The neural circuitry of the neocortex examined in the in vitro brain slice preparation

Brain Research, 243 (1982) 35-47 Elsevier Biomedical Press

35

The Neural Circuitry of the Neocortex Examined in the In Vitro Brain Slice Preparation CHRISTOPHER SHAW and TIMOTHY J. TEYLER* Department of Psychology, Dalhousie University, Halifax, Nova Scotia B3tt 4J1 (Canada) and (T. J. T.) Neurobiology Program, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272 (U.S.A.) (Accepted December 12th, 1981) Key words: neocortex - - cortex - - brain slice - - cortical slice - - neural circuitry - - evoked potential - - population response

The in vitro brain slice technique has been applied to the study of the neocortex. Cortical blocks were removed from adult rats deeply anesthesized with halothane, sectioned coronally at 400-700/~m, and placed in a brain slice chamber. Cortical slices typically showed spontaneous and evoked potential activity and normal histology for 8 h or longer. Single units and evoked potential recordings were made from different layers of the cortex using micropipettes. The evoked potentials to electrical stimulation of differing intensity, frequency, and from different cortical layers were analyzed. Evoked potential from all but the most superficial layers of the cortex showed a characteristic 6-component response to stimulation of nearby white matter. This evoked potential closely resembled cortical responses recorded in vivo by other investigators following afferent stimulation. The response amplitude of all components increased as stimulus intensity was raised. Radial movement of the recording electrode showed that components 1-3 had their largest amplitudes in the deepest cortical layers, component 4 reached its greatest amplitude and shortest latency in layer IV, and components 5 and 6 reached their greatest amplitudes in layers IV to II. The frequency following for various components was measured showing greater decline in amplitude for components 4-6 than 1-3. This, together with the results of previous investigators, suggests that the first 3 components represent afferent fiber input, while component 4 represents the first cortical response (layer IV). Components 5 and 6 represent later, additional cortical responses. Further support for the intracortical origin of component 4 was provided by lateral intracortical stimulation within layer IV, giving an evoked potential composed mostly of component 4. With lateral movement of the recording electrode in layer IV the evoked potential disappeared in under 1 mm, suggesting a fairly restricted afferent input to the cortex. The present results encourage the use of the cortical brain slice preparation as an appropriate model system in which to study cortical neural circuitry. INTRODUCTION I n recent years, the use o f the b r a i n slice p r e p a r a tion for in vitro studies o f n e r v o u s tissue has g r o w n e n o r m o u s l y . The a d v a n t a g e s o f this p r e p a r a t i o n are manifold20, 2a,29, a n d include direct o b s e r v a t i o n o f the tissue u n d e r study for precise spatial c o n t r o l o f s t i m u l a t i n g a n d r e c o r d i n g sites, a n d full c o n t r o l over the c h e m i c a l c o m p o s i t i o n , t e m p e r a t u r e a n d p H o f the external media. W i t h certain l i m i t a t i o n s (such as the relatively s h o r t life-time o f the slice), these a d v a n t a g e s m a k e the b r a i n slice p r e p a r a t i o n ideal f o r the d e t a i l e d s t u d y o f n e u r a l circuitry a n d p h a r m a c o l o g y o f different regions o f the n e r v o u s system. W e have a p p l i e d this technique to the n e o c o r t e x in an a t t e m p t to w o r k o u t some o f the basic circuitry * To whom correspondence should be addressed. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press

o f this structure. Previous studies b y o t h e r investigators show b i o c h e m i c a l a n d electrical activity in the cortical b r a i n slice f r o m v a r i o u s species 11,1z, 17,22,85,31. A d d i t i o n a l l y , the developing electrical activity a n d structure o f the cortex have been described for n e o n a t a l tissue culture p r e p a r a tions TM. However, we believe t h a t o u r results are the first detailed e v o k e d p o t e n t i a l study o f the n e o c o r t i c a l b r a i n slice p r e p a r a t i o n , a n d t h a t this p r e p a r a t i o n will be useful in e x p l o r i n g the intrinsic circuitry o f the neocortex. MATERIALS AND METHODS T h e basic techniques o f the b r a i n slice p r e p a r a tions have been described in detail elsewhere1,2,27, 2a.

36 Adult Long-Evans rats of both sexes (200-400 g) were used. Slices were taken from cortical areas between bregma and lambda, including visual, auditory and somatosensoty cortices as defined by Krieg 15. The electrical properties of the slices, at the present level of analysis, appeared to be quite similar regardless of the region of cortex ~rom which they were taken and will not be further distinguished by area. While variations in cell number and density do occur between neocortical areas 16, the functional structure of the neocortex generally appears to be very similar s. Additionally, in vivo studies in cat show essentially similar evoked potentials in different primary sensory cortices following stimulation of specific relay nuclei in the thalamus TM. The viability of cortical slices was found to be more dependent on dissection time than hippocampal slices, due perhaps to greater cortical susceptibility to anoxia, and several additional procedures were required to ensure optimally viable slices. Healthy slices could be obtained if the animal was first deeply anesthesized with halothane, the effects of which are rapidly reversible. Even healthier slices were obtained by immersing the animals in icewater, lowering the body temperature to 14-17 °C prior to or in place of halothane anesthesia. These procedures slow cortical metabolism, decreasing oxygen requirements during the actual slicing. Following anesthesia, a partial craniotomy was performed using standard surgical techniques. The dura was reflected and a block of cortex with underlying white matter was removed using a scalpel and curved spatula. The block was transferred to cold (0-4 °C), modified Earle solution 20 to remove surface blood, and then oriented for coronal sections on a Stoelting tissue slicer10. The block was sectioned at 400-700/~m and the slices rapidly transferred to the brain slice chamber. Slices rested on nylon nets, partially immersed in oxygenated Earle solution at 33-34 °C. The slices were kept moist and oxygenated by 95 ~ 02-5 ~ CO2 flowing through the chamber reservoir and over the surface of the slices. We could usually record spontaneous unit discharges and evoked population responses with stable waveforms for up to 8 h. The animal was usually maintained on deep halothane anesthesia for the duration of the experiment for possible removal of additional cortical blocks.

The cortical slices were allowed to reach to equilibrium in the chamber for at least one hour prior to recording. Single and multiple unit and field potential recordings were made using glass micropipettes filled with 1 M NaC1. Single and multiple unit electrode tip diameter measured 3-6/~m (7-10 Mf~ at 60 Hz) whereas field potential electrode tip diameter measured 15-20/zm (2-5 Mf~ at 60 Hz). Field potential electrodes so constructed are capable of recording field potentials generally uncontaminated with unitary discharges. Neural activity was conventially filtered, amplified and displayed. In all evoked potential records positivity is up. The reference electrode was located in the bottom of the pool. Signal averaging was done on a Dagan and permanent records made on an x-y plotter. Most evoked potential records represent two averaged responses. Electrical stimulation was delivered via 300 #m (o.d.) concentic bipolar electrodes 1. Stimuli were bipolar pulses of 0.1 ms duration, 1-30 V. Distances between stimulating and recording electrodes were measured optically with a graticule. Following recording, slices were fixed in 1 0 ~ formaldehyde, sectioned at 40 #m, and stained with cresyl violet for identification of stimulating and recording sites. RESULTS The appearance of a typical cortical slice is shown in Fig. 1. The coronally cut slice is crescent-shaped, measuring 2 × 4 mm, consisting of cortical gray matter and underlying white matter. Sections obtained from a similar slice 3 h post-operatively appear to

Fig. 1. The cortical brain slice in vitro. A coronal slice of rat cortex and white matter on a nylon net in a brain slice chamber.

37 INTACT

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Fig. 2. Histology of the cortical slice. A cortical slice, similar to the one illustrated in Fig. 1, was used for unit recording and then fixed 3 h post-operatively. Forty/~m thick sections were stained with creysl violet. The sections from the slice are compared to sections from an intact preparation (left) at different primary objective magnifications. Other than the presence of more red blood cells in the slice, the histologies as determined with the light microscope are essentially similar.

38

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The evoked response to afferent stimulation A characteristic 6-component population response, or evoked potential, was elicited in all but the most superficial layer of the cortex by electrical stimulation of the white matter. The evoked potential closely resembled potentials recorded in in vivo cat primary sensory cortex following electrical stimulation o f afferent pathways6,18, zl. The response consists of 3 early components (1-positive, 2-negative, 3positive) followed by two later, more prominent components (4-positive, 5-negative) and a final, variable positive component (6). In this paper we have followed the nomenclature of earlier workers 21. A representative layer IV evoked potential from the

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10V be normal at the light microscope level (Fig. 2). Spontaneous activity and injury discharges were frequently encountered when a microelectrode was inserted into a cortical slice. Fig. 3A shows an example of spontaneous extracellular unitary discharges. Single unit extracellular discharges could usually be reliably evoked by electrical stimulation of the white matter. In Fig. 3B stimulation of the subjacent white matter elicited unitary discharges recorded extracellularly from layer IV. The stimulus artifact is shown below a dot, followed by a short latency unitary response, which may represent antidromic invasion of layer IV neurons or fibers, and several later latency unitary discharges. These responses are of comparable latency to the population measures which form the bulk of this study. We did not see any rhythmic activity, analogous to the ECoG, in these cortical slices, perhaps because the volume of the tissue was insufficient to support endogenous rhythmic activity.

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Fig. 4. The evoked response to afferent stimulation. White matter stimulation elicits a characteristic evoked potential in the layer IV of the cortical slice. The 6 major components are labelled. Increasing stimulus intensity (V) increases the amplitude of all components. In this and all following figures positivity is up. Calibration: 5 ms, 0.5 mV.

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within layer IV. The results are shown in Fig. 7. All components of the response fell off rapidly with increasing distance from the stimulating electrodes, almost completely disappearing at a distance of about 1 ram. A similar experiment was performed to examine the 'vertical' components of the evoked response. The stimulating electrode was placed in the white matter while the recording electrode was moved radially from the deepest to the more superficial layers of the cortex. Fig. 8 illustrates this experiment. Components 1-3 reached their greatest amplitudes at recording site 1 (deepest layer). Component

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cortical brain slice to different stimulus intensities is shown in Fig. 4. All components of the responses increased with increasing stimulus intensity. Stimulus-response or input-output curves for the prominent negative component (5) of 3 cortical slices are shown in Fig. 5. The input-output functions seen here are similar to those seen in other tissues studied similarlyL To assess the viability of the cortical slice, a recording electrode was advanced in 50/~m steps through the slice within layer IV. Since the cortical slice was lying on its side the penetration was thus, parallel to the cortical surface. Identical stimulation was applied to the white matter. As can be seen from Fig. 6, the evoked response was present throughout the 400 /zm thick slice, but was largest in the center of the tissue. There thus appears to be an extensive region of healthy tissue in cortical slices. In order to examine the lateral spread of afferent input into cortical layer IV a stimulating electrode was placed in the white matter and a recording electrode placed in layer IV radially to it. The response to a fixed intensity electrical stimulus was recorded at this original site and laterally as the recording electrode was moved away in 200 # m steps

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Fig. 6. Depth profile of the cortical slice. The evoked potential to afferent stimulation in layer IV was recorded in 50/~m steps from the slice surface to bottom. Stimulus intensity: 6 V. Calibration: 5 ms, 1 mV.

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5 Fig. 7. The lateral spread of afferent input. The stimulating electrode was placed in the white matter and the evoked potential recorded in layer IV radially to it. Lateral movements in layer IV away from the original recording site gave smaller evoked potentials, which finally disappeared at about 1 mm. The vertical lines intersect components 2 and 5. Calibration: 5 ms, 1 mV. 4 reached its greatest amplitude at site 2 (layer IV). C o m p o n e n t s 5 and 6 reached their greatest amplitude at site 4. At a constant post-stimulus latency, portions o f the response appear to display an inversion between surface and depth. At site 4, the positive wave (component 4) is temporally contiguous

with the negative wave (component 5) recorded from site 1. Similarly, the negative wave (component 5) recorded at sites 3 and 4 is temporally contiguous with a positive wave recorded in position 1. These correspondences m a y reflect current dipoles or m a y indicate the existence o f activity propagated vertical-

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Fig. 8. Evoked potentials recorded in different cortical layers to radial afferent stimulation. Evoked potentials were recorded in layers I-VI showing changes in component shape, amplitude and latency. The responses at two sites lateral to site 4 were also measured. Calibration: 5 ms, 1 inV.

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ly through the cortex. The various components exhibited marked peak latency changes throughout the cortical slice. The early components, 1-3, showed latency changes as a function of radial distance from the stimulating electrode (Fig. 9). Component 1, for example, had a latency-to-peak of 0.74 ms at position 1 and a latency-to-peak of 1.37 ms at position 4. If components 1-3 represent fiber volley activity from a population of afferent and efferent fibers displaying a range of conduction velocities, then component 3 might expected to fractionate into sub-components as a function of distance. Inspection of the records at positions 4, 5 and 7 suggest that several subcomponents are emerging at this more remote recording location. Interestingly, the shortest latency subcomponent of waveform 3 is better represented in position 4, than in either positions 6 or 7, which are 200 # m lateral to position 4. This suggests that the afferent input to any point in the cortex is relatively narrow. Components 1 and 2 propagate radially through the tissue at about 1.0 m/s.

The longer latency components, 4-6, displayed a larger range of latency change as a function of cortical location. In comparing positions 1 and 4, component 5 changed from 4.20 ms to 6.51 ms and component 6 changes from 6.51 to 8.40 ms. Position 6, lateral to the radial penetrations, still displayed the large amplitude components 5 and 6, indicating that this activity is projected more laterally than the radial afferent projection. Components 3 and 4 were separable only at positions 4, 6 and 7, when sufficient distance allowed conduction velocity differences to become apparent. Components 4-6 all propagated through the tissue at the same relative rate of 0.4 m/s. At the most superficial site (5) most of the components have disappeared or been masked by a large positive-going wave. Lateral movements of the recording electrode from site 4 to sites 6 and 7 show a decrease in response amplitudes of all components as in the experiment illustrated in Fig. 7. Lateral sites generally display a longer latency to component peaks as compared to position 4. Further examination of the component latency differences at the various recording locations was accomplished through a regression analysis. Assuming a constant conduction velocity and a linear axon trajectory, the y-intercept represents the stimulation site. Given these assumptions, any unaccounted for time could represent the interposition of one or more synaptic delays. Table I presents the results of the least-squares linear regression analysis and the goodness of fit of that regression line to the data by the computed correlation coefficient. Correlation coefficients for all components indicate a good fit except for component 3. The low coefficient of correlation for this component is probably due to the difficulty of separating it from component 4 at short conduction distances. Discounting component 3, three groupings can be noted from Table I. The first group consists of components 1 and 2.

TABLE I Component no

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5 Fig. 10. The evoked response to afferent stimuli o f increasing frequency. Top left waveform is to same relative scale as other figures; top right is compressed on the time axis by a factor o f 5. All responses below are compressed. Waveforms on the left are at train onset, those at right are after 1 s o f continuous stimulation. F r o m top to bottom, frequencies are 1/s, I 0/s, 25/s, 50/s. Calibration: 5 ms, 1 inV.

44 The distance intercept of component 1 exactly coincides with the stimulating electrode location. This, along with the good linear regression fit, indicates a uniformly conducting (1.09 m/s) component originating at the stimulating electrode. Thus, component 1 probably represents the fastest conducting afferents activated by the stimulating electrode. Component 2 (component 3, if reliable, is also in this group) displays a slower conduction velocity and a different origin (intercept). These values may represent the slower electrotonic properties of smaller diameter afferents which may be either collaterals of the first component or separate axons. The second group consists of components 4, 5 and 6. All have negative origins indicating the imposition of one or more synaptic delays, thus supporting the evidence that these are polysynaptic responses. Their conduction velocity through the tissue is about 0.40 m/s. The frequency following capability of the different components was tested in order to distinguish between them. Fig. 10 shows the responses of the different components as the frequency increases from 1 to 50 Hz. Components 5 and 6 decline with increasing frequency more rapidly than component 2. In Fig. 11, the frequency following responses are plotted to graphically illustrate the difference between components 2 and 5. The evoked response to intracortical stimulation

The final series of experiments attempted to examine intracortical connections using intracortical stimulation and recording. In Fig. 12 an experiment of this kind is illustrated. Control stimulation of the white matter elicited the characteristic 6-component evoked potential from layer IV. Lateral stimulation within layer IV, however, produced a completely different evoked potential with a much broader component 4 and greatly reduced components 3 and 5. The use of waveform component designations 1-6, defined in terms of radial stimulation and recording, is, however, inappropriate for the kinds of lateral interactions seen here. Lateral stimulation, particularly within layer IV, may give rise to lateral inhibitory influences, which may be represented in the responses obtained. Stimulation of the superficial layers, either laterally or radially to the recording site, also markedly altered the waveform. These sites gave rise to short latency waveforms that

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resembled afferent volleys, followed by marked positive waveforms that may correspond to component 4. DISCUSSION

In summary, we note 3 general findings from this experiment. One, neural activity is reliably elicited and is propagated through the cortical tissue. Two, the evoked waveform was similar to that seen in other mammalian cortices, and in particular, rat cortex3, 30. Third, the lateral spread of neural activity was more limited than the radial spread of activation, a finding consistent with the columnar hypothesis. While these results were obtained from the rat, we have obtained essentially similar results from slices of cat neocortex. The multi-component evoked potential that we see in the deeper cortical layers following white matter stimulation is quite similar to surface cortical potentialsirecorded in vivo in primary sensory cortices of the cat to stimulation of afferent pathways or specific thalamic relay nucleP, 18ml,2~. This suggests that the in vitro cortical brain slice retains the basic, normal electrophysiology of the cortex. The presence of spontaneous and evoked single unit activity, in addition to the apparently normal histology, ~"further support this view. While it is undeniable that the cortical brain slice can never be completely

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5 Fig. 12. The evoked response to intracortical stimulation. The recording electrode was placed in layer IV. The stimulating electrode was in the white matter (right middle) or within the cortex. Calibration: 5 ms, 1 mV. 'normal', the present results suggest that the use of the cortical slice is justified in a model system's approach 2s to the problems of cortical circuitry and pharmacology. A few tentative conclusions concerning cortical circuitry can be reached on the basis of the present results. Evoked potential components 1-3 are largest at the site closest to the stimulating electrode (Fig. 8). This might be expected from previous in vivo studies which presented evidence in support of

the view that these components represent the afferent input to the cortex, perhaps composed of fibers of different conduction velocities6, 21. Further support for identifying components 1-3 with the afferent input fiber waves comes from the frequency following experiments (Figs. 10, 11) showing that component 3 follows to a higher frequency than components 4-6. In addition, components 2 and 3 have a faster conduction velocity than the later components.

46 Component 4 reaches its maximum amplitude and shortest latency in layer IV (Fig. 8). This, together with the in vivo evoked potentials studiesZ,2a,2a, suggests that this component represents the first wave of cortical origin following afferent input to this layer lz. Further support for this conclusion is provided by the lateral intracortical stimulation experiments illustrated in Fig. 12. Lateral stimulation within this layer produces an evoked potential composed almost entirely of what appears to be component 4. This component itself may be a complex response of many cell types. Anatomical studies in cat visual cortex have shown at least 7 cell classes, 6 of these receiving afferents from the lateral geniculate nucleus 9. An evoked response much like this has been shown following intracortical lateral stimulation in cat motor cortex z although the layers were not identified 4. The afferent input to layer IV appears to be fairly restricted laterally (Fig. 7) with component 4 disappearing in 1 mm or less from the stimulation site in the white matter. This result is consistent with the columnar organization hypothesis of neocortex. Component 5, a large negative wave, appears to reach its maximum amplitude in more superficial layers of the cortex (layers IV-II). Component 5, together with component 4, may represent the primary post-synaptic response of intracortical elements to afferent stimulation. The lateral distribution of component 5 is somewhat wider than components 1-3, suggesting a horizontal projection of information within cortical layers that is roughly orthogonal to afferent input. Component 6, is considerably more variable in amplitude than the other components and may represent additional, polysynaptic activity of a complex nature elicited by the afferent volley. The nature or origin of the positive-going potentials elicited in the most superficial layer recordings are unclear, although they may represent a longer latenREFERENCES 1 Alger, B. E. and Teyler, T. J., A monosynaptic fiber track studied in vitro: evidence of a hippocampal CA1 associational system, Brain Res. Bull., 2 (1977) 355-365. 2 Alger, B. E. and Teyler, T. J., Long-term and short-term plasticity in the CAI, CA3 and dentate regions of the rat hippocampal slice, Brain Research, 110 (1976) 463-480. 3 Allison, T. and Hume, A. L., A comparative analysis of short-latency somatosensory evoked potentials in man,

cy component 4, or the polarization of apical dendrites of cells located in the deeper cortical layers 24. The analysis of the component peak latency changes as a function of recording position appear to indicate that the early components, 1-3, propagate through the tissue at about 1.0 m/s in a relatively narrow band. Components 4-6 propagate more widely through the tissue at about 0.4 m/s. The dynamic nature of the component latency changes apparent from cortical depth to surface make difficult a source-sink analysis of this activity utilizing these recording techniques. We believe these data may support the idea that a wave of neural activity is sweeping up through the tissue rather than only reflecting differences in static source-sink relationships 14. Current-source-density experiments are underway to clarify this question. The large evoked potential resembling component 4, recorded in layer IV to stimulation of the superficial layers (Fig. 12) is not understood. One possible explanation for this is the known afferent connection from pyramidal cells in layers II and 1II onto cells in layer V 5,x9. Experiments in progress should enable a better understanding of this question and the complex intracortical circuitry. We suggest that the unique features of the brain slice preparation can be utilized to advantage in probing the circuitry of the neocortex. ACKNOWLEDGEMENTS This work was partially supported by grants from the Killam Foundation and the National Institutes of Health (NRSA) IF 32 EY 05393-01 (C.S.), and research grants from the National Science Foundation and National Institute of Health (T. J. T.). We thank N. Chiaia and A. Kulics for comments and Ms. Carol McAulay and the Word Processing Center for typing this manuscript. monkey, cat and rat,. Exp. NeuroL, 72 (1981) 592-611. 4 Asanuma, H. and Ros6n, I., Spread of mono- and polysynaptic connections within cat's motor cortex, Exp. Brain Res., 16 (1973) 507-520. 5 Butler, A. B. and Jane, J. A., Interlaminar connections of rat visual cortex: an ultrastructural study, J. comp. Neurol., 174 (1977) 521-534. 6 Chang, H.-T. and Kaada, B. An analysis of primary response of visual cortex to optic nerve stimulation in cats, J. NeurophysioL, 13 (1950) 305-318.

47 7 Crain, S. M. and Bornstein, M. B., Bioelectric activity of neonatal mouse cerebral cortex during growth and differentiation in tissue culture, Exp. Neurol., 10 (1964) 425-450. 8 Creutzfeldt, O. D., Generality of the functional structure of the neocortex, Naturwissenschaft, 64 (1577) 507-517. 9 Davis, T. L. and Sterling, P., Microcircuitry of cat visual cortex: classification of neurons in layer IV of area 17, and identification of the patterns of lateral geniculate input, J. comp. Neurol., 188 (1979) 599-628. 10 Duffy, C. J. and Teyler, T. J., A simple tissue slicer, Physiol. Behav., 14 (1975) 525-526. 11 Franck, G., Brain slices. In G. H. Bourne (Ed.), The Structure and Function of Nervous Tissue, Vol. IV, 1972, pp. 417-466. 12 Garey, L. J. and Powell, T. P. S. An experimental study of the termination of the lateral geniculocortical pathway in the cat and monkey, Proc. roy. Soc. B, 179 (1971) 41-63. 13 Gutnick, M. J. and Prince, D. A., Dye coupling and possible electrotonic coupling in the Guinea Pig neocortical slice, Science, 211 (1981) 67-70. 14 Kennedy, C., des Rosiers, M. H., Sakurada, O., Shinohara, M., Reivich, M., Jehle, J. W. and Sokoloff, L., Metabolic mapping of the primary visual system of the monkey by means of the autographic (14C)deoxyglucose techniques, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 4230-4234. 15 Kreig, W. J. S., Connections of the cerebral cortex: I. The albino rat. A. Topography of the cortical areas, J. comp. Neurol., 84 (1946) 221-275. 16 Kreig, W. J. S. Connections of the cerebral cortex: I. The albino rat. B. Structure of the cortical areas, J. comp. Neurol., 84 (1946) 277-323. 17 Kuhnt, U. and Schaumberg, R., Spontaneous and evoked activity in slices maintained in vitro from the lateral geniculate body and the cerebral cortex of the guinea pig. In O. Creutzfeldt (Ed.), Afferent and Intrinsic Organization of Laminated Structures in the Brain, Europ. Brain Res., Suppl. 1, 1976, pp. 394-396. 18 Landau, W. M. and Clare, M. H., A note on the characteristic response pattern in primary sensory projection cortex of the cat following a synchronous afferent volley, Electroenceph. clin. NeurophysioL, 8 (1956) 457-464.

19 Lund, J. S. and Booth, R. G., Interlaminar connections and pyramidal neuron organization in the visual cortex, area 17, of the macaque monkey, J. comp. Neurol., 159 (1975) 305-334. 20 Lynch, G. and Schubert, P., The use of in vitro brain slices for multidisciplinary studies of synaptic function, Ann. Rev. Neurosci., 3 (1980) 1-22. 21 Malis, L. I. and Kruger, L., Multiple response and excitability of cat's visual cortex, J. NeurophysioL, 19 (1956) 172-186. 22 Mansfield, R. J. W. and Simmons, L. K., Intrinsic processing in the visual cortex of primates, Neurosci. Abstr., 5 (1979) 795. 23 Mitzdorf, U. and Singer, W., Prominent excitatory pathways in the cat visual cortex (A17 and A18): a current source density analysis of electrically evoked potentials, Exp. Brain Res., 33 (1978) 371-394. 24 Pollen, D. A., On the generation of neocortical potentials. In H. H. Jasper, A. A. Ward and A. Pope (Eds.), Basic Mechanisms of the Epilepsies, Little and Brown, Boston, 1969, pp. 411-420. 25 Richards, C. D. and Mcllwain, I-I., Electrical responses in brain samples, Nature (Lond.), 215 (1967) 704-707. 26 Seil, F. J. and Leiman, A. L., Development of neocortex in tissue culture. In O. Creutzfeldt (Ed.), Afferent and Intrinsic Organization of Laminated Structures in the Brain, Europ. Brain Res., Suppl. 1, 1976, pp. 249-254. 27 Teyler, T. J. and Alger, B. E., Monosynaptic habituation in the vertebrate forebrain: the dentate gyrus examined in vitro, Brain Research, 115 (1976) 413-425. 28 Teyler, T. J., Plasticity in the hippocampus: a model systems approach. In A. H. Riesen and R. F. Thompson (Eds.), Advances in Psychobiology, VoL llI, Wiley, New York, pp. 301-326. 29 Teyler, T. J., The brain slice preparation: hippocampus, Brain Res. Bull., 5 (1980) 391-403. 30 Wiederholt, W. C. and Iragui-Madoz, V. J., Far-field somatosensory potentials in the rat, Electroenceph. clin. Neurophysiol., 42 (1977) 456-465. 31 Yamamoto, C. and Kawai, N., Origin of the direct cortical response as studied in vitro in thin cortical sections, Experientia, 23 (1967) 821-822.