Brain Research, 54() (199i) 14 2~{

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Postnatal development of neuronal connections in cat visual cortex studied by intracellular recording in slice preparation Yukio Komatsu and Michiyo Iwakiri Department of Physiology, Kyoto Prefectural University of Medicine, Kyoto (Japan) (Accepted 21 August 1990)

Key words: Visual cortex; Area 17; Postnatal development; Cat; Slice preparation; Inhibitory postsynaptic potential; Excitatory postsynaptic potential

Postnatal development of neuronal connections in cat visual cortex (area 17) was studied in slice preparations obtained from kittens aged 1-18 weeks after birth and adult cats by recording intracellularly excitatory (EPSP) and inhibitory postsynaptic potentials (IPSP) evoked in cortical cells by stimulation of white matter. The EPSPs were already present in all cells at 1 week of age. Their efficiency assessed by their maximum rate of rise was low initially and increased progressively with age. In contrast, the IPSPs were absent in half of the cells at 1 week and almost all of the cells came to demonstrate inhibition by 9 weeks except for a few layer II-III cells. At all ages about three-quarters of the IPSPs had GABAA-mediated early and GABAB-mediated late components with different time courses, reversal potential and sensitivity to GABA antagonists, while the remaining IPSPs had only the early component. The efficiency of both IPSPs assessed by the associated conductance increase showed an increase of more than twice from 1 to 5 weeks, reaching the same level as adults. The time course of the development of inhibition demonstrated in this study paralleled the time course of the development of selective visual responsiveness in cortical cells, suggesting that the postnatal maturation of inhibitory connections is a basis of maturation of visual responsiveness. INTRODUCTION D e v e l o p m e n t a l studies of neuronal responsiveness in the visual cortex over the last two decades have established that the cortical circuitry is very i m m a t u r e at birth 1"5"6"9"1°'19'27'55. The response of the visual cortical cells to visual stimulation is w e a k e r and less selective in n e w b o r n animals than in adult animals. The photic responsiveness rapidly matures during the early postnatal p e r i o d and reaches the adult type by the end of 2 months. N e u r o a n a t o m i c a l evidence has been p r o v i d e d for the postnatal m a t u r a t i o n of cortical circuitry which may underlie the d e v e l o p m e n t of visual responsiveness. Visual cortical cells of newborn animals exhibit very small s o m a t a , short dendrites, and rather few spines. All of these morphological features show rapid d e v e l o p m e n t during the early postnatal p e r i o d 7'31'41-43'45"47'50. Electron microscopic studies have d e m o n s t r a t e d that the n u m b e r of synapses is rather few in newborn animals and increases during this period 14'32'44"57. Evidence suggesting elimination of the a b e r r a n t connections has also been r e p o r t e d 3°'48"56. These findings indicate that extensive reorganization of the visual cortical networks occurs during the early postnatal period, involving both the

formation and the elimination of the excitatory and inhibitory synaptic connections. H o w e v e r , there have b e e n few reports on electrophysiological studies d e m o n s t r a t i n g the d e v e l o p m e n t of excitatory and inhibitory transmission in the cortex. The aim of the present study was to investigate the development of the cortical circuitry by using slice preparations, which provides stable recording of the synaptic potentials e v o k e d in cortical cells from white matter. The results indicate that excitatory transmission has a l r e a d y been established at birth, and that inhibitory transmission develops later during the postnatal period, which m a y be the basis for the d e v e l o p m e n t of the response selectivity of visual cortical cells. A part of the present results have been published 35.

MATERIALS AND METHODS

Preparation Fifty-two kittens at ages of 1 (1-7 days), 3 (15-21 days), 5 (29-35 days), 9 (57-63 days) and 18 (120-126 days) weeks after birth and 9 adult cats (older than 1 year) were used in the study. A block of visual cortex (area 17) was dissected from the brain of the animals under deep anesthesia induced by sodium pentobarbital (50 mg/kg). Coronal slices (width 0.4 mm) were prepared using a Vibratome

Correspondence: Y. Komatsu, Department of Physiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602, Japan. 0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)

15 (Oxford) or Microslicer (Dosaka, DTK-1000) and incubated in Krebs-Ringer solution ((in mM): NaCI 124, KCI 5, KH2PO 4 1.24, MgSO 4 1.3, CaC! 2 2.4, NaHCO 3 26, glucose 10) saturated with a mixture of gas with 95% 0 2 and 5% CO 2 at 33 °C for 1 h. After the incubation, a slice was placed in a submerge type recording chamber and perfused with the same solution as used for incubation (rate of perfusion, 1-3 mi/min). The drugs used in the study were bicuculline methiodide (Pierce) and phaclofen (Tocris neuramin).

Stimulation and recording A glass microelectrode filled with 2 M potassium methylsulfate or 3 M potassium acetate (electrical resistance 40-150 MO) was mounted on a 3-dimensional oil-driven micromanipulator (Narishige MO-103), one axis of which was aligned with the cortical columnar structures. Cortical cells were penetrated in layers II-VI along the cortical column (dotted line in Fig. 1A), and intracellular responses were studied. A conventional bridge circuit was used to record the membrane potential during current injection (amplitude 0.1-2 nA, duration 200--800 ms) through the recording microelectrode. To characterize the postsynaptic potentials, two pairs of bipolar stimulating electrodes made of Teflon-coated platinum-iridium wires (interpolar distance 0.15 mm, diameter 0.1 mm) were placed in white matter on the extension of the recording track, so that they stimulated the same axons: one of the electrodes was placed roughly at the border between layer VI and white matter and the other 0.5-0.7 mm distant from the border 36 (sl and s2 in Fig. 1A). Constant-current pulses (intensity 0.1-4.5 mA, duration 100/~s)

Histology At the end of the recording experiment the recording microelectrode was replaced with another electrode containing 2% Pontamine sky blue, and recording sites were marked for two cells by electrophoretic dye injection (tip negative, 5/~A, 10 min) according to the micromanipulator readings. The location of the marked sites was determined on histological sections stained with Cresyl violet. The location of other cells was determined in the reconstructed recording track according to the micromanipulator readings, by

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were applied to the stimulating electrodes from a stimulator. Stimulation was applied at low frequencies (0.1-0.2 Hz) to avoid cumulative effects such as potentiation or depression of synaptic transmission36. The latencies of postsynaptic responses were measured with the distal as well as the proximal electrodes to obtain the conduction velocity of afferent impulses and central delays, that is the time spent in synaptic transmission after afferent impulses arrived at axon terminals35'36. To calculate the conduction velocity, the difference in latencies of the excitatory postsynaptic potentials (EPSPs) evoked by the two stimulating electrodes was divided by the distance between the two electrodes. The central delay was determined by subtracting the conduction time of the afferent impulses, which was estimated from the calculated conduction velocity, from the EPSP latency. The EPSP latencies were determined from diverging points of the superimposed traces of intracellular and extracellular responses (solid and dotted lines in Fig. 1B-F).

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Fig. 1. Postsynaptic potentials evoked in cortical cells by white matter stimulation. A: experimental arrangement, m, recording electrode, sl and s2, stimulating electrodes. WM, white matter. B-F: average traces (n = 5) of postsynaptic potentials recorded from cells sampled from an adult cat and 9-, 5- and 1-week-old kittens. The superimposed interrupted and dotted lines show intracellular responses during the hyperpolarization of membrane potential and extracellular responses recorded after the withdrawal of the recording electrode from the cells. Resting membrane potentials were -55 (B), -54 (C), -57 (D), -55 (E) and -55 mV (F). Hyperpolarizing currents were 0.6 nA for B,C and 0.4 nA for D,E. The intensity of white matter stimulation was 0.5 mA for B,C and 1 mA for D-F. Voltage calibration of 5 mV applies to all traces. Time calibration of 3 ms applies to B-D.

16 making allowances for the shrinkage of the histological section which was determined as the ratio of the distance between the marked sites and that in the manipulator readings.

RESULTS

Postsynaptic potentials The intracellular study of postsynaptic potentials was c o n d u c t e d in 479 cells with a resting m e m b r a n e potential d e e p e r than - 5 0 mV. Stimulation of white m a t t e r e v o k e d a depolarizing response followed by a hyperpolarizing response in all cells (85/85) sampled from adult cats (Fig. 1B), and in almost all cells of 9- (75/78) and 18-week-old kittens (87/90) (Fig. 1C). The rising slope of the depolarizing response was slightly increased when m e m b r a n e potential was h y p e r p o l a r i z e d by current injection through the recording electrode. H o w e v e r , the change was so small that the rising phase of the two responses was almost indistinguishable in the s u p e r p o s e d traces (solid and i n t e r r u p t e d lines in Fig. 1 B - C ) . In contrast, the hyperpolarizing response was reversed by m e m b r a n e

hyperpolarization. This voltage d e p e n d e n c e indicates that the response is an EPSP followed by an inhibitory postsynaptic potential (IPSP). The E P S P - I P S P sequence has been r e p o r t e d in the visual cortical cells of adult cats with stimulation of the lateral geniculate nucleus ( L G N ) 52. Similar but slower postsynaptic potentials were observed in about t h r e e - q u a r t e r s of the cells at 3 (48/67) and 5 weeks (63/82) (Fig. 1D) and in less than half of cells (33/77) at 1 week (Fig. 1E). H o w e v e r , in the rest of cells EPSPs were p r o d u c e d in isolation from the IPSPs (Fig. 1F). Responses always started with EPSP in this study, in which cells were all sampled only from the region along the d o t t e d line in Fig. 1A, whose afferents were supposed to be effectively activated by stimulating electrodes placed in white matter. H o w e v e r , cells located far from this region often started with IPSP in response to stimulation of white m a t t e r , although these d a t a are not included in the results. Stimulation of white m a t t e r usually p r o d u c e d in cortical cells (about 80%) complex IPSPs containing early

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50 ms Fig. 2. Complex and simple IPSPs. A: average traces (n = 5) of postsynaptic potentials induced in a layer II-III cell by white matter stimulation in a sample from a 5-week-old kitten while the membrane potential was changed to the value attached to the left of each trace. Resting membrane potential, -56 mV. B: similar to A but for a layer IV cell sampled from another 5-week-old kitten. Resting membrane potential, -61 mV. C: amplitude of IPSP (ordinate) plotted as a function of membrane potential (abscissa). The data were obtained from the cell shown in A. Circles, e-IPSP; squares, I-IPSP. Amplitude of e-IPSP and I-IPSP was measured 15 and 110 ms (indicated by open and filled arrows in A) after white matter stimulation, respectively. D: similar to C but for the cell shown in B. Time calibration is common to A and B.

17 (e-IPSP) and late components (I-IPSP) that showed a difference in time course, reversal potential and sensitivity to ),-aminobutyric acid ( G A B A ) antagonists. Fig. 2 A illustrates the complex IPSP recorded from a layer I I - I I I cell of a 5-week-old kitten. The e-IPSP curtailed the preceding EPSP, and peaked at 15 ms (open arrow) after the stimulation, while the I-IPSP started more slowly than the e-IPSP, and reached a peak at 110 ms (filled arrow). In the experiment where the membrane potential was changed by current injection (Fig. 2A, C), the e- and 1-IPSPs showed different reversal potentials. At the resting m e m b r a n e potential (-56 mV), both components were of the hyperpolarizing form, and they decreased as the m e m b r a n e was hyperpolarized. The e-IPSP was reversed to the depolarizing form between the membrane potentials o f - 6 2 and - 6 9 mV, while the I-IPSP was reversed between -69 and -83 mV. In a small percentage of cells (less than 20%), stimulation of white matter induced a simple IPSP with

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only one component (Fig. 2B, D). This IPSP is apparently identical to the e-IPSP of complex IPSP since the time to peak (20 ms) and the reversal potential (-64 mV) were almost the same as those for the e-IPSP. The difference between e- and I-IPSP was characterized by bath application of bicuculline methiodide and phaclofen, which are specific antagonists for G A B A A 8' 16.38 and G A B A B receptors 17"24"34,respectively. Bicuculline methiodide (1/~M) significantly decreased the amplitude of e-IPSP to about half that of the control, but no significant changes occurred in the I-IPSP, EPSP or input resistance (Fig. 3A, C). In contrast, phaclofen (400 #M) decreased the I-IPSP to less than one fifth of the control without affecting other parameters (Fig. 3B, D). These results indicate that the e-IPSP is mediated by G A B A A receptors, while the I-IPSP is mediated by G A B A a receptors.

Development of excitatory synaptic connection Connectivity with white matter axons. Excitatory syn-

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R I I P S P EPSP Fig. 3. Effect of GABAA and GABA a antagonists on IPSPs. A: average traces (n = 5) of postsynaptic potentials evoked by white matter stimulation in a layer II-III cell sampled from a 5-week-old kitten. Upper trace, control. Middle trace, 15 min after application of 1/zM bicuculline methiodide. Lower trace, superimposed responses of the upper and the middle traces. B: similar to A but for application of phaclofen (400/zM) to another layer II-III cell. Time calibration is common to A and B. C: summary of effect of bicuculline methiodide on 6 layer II-III cells sampled from 5-week-old kittens. Ordinate, amplitude of e- or I-IPSP, slope of EPSPs or input resistance (R) represented by percentage for control. Asterisks indicate that the effect is statistically significant (P < 0.05, paired t-test). D: similar to C but for the effect of phaclofen studied in 5 layer II-III cells sampled from 5-week-old kittens.

18 aptic connectivity of cortical cells with the white matter axons was studied by applying stimulation through a pair of electrodes placed in white matter (Fig. 1A). During all of the developmental stages, central delays were composed of two groups separated at the border value of 1-1.1 ms (dotted lines in Fig. 4A), which may represent the mono- and polysynaptic transmission as reported for EPSPs evoked by L G N stimulation in visual cortical cells of adult cats ~'52. The frequency dependence of EPSPs supported the reliability of the identification of connectivity based on the central delay. The onset of EPSPs with monosynaptic central delays was almost constant (difference < 0.3 ms, n = 9) to stimulation at 0.1-10 Hz, although the EPSP was smaller at high than low frequencies. In contrast, the onset of EPSPs with polysynaptic central delays delayed (0.6-4 ms, n = 8) with the increase of stimulation frequency. The delay in polysynaptic cells at higher frequency may reflect the longer latency of orthodromic action potentials in the cells presynaptic to the recorded cells due to the reduction of EPSP.

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The cells with monosynaptic central delays (hatched columns in Fig. 4A) predominated over those with polysynaptic central delays (blank columns) at all ages. The percentage of polysynaptic cells was shown to change with age. It was smallest (10%) at I week, reached maximum (50%) at 3 weeks, and decreased slightly at 5-18 weeks (29-35%) and further in adults (14%). In addition, laminar differences were found in the postnatal changes in the proportion of polysynaptic cells (Fig. 4B). The polysynaptic cells were rare and confined to layers l V - V I at 1 week. During 1 and 3 weeks, the proportion of polysynaptic cells increased in all layers. Thereafter, it remained unchanged in layers I I - l I I , V and VI until 18 weeks, while in layer IV it decreased monotonically and fell to zero by 18 weeks. No further change was detected in layers I I - I I 1 and IV, but polysynaptic cells were not found in layers V and VI in adult cats. Maximum rate of rise of EPSP. To assess the efficiency of excitatory synaptic transmission the maximum rate of rise (MRR) in EPSPs produced by supramaximal stim-

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Fig. 4. Central delay of EPSP. A: frequency histograms of central delay of EPSP in cells sampled from kittens aged 1, 3, 5, 9 and 18 weeks and adult cats. The interrupted line indicates the upper border of the monosynaptic central delay. Shaded and open columns indicate monoand polysynaptic central delays, respectively. B: laminar distribution of monosynaptic and polysynaptic cells. Shaded and blank columns represent the number of cells responding with monosynaptic EPSP and that with polysynaptic EPSP, respectively.

19 ulation was measured in monosynaptic and polysynaptic cells. At 1 week, the MRRs of monosynaptic EPSPs were commonly small in all layers; about one-third of the adult value (Fig. 5A). They all increased rapidly between 1 and 5 weeks and slowly thereafter up to the adult level. A statistical test showed that the difference of mean value of MRRs between young kittens (1, 3 and 5 weeks) and adults was significant (P < 0.05, t-test) in most layers but insignificant (P > 0.05) between old kittens (9 and 18 weeks) and adults in all layers. The polysynaptic EPSPs also showed an increase in MRRs with the time course similar to that in monosynaptic EPSPs, while some laminar differences were noticed (Fig. 5B). In layer I I - I I I (triangles), the M R R of polysynaptic EPSPs was very small at 3 weeks; one-sixth of the adult value and half of the M R R of monosynaptic EPSPs at 1 week. It increased very steeply during 3-5 weeks and continued to increase, although less steeply, during the rest of stages. The rate of increase in the later stages (9-18 weeks) was steeper in polysynaptic than in monosynaptic EPSPs. Layer IV cells (open circles) showed very similar changes in M R R to layer I I - I I I cells, although polysynaptic cells were absent in stages later than 18 weeks. The development in layers V (squares) and VI (filled circles) seemed to start earlier than those in layers I I - I I I and IV: the MRRs were more than twice larger in layers V and VI than in layers I I - I V at 3 weeks (P < 0.05, t-test). The following development proceeded gradually up to 9 weeks in layer VI and up to 18 weeks in layer V at almost the same rate as in the later stages in layers I I - I I I . Conduction velocity of afferent impulses. Conduction velocity of afferent impulses was determined in 213 monosynaptic cells. Layer IV was studied by separating it into sublayers, IVab and IVc, because it is known that x- and y-geniculate axons terminate preferentially to layer IV~ and IV,b, respectively 12'18'2°'22'29'39. The conduction velocity was very slow at 1 week and increased gradually until adulthood in all layers, in agreement with the anatomical observation that myelination begins at about 1 month after birth and is not completed by 6 months 25"4°. The estimation of conduction velocity seems to be reliable at least in cells sampled from kittens younger than 9 weeks since the conduction velocity was slow enough to be determined by the rather small distance between two stimulating electrodes. In older animals, however, the values may be only rough estimates. Some laminar differences in the development were demonstrated (Fig. 6). In layer IVab cells conduction velocity was extremely slow (0.5 m/s) at 1 week. It progressively increased throughout all stages of development up to the adult level, but the rate of increase was

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smaller in the 1-3 week period than in later weeks. Similar time course of increase occurred in layer IVc, but at each age the value was always smaller in layer IV c than in layer IVab. These differences were all statistically significant (P < 0.05, t-test) except that for the 3 weeks where the sample population was small. A similar progressive increase was found in the conduction velocity in layers V and VI. Delayed development occurred in layer I I - I I I . The conduction velocity remained slow (about 1 m/s) even at 18 weeks when it began to increase and eventually reached a value similar to that in other layers in adult cats.

Development of inhibitory synaptic connection Proportion of cells with IPSP. The presence of inhibitory connection was tested in 479 cells, including 305 and 174 cells where central delays were determined and were not determined, respectively. The IPSPs were small or even reversed in cells with relatively deep resting mem-

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brane potentials. Therefore, if the IPSP was not evident in the resting membrane potential, the presence of IPSP was tested under membrane potentials depolarized up to a b o u t - 5 0 mV. At 1 week, the cells exhibiting IPSPs (hatched and filled columns in Fig. 7) were less numerous than those without IPSPs (blank columns) in all layers except for layer V. The percentage of the former cells was less than 40% in layers I I - I I I , IV and VI, but was 65% in layer V. It increased during the period of 1-5 weeks and reached close to 100%, except for layer I I - I I I where it was still below 70% at 5 weeks. IPSPs were found in almost all cells at 9 and 18 weeks except for a fraction of cells in layer I I - I I I , and in all cells in the adult cats. The complex IPSP was predominant over the simple IPSP in all layers at all ages except for 1 week. At 1 week the number of cells with complex IPSP (filled columns in Fig. 7) was almost the same as those with simple IPSP (shaded columns) in all layers. In kittens older than 3 weeks, however, the percentage of cells with complex IPSP was very high; more than 75% except for the 53% of layer IV at 3 weeks. In adults it decreased slightly (60-80%). Conductance change associated with IPSP. The efficiency of inhibition was assessed by the conductance increase associated with e- and I-IPSP. The IPSP conductance was calculated from the slope of plots of

membrane potential at the peak of e- and I-IPSP vs injected current, minus the resting conductance 13. The conductance changes of IPSP were measured from the response evoked by just subthreshold stimulation to elicit orthodromic action potentials to avoid contamination of afterhyperpolarization. Analysis was done mostly in cells

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21 sampled from layers I I - I I I and IV. Since no substantial difference was demonstrated between the two layers, the results were presented together in Fig. 8. Inhibitions were weaker in newborn than older animals. Although the values varied considerably between cells, the conductance of e-IPSP was significantly smaller for the kittens at the 1-3 weeks than for the old animals (Fig. 8A). Similarly, the conductance of I-IPSP was significantly smaller for 1-3 weeks than for adults (Fig. 8B). The reversal potentials of the e- (mean - 6 4 mV) and I-IPSPs (-76 mV) both remained unchanged during all of the developmental stages (Fig. 8C).

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resistance was maximum (about 60 MS9) at 1 week and decreased to the adult level, about half of the initial value, by 9 weeks (35 MSg) (Fig. 9A). A similar change occurred in the m e m b r a n e time constant: it was maxim u m (about 40 ms) at 1 week and reached the adult level (about 20 ms) by 9 weeks (Fig. 9B). These changes in passive m e m b r a n e properties may explain, at least partly, the slow time course of postsynaptic potentials in young animals. DISCUSSION

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Fig. 8. Conductance increase associated with IPSPs and reversal potentials of IPSPs. A: conductance increase (mean + S.D.) associated with e-IPSP is plotted against age. B: similar to A but for I-IPSP. C: reversal potentials of e-IPSP (open squares) and I-IPSP (filled squares). The number of cells are 6, 14, 10 and 4 for 1-3, 5, 9 and 18 weeks, and 10 for adults. Asterisks indicate that the value is significantly different from the adult value (P < 0.05, t-test).

Postsynaptic potentials The present study demonstrated that white matter stimulation produced postsynaptic potentials in visual cortex cells of slice preparations, which were similar to those evoked in adult visual cortical cells by L G N stimulation in vivo 52. White matter stimulation evoked an EPSP followed by an IPSP in most cells in old kittens and

22 adult cats. In newborn kittens half of the cells showed a similar EPSP-IPSP sequence of responses, although the rest of the cells exhibited EPSPs but lacked IPSPs. Most IPSPs were composed of both early and late two components (e- and I-IPSP). The e- and I-IPSP were mediated by G A B A A and G A B A B receptors since they were selectively reduced by a G A B A A receptor antagonist, bicuculline 8'16'38, and by a G A B A B antagonist, phaclofen 17'24'34, respectively. The observation that reversal potential was deeper for the 1-IPSP than for the e-IPSP suggests that the I-IPSP is mediated through K channels while the e-IPSP is mediated through CI channels as indicated in e- and I-IPSPs of pyramidal cells in hippocampus, piriform cortex and neocortex, and LGN cells 2"13"15"21"23"46'53. The ubiquitous laminar distribution of cells with both e- and I-IPSP coincided with the fact that G A B A A and G A B A B receptors both distribute rather evenly throughout all layers of cerebral cortex 8.

aptic), especially in layer 11-III. This may be related to the late maturation of visual responsiveness ~'55 and long critical period of plastic changes in the visual responsiveness of layer I I - I I I cells 4"ca. These postnatal changes in excitatory synaptic transmission may well be explained by the change in the number of synapses. The low efficiency in newborn kittens may be ascribable to the small number of synapses possessed by each cortical cell 14"41"57. The M R R increased the most during the first 2 months, which is the period of new synapse formation in cat visual cortex TM 41.57. However, the later slow increase of EPSP efficiency does not agree with the decrease in the number of synapses in each cell in the later stage of development al" 57 This discrepancy could be explained if individual synapses remaining in later stages are strengthened while the number of synapses decrease, so that the total efficiency still slowly increases in the later stage of development.

Development of excitatory synaptic connection All cortical cells received excitatory synaptic inputs even in newborn kittens, which agrees with the morphological results showing that LGN axons terminate in visual cortex already at birth 26"33. However, there was a difference in the connectivity between 1-week-old kittens and older animals. At 1 week, most cells responded with monosynaptic EPSPs to white matter stimulation, while in older kittens about one-quarter of the cells lacked monosynaptic EPSPs and received polysynaptic excitation. The increase in proportion of polysynaptic cells occurred mostly in layer I I - I I I . This change may reflect reorganization of the termination area of LGN axons in visual cortex. Morphological and electrophysiological examinations have demonstrated that LGN axons terminate not only in layers IV and VI, which are the main termination area in adults, but also in layers I - I I I in newborn kittens 33'37. In adult cats y-geniculate axons terminate preferentially in layer IVab and x-geniculate axons terminate in layer IV c or in both layers IVab a n d IWc11'18'2°'22'29. The present analysis of the conduction velocity of afferent impulses indicates that differential termination of x- and y-geniculate axons is already established in an early postnatal stage, since the conduction velocity of afferents was faster in layer IV~b cells than in layer IVc cells at all age groups. Although all cortical cells had already established excitatory synaptic connections with their afferents at birth, the efficiency of synaptic transmission assessed by M R R of EPSP was extremely lower in newborn kittens than in mature animals. The development of synapses between cortical afferents and cortical cells (monosynaptic) preceded that of intracortical synapses (polysyn-

Development of inhibitory synaptic connection IPSP was induced in only a part of the cells in newborn kittens by white matter stimulation, whereas EPSPs were induced in all cells. In addition, the effectiveness of IPSPs assessed by IPSP conductance was less in newborn animals than in older kittens. This late development of inhibition in comparison with excitation seems to account for the fact that visual cortical cells could respond to visual stimulation but lacked selective responsiveness in newborn animals 5'6"1°'19, since the selective responsiveness of adult cortical cells to orientation and direction of visual stimulation is lost with the elimination of inhibition due to the application of G A B A blockers 5~'54. This inference is supported by the fact that the most steep increase in the proportion of cells with IPSPs occurred b.etween 1 and 5 weeks, which corresponds to the period showing a steep increase in the proportion of cells revealing orientation selectivity 1'5'6'1°'19"55. Furthermore, the laminar difference in development of inhibition was almost the same as that of selective responsiveness 1 55. Therefore, it is likely that postnatal development of selective responsiveness in visual cortical cells is mainly based on the maturation of cortical inhibition. It is well established that the inhibition of cortical cells by G A B A A receptors contributes to construction of selective visual responsiveness 51"54. The observation that G A B A B receptor agonist baclofen also inhibits the spontaneous and visually evoked activity of visual cortical cells 3, may suggest that G A B A B receptors also contribute to selective responsiveness. This view is supported by the observation that GABAa-mediated I-IPSP also developed in parallel with the selective responsiveness of visual cortical cells.

23

Acknowledgements. This work was supported by Grants-in-Aid for Scientific Research Projects 01570071 and 02679956 from the

Japanese Ministry of Education, Science and Culture.

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