthermore, responsive areas were virtually round during blockade of gabergic inhibition, as also suggested by intracellular recordings (Creutzfeldt et al., 1974) as well as by the response width in non-optimal
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o r i e n t a t i o n s d u r i n g g l u t a m a t e e x c i t a t i o n ( H e s s a n d M u r a t a , 1974). It is t h e r e f o r e r e a s o n a b l e to c o n c l u d e that i n t r a c o r t i c a l i n h i b i t i o n m a k e s visual cortical n e u r o n s m o r e o r less selectively r e s p o n s i v e to m o v i n g line stimuli o f c e r t a i n o r i e n t a t i o n a n d d i r e c t i o n . It r e m a i n s still a r i d d l e , h o w e v e r , w h y t h e i n h i b i t o r y m e c h a n i s m r e s p o n s i b l e for o r i e n t a t i o n sensitivity a p p e a r s to b e m o r e r e s i s t a n t to r e m o v a l o f g a b e r g i c i n h i b i t i o n t h a n t h a t for d i r e c t i o n sensitivity. T h e s t r o n g e r r e s i s t a n c e o f o r i e n t a t i o n sensitivity to d i s i n h i b i t i o n m a y i n d i c a t e t h a t t h e i n p u t to c o r t i c a l n e u r o n s a l r e a d y has an o r i e n t a t i o n a l bias as s u g g e s t e d by r e c o r d i n g s in t h e r e t i n a ( H a m m o n d , 1 9 7 4 ) a n d t h e l a t e r a l g e n i c u l a t e b o d y ( C r e u t z f e l d t a n d N o t h d u r f l , 1978). T h e fact t h a t s o m e n e u r o n s a r e n o t a f f e c t e d at all b y e i t h e r drug, suggests t h a t at least s o m e o f t h e m m a y b e s e c o n d o r d e r c o r t i c a l n e u r o n s , w h i c h a r e d r i v e n by o r i e n t a t i o n / d i r e c t i o n sensitive first o r d e r neurons.
Acknowledgements. W. Eckart, who developed the computer programs and collaborated in the design and performance of the experiments, died in September 1977, 6 months after completion of the experimental work. We gratefully acknowledge his ingenious and inventive contributions. We also thank Drs. R. Hess and B.B. Lee for helpful advice during the experiments and preparation of the manuscript.
References Benevento, L.A., Creutzfeldt, O.D., Kuhnt, U.: Significance of intracortical inhibition in the visual cortex. Nature 238, 124-126 (1972) Bishop, P.O., Coombs, C.S., Henry, G.H.: Receptive fields of simple cells in the cat striate cortex. J. Physiol. 231, 31-60 (1973) Blakemore, C., Tobin, E.A.: Lateral inhibition between orientation detectors in the cat's visual cortex. Exp. Brain Res. 15, 439-440 (1972) Creutzfeldt, O.D., Ito, M.: Functional synaptic organization of primary visual cortex neurons 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) Creutzfeldt, O.D., Nothdurft, H.C.: Representation of complex visual stimuli in the brain. Naturwissenschaften 6, 307-318 (1978) Curtis, D.R., Duggan, A.W., Felix, D., Johnston, G.A.R.: GABA, bicuculline and central inhibition. Nature 220, 1222-1224 (1970) Curtis, D.R., Duggan, A.W., Felix, D., Johnsto n, G.A.R., McLennan, H.: Antagonism between bicuculline and GABA in the cat brain. Brain Res. 33, 57-73 (1971) Curtis, D.R., Felix, D.: The effect of bicueulline upon synaptic inhibition in the cerebral and cerebellar cortices of the cat. Brain Res. 34, 301-321 (1971) Curtis, D.R., Johnston, G.A.R.: Amino-acid transmitters in the mammalian central nervous system. Ergebn. Physiol. 69, 98-188 (1974) Hammond, P.: Cat retinal ganglion cells: size and shape of receptive field centres. J. Physiol. (Lond.) 242, 99-118 (1974) Hess, R., Murata, K.: Effects of glutamate and GABA on specific response properties of neurones in the visual cortex. Exp. Brain Res. 21, 285-297 (1974) Hess, R., Ostendorf, D., Sanides, D., Negishi, K., Creutzfeldt, O.D.: Neural connections in the visual cortex as revealed by intracortical chemical stimulation. In: Afferent and Intrinsic Organization of Laminated Structures in the Brain, O. Creutzfeldt (Ed.), Exp. Brain Res. Suppl. 1, 384-388 (1976)
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Hubel, D.H., Wiesel, T.N.: Receptive fields, binocular interaction and functional alcchitecture in the cat's visual cortex. J. Physiol. (Lond.) 169, 106-154 (1962) Hubel, D.H., Wiesel, T.N.: Receptive fields and functional architecture in two non-striate visual areas (18 and 19) of the cat. J. Neurophysiol. 28, 229-289 (1965) Iversen, L.L.: Role of transmitter uptake mechanisms in synaptic neurotransmission. Br. J. Pharmacol. 41, 571-591 (1971) Iversen, L.L., Mitchell, J.F., Srinivasan, V.: The release of y-aminobutyric acid during inhibition in the cat visual cortex. J. Physiol. (Lond.) 212, 519-534 (1971) Karlsson, A., Fonnum, F., Malthe-S~renssen, D., Storm-Mathisen, J.: Effect of the convulsive agent 3-mercaptopropionic acid on the levels of GABA, other amino acids and glutamate decarboxylase in different regions of the rat brain. Biochem. Pharmacol. 23, 3033-3061 (1974) Lamar, C., Jr.: Mercaptopropionic acid: A convulsant that inhibits glutamate decarboxylase. J. Neurochem. 17, 165-170 (1970) Mitchell, J.F., Srinivasan, V.: Release of SH-y-aminobutyric acid from the brain during synaptic inhibition. Nature 224, 663-666 (1969) Ostendorf, D., Hess, R.: Antagonism between GABA and bicuculline in visual cortical neurones. Is GABA mediating synaptic inhibition? Eur. J. Physiol. 335, (Suppl.) R 98 (1975) Pettigrew, J.D., Daniels, J.D.: GABA antagonism in visual cortex: different effects on simple, complex, and hypercomplex neurons. Science 182, 81-83 (1973) Rodriguez de Lores Arnaiz, G., Alberici de Canal, M., DeRobertis, E.: Alteration of GABA system and Purkinje cells in rat cerebellum by the convulsant 3-mercaptopropionic acid. J. Neurochem. 19, 1379-1385 (1972) Rodriguez de Lores Arnaiz, G., Alberici de Canal, M., Robiolo, B., Mistrnrigo de Pacheco, M.: The effect of the convulsant 3-mercaptopropionic acid on enzymes of the y-aminobutyrate system in the rat cerebral cortex. J. Neurochem. 21, 615-623 (1973) Rose, D., Blakemore, C.: Effects of bicuculline on functions of inhibition in visual cortex. Nature 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) Sillito, A.M.: Inhibitory processes underlying the directional specificity of simple, complex and hypercomplex cells in the cat's visual cortex, J. Physiol. (Lond.) 271, 699-720 (1977) Sillito, A.M.: The use of iontophoretically applied bicuculline to investigate inhibitory mechanisms in the visual cortex. In: O.D. Creutzfeldt (Ed.). Afferent and intrinsic organization of laminated structures in the brain. Exp. Brain Res. Suppl. 1, 389-393 (1977) Sprince, H., Parker, C.M., Josphs, J. A., Jr., Magazino, J.: Convulsant activity of homocysteine and other short-chain mercapto acids. Ann. N.Y. Acad. Sci. 166, 323-325 (1969) Storm-Mathisen, J., Fonnum, F., Malthe-Sorenssen, D.: GABA uptake in nerve terminal. In: G A B A in Nervous System Function, E. Roberts, T.N. Chase, D. Tower (Eds.), pp. 387-394. New York: Raven Press 1975 Tsumoto, T., Creutzfeldt, O.D., Legendy, C.R.: Functional organization of the corticofugal system from visual cortex to lateral geniculate nucleus in the cat (with an appendix on geniculo-cortical monosynaptic connections). Exp. Brain Res. 32, 345-364 (1978) Tsumoto, T.: Inhibitory and excitatory binocular convergence to visual cortical neurons of the cat. Brain Res. (in press) Wu, J.-Y.: Purification, characterization, and kinetic studies of GAD and GABA-T from mouse brain. In: GABA in Nervous System Function, E. Roberts, T.N. Chase, D.B. Tower (Eds.), pp. 7-55. New York: Raven Press 1975 Received June 22, 1978
