Documenta Ophthahnologica 43,1 : 91-99, 1977 SOME INNOVATIONS IN MICROPHYSIOLOGICAL TECHNIQUES - THEIR APPLICATION TO THE STUDY OF THE VISUAL SYSTEM NETWORK B. BLUM

(Tel Aviv, lsrael) The visual system operates on the basis of multiple modes of spatio-temporally encoded signals. Therefore, studies of its network properties must be concerned with a variety of transfer functions in the time and space domaines (Ratliff et al., 1970; Grusser, 1972). Some of the encoding of sensory information, including visual, is believed to be in the form of a frequency code. Rate of spiking or of bursts of spikes and the frequency of spikes in a burst as weU as the burst duration were shown to be contributory to the information transfer capacity of a nerve channel (Hensel et al., 1970; Grusser, 1972). It is rather significant to this hypothesis that modifiability of such spike patterns by specific pre-conditioning stimuli has been shown (Grusser, 1972; Blum et al., 1972d). It has also been suggested that the spike frequency code may serve as a signal for the short-term, whereas the patterned bursts serve as signal for the long-term (Blum et al., 1975). While the encoding of signals may be expressed as specific time relationships among spikes in a single nerve channel (Blum et aL, 1975; Perkel et al., 1967), such relationships among channels are perhaps of a still greater significance (Jassik-Gerschenfeld, 1966; Perkel et al., 1967; Blum, 1972). This is because of the greater complexity of most C.N.S. encoding which is often at the same time both temporal and spatial, and because it is largely based on conditional probabilities of correlated discharges arriving through different channels. Axonal branchings which are so common in the C.N.S. serve perhaps as delay lines in generating the above-mentioned time relationships (Blum, 1971). In addition, an innate membrane mechanism (Fuortes, 1971) must also play an important role in such a function. Furthermore, in the longterm encoding, delayed inhibitions and facilitations have been shown to play crucial roles in the signal patterning processes (Rathff et al., 1970; Blum, 1975). Conceivably, b o t h network and membrane-related mechanisms act in coordination, so that different spike trigger zones, each with specific characteristics and each receiving specific inputs, interactively generate the

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encoded signal engram. It is at these post-synaptic zones that perhaps the effects of pre-conditioning stimuli on the encoding process take place. Studies were carried out on the possibility that axonal branching provides the structural basis for the generation of cross-correlated signals (Perkel et al., 1967). These have actually shown that, possibly due to the fall in the factor of safety at the branching point or to some other control mechanism, there is an intermittency in impulse propagation through different axonal branches (Blum, Project report to the Ford Foundation, 1969). This phenomenon apparently allows for rates of impulse propagation which vary with the mode of discharge of the parent neurone (Blum, 1972a, b). It has been shown on a visually-related pathway, i.e., one leading from S.C. to DLGN, that rates of impulse propagation in that pathway are different when the cell spontaneously discharges as compared to its discharge by light flashes (Blum et al., 1972b). Attention was thus drawn to the possibility of utilizing such an approach to study whether and how impulses relayed in this manner through axonal branchings may be patterned into numerous inter-dependent, cross-correlated stochastic signals on which the activation of a way-station or of an end-target will depend. Spatial factors in such processing of visual information have been referred to in the 'columnar hypothesis' (Mountcastle, 1957a; Hubel & Weisel, 1962), in the 'domaine concept' of Shell (1956), and in the hypothesis on layered structure of nuclei in the C.N.S. Nonetheless, it is accepted that this factor has not been given sufficient attention. Based on the above-mentioned considerations, techniques for the study of the visual system network, and specifically of the role of the S.C., were designed according to the following strategies: MULTIPLEUNIT TECHNIQUE This approach, despite innate difficulties, is becoming increasingly utilized. Our method (Blum et al., 1965), permitted the simultaneous study of at least four single units 200mu to 7ram apart, thereby enabling concurrent study on single units and on their surround. ! Some of the logic repertoire of the C.N.S. was investigated including and-gate and or-gate logic (Blum, 1972c). The possibility to study both temporal and spatial inter-relationships is, however, the main advantage of this approach, THE ANTIDROMICMETHODFOR IDENTIFICATION OF DIRECT CONNECTIONS While anatomic methods are available for the determination of nerve projec92

tions, this approach allows study only of pathways in isolation. This is the case even with the recent advances whereby a neurone with its axonal branching can be visualized (LaVail et al., 1972; Nauta et al., 1974; Maciewicz, 1974). With microphysiological techniques the living animal as well as nerve pathways within the context of a neuronal network can be studied. Improvements in the antidromic method will be described below which permit the characterization of dynamic properties of pathways, including the precise determination of the way these serve as conduction pathways (Blum, 1972a, b). In Fig~ 1 the antidromic method is explained. Unit response are recorded by a micro-electrode and stimulation is given to different sites, obtaining two types of responses - presumably one antidromic and the other orthodromic. Differentiation between the two is made on the basis of the following criteria (Shimazu et al., 1965): 1) spike shape (see Fig. 2) is useful in such a differentiation, both in extracellular as in intracellular studies provided that correct recording conditions are used; 2) constancy of response and of latency cannot be regarded as reliable, by themselves, since these are satisfied not only by an antidromic but also by an orthodromic monosynaptic unit response; 3) a response with 100% fidelity to a high rate of stimulation ie.g. 150/sec.) is a reliable criterion in the mammalian C.N.S. characterizing an antidromic response; 4) the test of collision is equally reliable (Darian-Smith et al., 1966; Blum, 1971). This test was developed independently by us and other investigators for the identification of antidromic unit responses as follows: The unit is evoked a short time after its spontaneous discharge. If this time interval is made shorter than the evoked unit response latency, and if the latter is antidromic, the impulse resulting from the spontaneous discharge is annulled by that resulting from the stimulus. On the basis of this annulment it is concluded that the stimulus was delivered to an axon derived from the cell from which the recording was taken. USE OF COLLISION PHENOMENON IN DETERMINING THE PROPERTIES OF A NERVE PROJECTION AS A PATHWAY FOR IMPULSE PROPAGATION Using the response to high rate of stimulation as a criterion for the identification of antidromic response, the collision test may be utilized on the same pathway to estimate the probability rate for impulse propagation through the pathway (Blum, 1972a, b). 93

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Fig. 1. The antidromic method for the microphysiological definition of direct neuronal connections. At (M) is a microelectrode coupled with a neurone for intra- or extra-cellular recording. At (SI) is a stimulating electrode located at a region which presumably contains axonal (EF) endings of the same neurone. Stimulation through this electrode would initiate an impulse ha the 'antidromic' (A) direction shown by the arrow. At SII is another stimulating electrode located at input fibers (AF) to that neurone. Stimulation through SII would synapticaUy excite the neurone to initiate an 'orthodromic' (O) impulse in the direction of the corresponding arrow. (Col) is a collateral fiber branching off from the neuronal axon.

This is d o n e b y observing t h e t i m e s of o c c u r r e n c e a n d o f failure of collision in a long series o f c o n s e c u t i v e l y e x e c u t e d tests o f collision. P a t h w a y s f r o m SC t o o t h e r visual areas were studied in this way. D i f f e r e n t rates were s h o w n for t h e p a t h w a y f r o m SC to D L G N w i t h s p o n t a n e o u s n e u r o n a l dis-

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charges as compared to the discharge of the neurone by light. Also different rates were shown in collateral branches of SC neurones one leading to DLGN as compared to the one leading to the pulvinar nucleus. Thus an experimental confirmation was obtained to an assumption often made intuitively of dependence of the relay of impulses on signal value, or that signals originating from the same source may vary when relayed into different channels. SOME OF THE DIRECT CONNECTIONS OF THE S.C., ITS LAYERED AND DOMAINE ORGANIZATION A STUDYWITH THE ANTIDROMICMETHOD AND WITH PAIRED STIMULATION As is shown in Fig. 2 unit responses that were recorded from S.C. neurones and characterized as antidromic provided information on the outputs of these neurones. These included outputs to the ipsilateral DLGN and pulvinar nuclei, including collateral outputs to these and bilateral outputs to the CM nucleus. In addition S.C. cells were defined with fast projections to the cortical visual area 18 and collaterals to the occulomotor nucleus (III). Monosynaptic unit responses that were obtained in S.C. following stimulation of the DLGN of either hemisphere of pulvinar nucleus and of visual cortical area 18 of the same hemisphere aided in definition of inputs to S.C. neurones. Anatomical data available on these connectivities only helped in better designing the experiments but were insufficient by themselves. It was only with the addition of the microphysiological results that it was possible to attempt drawing a schematic of the network composed of these connectivities (Figs. 2 & 3; see also Blum et al., 1975). The data also provided information on the topographic organization of S.C. Based on the locations of the neurones from which the above-mentioned responses were recorded, some aspects of the relationship of the layer organization of S.C. to its processing of information could be deduced. It may be concluded that the view that the first stage of input reception resided in the superficial layer is only partially true for the foUowing reasons. Optic tract inputs reach not only the superficial layer but also the intermediate layer. Furthermore, the superficial layer was also shown to receive inputs from the cortical visual area 18. This input was shown to be concerned at least in part with modifications of the encoding of the optic tract input in this S.C. layer (Blum et al., 1972). Other functions which have been proposed for this cortical input were concerned with disparity correction (Hassler, 1972; Blum et al., 1975). The intermediate layer according to the present experiments contains ceils with occulomotor function as weU as neurones with

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DLGN

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Fig, 2. Antidromic and orthodromic unit responses recorded in SC by which outputs and inputs to their neurones are defined respectively. In (A) are shown an antidromic (au) and orthodromic (ou) unit responses (u) to DLGN stimulation. Note differences in spike shapes. In (B), (C) and (D) orthodromic unit responses showing failure of collision (B) and lack of constancy of latency values (C, D) although the constancy of th e responses suggests a monosynaptic unit response. The diagram illustrates this two-way connection between SC and DLGN. Note the fibers mediating antidromic (A) and orthodromic (B, C, D) responses.

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Fig. 3A. Antidromic unit responses obtained in SC foUowing stimulation of the center median nucleus (CM) of ipsilateral (on left) and of contralateral (on right) hemispheres. Lower tracings in each case provide collision evidence for the antidromicity of the responses. Fig. 3B. Unit responses obtained orthedromically in SC following stimulation of DLGN (left column) at 50% response rate and reduction of the latter to 0% by preconditioning train stimuli to the ipsilateral CM (column on right). A digram drawn to explain the observations in (B) showing projections from SC to CM of either hemi-

sensory visual function. The relationship between these has not yet been defined. Sublayers of this strata were shown to contain outputs to and from DLGN and the pulvinar nucleus, the latter in what appears to be a domaine to domaine connection. Also collaterals to both nuclei were shown to originate in cells of this intermediate layer. Other cells of that layer were shown to send projections to area 18 and collaterals to the occulomotor nucleus III. Although no specific data were obtained on interactions between S.C. layers our results do not conflict with the view that information undergoes processing en route from the superficial layer of S.C. inwards (Mcllwain, 1970, 1971 ; Wurtz, personal communication, 1975; Godel, Ph.D. thesis, Tel Aviv University, 1975). Some of the data on connectivities was utilized to study their interactions. CM outputs, for example were shown to have collaterals which apparently participated in determining response levels of some S.C. neurones. A sample is illustrated in Fig. 3 in which it is shown that CM stimulation effects apparently through such collaterals a reduction of response level to monosynapfic activation of S.C. neurone by DLGN stimulation from 50Z to 07o. In summary, consideration was given to techniques required for the analysis in space and time of visual data processing. Some of our approaches as well as some of others were described. REFERENCES Abeles, M. Travel into the brain. In: Intnl. sympos, on signal analysis and pattern recognition in biol.-med.-eng., Haifa, July (1974). Blum, B. & B. Feldmann. A micro-drive for the independent manipulation of four microelectrodes. 1EEE Trans. Biomed. Eng., BMF 12:121 (1965). Blum, B., L.M. Halpern & A.A. Ward. Microelectrode studies of the afferent connections and efferent projections of neurones in sensorimotor cortex of the cat. Experi. mentalNeurology 20:156 (1968). Blum, B. Microphysiological characterization of output channels and of impulse propagation from the sensorimotor cortex including through pyramidal tract neurones' collaterals to the center median nucleus of the cat and of the monkey, lntnl. J. Neurology. Special Issue on the 'Thalamus' ( 1971). Blum, B. Rates of nerve impulse propagation as output characteristics - a study on pyramidal tract neurones of cat and monkey. Kybernetik 10:220 (1972). Blum, B., V. Godel, S. Gitter & R. Stein. Impulse propagation from photically discharges neurones in the visual system. Pflug. (European) Arch. Physiol. 332:38 (1972b). Blum, B, Logic Operations in the central nervous system - Implications for information transfer mechanisms. Kybernetik 11:170 (1972c). Blum, B., V. Godel, S. Gitter & R. Stein. Unit responses and interactions at superior collicusus first stage of input reception. Experientia 28:1440 (1972d). Blum, B, Interactions of visual pathways at the level of the superior coUicusus. In: Prec. of XIII Iscerg, Doe. Ophthal. Prec. Series (in press).

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Creutzfeldt, O., G.M. Innoccenti & D. Brooks. Vertical organization in the visual cortex (area 17) in the cat. Exp. Brain. Res. 2 1 : 3 1 5 (1974). Darian-Smith, I. & T. Yokota. Cortico-fugal effects on different neuron types within the cat's brain stem activated by tactile stimulation of the face. J. Neurophysiol. 29: 185 (1966). Fuortes, M.G.F. Repetitive activity of excitable membranes. Proc. XXV. Inml. Congress Physiol. ScL 8:41 Munich (1971). Grusser, O.J. Information Theorie und die Signalverbreitung in den Sinnesorganen und im nervensystem. Naturwis. 5 9 : 4 3 6 (1972). Hassler, R. In: J. Dichgans & G. Bizzi, Cerebral control of eye movements and motion perception. Bibliot. OphthaL, S. Karger, Basel (1972). Hensel, H. & R.D. Wurster. Static properties of cold receptors in nasal area of cats. J. Neurophysiol. 33:271 (1970). Hubel, D.H. & T.N. Wiesel. Receptive field, binocular interaction and functional architecture in the cat's visual cortex. J. Physiol., London 160:106 (1962). Jassik-Gerschenfeld, D. Activity of somatic origin evoked in the superior colliculus of the cat. Extl. Neurol. 16:104 (1966). La Vail, J.H., K.R. Winston & A. Tish. A method based on retrograde intra-axonal transport of protein for identification of cell bodies of origin of axon terminating within the C.N.S. Brain Res. 5 8 : 1 4 1 6 (1972). Maciewitz, R.J. Afferents to the lateral suprasylvian gyrus of the cat traced with horseradish peroxidase. Brain Res. 7 8 : 1 3 9 (1974). Mcllwain, J.T. & H.L. Fields. Superior colliculus: Single unit responses to stimulation of visual cortex in the cat. Science 170:1426 (1970). Mcllwain, J.T. & H.L. Fields, Interactions of cortical and retinal projections in single neurons of the cat's superior colliculus. J. Neurophysiol. 3 4 : 7 6 3 (1971). Mountcastle, V.B. Modality and Topographic properties of single neurons of cat's somatic sensory cortex. J. Neurophysiol. 2 0 : 4 0 8 (1957). Mountcastle, V.B., P.W. Davies & A.L. Berman. Response properties of neurones of cat's somatic sensory cortex to peripheral stimuli. J. Neurophysiol. 2 0 : 3 7 4 (1957b). Nauta, H.J., M.B. Pritz & R.J. Lasek. Afferents to the rat caudo-putamen studied with horseradish peroxidase. An evaluation of a retrograde neuro-anatomical research method. Brain Res. 6 7 : 4 9 (1974). Perkel, D.H., G.L. Gerstein & G.P. Moore. Neuronal spike trains and stochastic point processes. II. Simultaneous spike trains. Biophys. J. 7 : 4 1 9 (1967). Phillips, C.G. Intracellular records from Betz ceils in the cat. Quart. J. exp. Physiol. 41 : 58 (1956). Ratliff, F., B.W. Knight & N. Milkman. Super position of excitatory and inhibitory influences in the retina of limulus: Effect of delayed inhibition. Proc. Nml. Acad. ScL 67:1558 (1970). Shimazu, H., N. Yanagisawa & B. Garoutte. Cortical transmission in the cat. Jap. J. Physiol. 15:101 (1965). ShoU, D.A. The organization of the cerebral cortex. Wiley, N.Y. (1956). Walloe, L. Transfer of signals through a second order sensory neuron. Instit. of Physiol., Univ. of Oslo. Press (1968). Wurtz, R.H, Responses of striate cortex neurons to stimuli during rapid eye movements in the monkey, k Neurophysiol. 32:975 (1969). Author's address: Department of Physiology and Pharmacology The Sackler School of Medicine Tel Aviv University Tel Aviv Israel

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