Contains reports on four research projects.Bell Telephone Laboratories, Inc.U.S. Air Force (Aeronautical Systems Division) under Contract AF 33(615)-1747National Aeronautics and Space Administration (Grant NsG-496)National Institutes of Health (Grant MH-04737-05)National Science Foundation (Grant GP-2495)The Teagle Foundation, Inc

Frenk, S.

Lettvin, Jerome Y.

Maturana, Humberto R.

Pickard, W. F.

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XIX. NEUROPHYSIOLOGYW. S. McCulloch Susan Hurley Barbara C. PickardM. Blum K. Kornacker W. F. PickardJ. E. Brown J. Y. Lettvin W. H. PittsE. E. Fetz Diane Major T. G. SmithS. Frenk T. McLardy A. TaubR. C. Gesteland L. M. Mendell P. D. WallB. Howland S. A. Papert Barbara G. WickelgrenA. MAMMALIAN NODES OF RANVIER1. RECORDING METHODSAbout eight years ago, we spent a few months penetrating with microelectrodes intolarge myelinated fibers in normally circulated dorsal roots of the cat. At that time, theevidence suggested that the entry was at or near a node of Ranvier rather than throughthe myelin sheath. An electrode would stay in a single fiber very often for an hour ormore with no deterioration in the electrical behavior of the fiber. Membrane potentialswere usually ~70-80 my and the height of the nerve spike ~100-120 my, with a durationof 0.5 msec or less. It seemed that such a preparation would be useful in studying mem-brane properties on mammalian nerve; however, for several reasons, the work was putaside. Since that time, nobody else seems to have interested himself in this problem or tohave discovered how easy the preparation is. Therefore two members of our group haverevived the unreported method and W. Pickard is developing it further.We use ordinary micropipettes with tips of 0. 3 micron (I. D.), or less, and filled with3 molar KC1 in the usual fashion. These tips exhibit a resistance, in tissue fluids, 10-15 megohms, and the capacitance across the pipette wall in the fluid is ~1 pfd/mm lengthof pipette independent of diameter (because in drawing the pipette the I. D./O. D. ratiotends to remain constant). Such a probe is connected to a conventional DC amplifier ofhigh input resistance and low leakage current, which also has an adjustable "negativecapacitance" compensation at the input.A young cat's spinal cord is exposed in the usual way, grounded, and bathed inmineral oil. Over a small patch of dorsal root the arachnoid is removed between ves-sels. Into this patch we thrust the microelectrode and, with a little manipulation, impalea large fiber. This is seen as a sharp change of approximately -70 my with respect toground. We stimulate the fiber through the recording electrode and cancel the stimulusartefacts in the following way: When a trapezoid of voltage is differentiated it yields twosquare pulses of current in opposing directions. The magnitude of the current througha fixed capacitor depends on the rate of change of the voltage, the duration depends onThis work is supported in part by the Bell Telephone Laboratories, Inc. ; TheTeagle Foundation, Inc.; the National Science Foundation (Grant GP-2495); the NationalInstitutes of Health (Grant MH-04737-05); the U.S. Air Force (Aeronautical SystemsDivision) under Contract AF 33(615)-1747; and the National Aeronautics and SpaceAdministration (Grant NsG-496).QPR No. 78 279(XIX. NEUROPHYSIOLOGY)the duration of the ramp functions used to generate the trapezoid, and the intervalbetween the two pulses is the interval between the two ramp functions. Thus we can con-trol all of the parameters of a stimulus. We set the square pulses of current at-9~10 amp, 0. 1-msec duration, and 10-msec interval. These square current pulses yieldsquare voltage swings whose magnitude measures the resistance through the electrodethrough the electrode to ground, and whose rise and fall times measure the capacitiveshunt to ground across the electrode wall. The rise times can be adjusted to ~5 secby means of the negative capacitance without introducing distortion. These square volt-age pulses can be balanced out in a push-pull amplifier by an analogue of the electrodeimpedance which is also capacitively connected to the trapezoid generator. Since, forsuch low currents, the KC1 pipette shows no severe nonlinearities, the cancellation ofthe induced voltage changes across the electrode can be done extremely well in a push-pull amplifier. As one penetrates a nerve fiber the cancellation that served so well inthe external fluid ceases to be effective because the resistivity to ground changes as soonas the tip of the electrode is up against a high-resistivity barrier such as membrane.When the membrane is traversed the cancellation is again effective as long as the elec-trode tip lies in axoplasm (which has about the same resistivity as the external fluids)and is not touching the other side of the fiber because of having been pushed too far. Byfine adjustment after penetration, the tip can be positioned directly in the center of thefiber by using electrical monitoring. But the resistivity across a nodal membrane isseveral megohms and the capacitance is a few picofarads. Furthermore, the axoplas-mic bridges to the nodes on either side are each several megohms. Thus, riding on topof the compensated signal there will appear an additional signal caused by the node mem-brane, and it will have a longer rise time than the compensated pulse. The net resultof suitable compensation is that it is possible to measure the so-called electrotonic prop-erties of the node directly. This current level is frequently sufficient to stimulate thenode and one sees the nerve spike. The threshold for most nodes is fairly low and atno time is it necessary to use currents that take the electrode into nonlinear operation(e. g., currents greater than 10 - 8 amp). It is possible, by using such an arrangementto measure the changes in impedance during the spike and many other properties of nerv-ous membrane, and we have done so.2. MATHEMATICAL AXONOLOGYIf the medullated axon is idealized as a periodic structure composed of lengthsof lossy passive transmission line (internodes) coupled by electrically short lengthsof active transmission line (nodes), then it is possible to derive by classical meansan equation for the temporal variation of the nodal voltage. This resulting equationisQPR No. 78 280(XIX. NEUROPHYSIOLOGY)2s e6 - 1 1 j+ J o(w) e Ot d =rl + r2 e + 1 -dVbc dt + bGNa[Vo+VNa] + bGK[Vo-VK] + bGL[Vo-Vo],where V (t) is nodal voltage, ' ° nodal resting potential, b nodal length, the Vj severalionic battery potentials, the G.(t) several nodal ionic conductances per unit length, Cnodal shunt capacitance per unit length, rl and r2, respectively, the resistance per unitlength of the extracellular solution and the axoplasm, 62 = (r 1 +r2)g, where g is the shuntconductance per unit length of internode, a is the internodal length, andJ () = -2 a [cosh ya - cos Wo],o a(rI+r2) o sinhyawhere D (w) is the Fourier transform of Vo (t),2 = (rl+r)(g+jc), where c is the inter-nodal shunt capacitance per unit length, and Or = (a+b), where 0 is the pulse conductionvelocity along the axon.It can be shown that this equation for nodal voltage does not, for many axons, approx-imate a linear second-order differential equation even if the several ionic conductancesare explicitly known as functions of time.By a limiting process, this equation can be simplified to yield the usual equation forpropagation along an unmyelinated axon. This new equation can be manipulated to yieldan approximate expression for the conduction velocity. For squid giant fibers theexpression for 0 reduces too1= 3 VT()1 1/4C (F 2 2 o T)where is the mean maximum rate of use of the axon's sodium conductance per0unit length, C is the capacitance per unit length, (rl+r2 ) is the total resistance perunit length of axoplasm and intercellular medium, and VT is the threshold voltage. Thisformula gives numerical predictions that agree well with experimental data.A detailed account of these researches will appear in a paper that is now being pre-pared for submission to the Journal of Theoretical Biology.W. F. PickardB. UNMYELINATED FIBERS IN THE DORSAL ROOTS OF CATSWith Dr. R. C. Gesteland, who is leaving our laboratory and will be greatly missed,we have developed a method for recording the activity of unmyelinated fibers in the dor-sal roots of mammals. He will take this technique to Northwestern University where heQPR No. 78 281(XIX. NEUROPHYSIOLOGY)plans to do a major study on these fibers. Briefly, it is this: When a cat is preparedas for the study of nodes, and the arachnoid is cleaned off a patch of root, one uses,instead of a micropipette, a Dowben-Rose electrode consisting of a glass capillary filledwith Wood's metal and plated at the tip with a cap of gold on which Pt black is deposited.When such an electrode just touches the surface of the root, not only can one record thefast transients caused by myelinated fibers, but also much slower diphasic and triphasictransients, lasting 10 msec or so. These slower transients can be increased in fre-quency by manipulation of the viscera or by pressing on the skin, and so on. They seemvery frequently to be autonomic (or enteroceptive) but sometimes are somatic. If theelectrode is pressed down so as to penetrate the root, then these transients disappearand never return when the pressure is relieved, although the fast transients recover. Wehave seen such sensitivity to pressure in other unmyelinated small-fiber systems such asthe olfactory nerve of the frog or the optic nerve of the octopus. Presumably it is thissensitivity that has prevented the prior discovery of such transients by other workers.We had hit upon this technique three years ago and laid it aside until one of us felt hecould handle it seriously. It certainly beats the more exacting and tedious method ofdissecting down small bundles.J. Y. LettvinC. OLFACTION IN THE FROGOur study of the frog's nose is to appear soon in the Journal of Physiology. WithR. C. Gesteland as principal investigator, we report some new observations on first-order olfactory elements studied both individually and en masse. What we found wasthis:(i) Most of the axons of the olfactory neurons have a resting discharge; the proba-bility of an impulse occurring at any moment is not extremely low.(ii) This probability of discharge can be increased, in any fiber, by some odors anddecreased by others; that is to say, the response has two directions.(iii) For any odor that causes a response, its effect during insufflation may be eitherthe same or opposed to that produced by stopping the odor.(iv) For any fiber, the effect of a variety of odors can be ordered in terms ofincreasing or diminishing the probability of firing during and following the presentationof the odor.(v) The probability that two fibers will show the same order decreases steadily withthe number of odors used. That is to say, if we use two odors that produce the sameresponse in each of two fibers, a short search reveals a fiber that responds differentlyto the odors.(vi) Analysis of the voltage transient known as the electro-osmogram, a "slow wave"very like the electroretinogram and others recorded from sensory tissue, reveals thatQPR No. 78 282(XIX. NEUROPHYSIOLOGY)there are two processes governing the course of the transient. One can be regarded asa current-generating process across the receptor membrane and the other as a shuntingprocess. The interaction of these two processes accounts for the families of transientshapes that we see. The two processes that are actually involved are unknown, but theyare equivalent formally to what we have described; i. e., they are not similar processessimply of opposing sign.(vii) That there are two processes involved in changing impedance across the olfac-tory mucosa during stimulation can be shown at certain frequencies of applied currentthat depend on electrode parameters. At these frequencies the phase angle and magnitudeof resulting voltage swings can fluctuate independently, the amount depending on thestimulus.(viii) The two directions of the response in the single fiber, the two processesinvolved in generating the electro-osmogram, and the two processes revealed in theimpedance measurement have been taken by us to be different representations of a dualprocess in the receptors. The coincidence of results may be fortuitous, for we haveproved no causal relations; but the results are suggestive of what we suspect.(ix) This view leads to regarding the olfactory receptors as formally equivalent toneurons elsewhere in the nervous system, with the sensitive sites acting as excitatory andinhibitory postsynaptic membrane patches do in central neurons.We do not regard every olfactory neuron as different in its smelling order from everyother neuron (and there are several million in each nose). Rather, there must be somegroupings if we extend our olfactive experience to the frog. Such groupings are, however,unlikely to be uncovered by a fiber-to-fiber search when one considers how many smellsmust be used per fiber to establish groups.(x) Olfactory space ceases to be comparable to other sensory spaces if, as nowseems possible, we must deal with what may at first appear as arbitrary ordering prin-ciples. Notions of distance in such a space may only be locally definable and this wouldbe in accord with the psychological data.(xi) Finally, as a practical matter, Gesteland has shown that, by using the electro-osmogram, and the phase angle and amplitude of the voltage fluctuations resulting fromthe application of an oscillating current to the mucosa, it is possible to make clean dis-tinctions between many different odors. This trivariant signature, however usefulempirically, implies nothing about the relation of its components to the olfactory process.J. Y. LettvinD. RETINAL STUDIES IN THE FROGThe ganglion cells in frog retina were incompletely described by two of us severalyears ago. We were more concerned with the short-time invariants, neglecting the moresecular processes and only sketching possible generative events. We put off the problemQPR No. 78 283(XIX. NEUROPHYSIOLOGY)of characterizing these cells more completely until we would have a chance to worktogether again. This we have been able to do this spring. We chose the simplest ele-ment to analyze - the "off" fibers of Hartline (our "dimming" detectors). These unitsare characterized by a vigorous, rhythmic and prolonged response to the darkening oftheir receptive fields, and an interruption of that discharge, a complete impression, byrebrightening. At least this is the way Hartline, Barlow, and ourselves separatelyreported. Yet this description, however simple, raises some definite questions: Whatgoverns the rate and length of the off-response if it is a function of the size of the stepto darkness? Is the response characterized by one time constant or by several? Areall off-fibers alike in most respects, or are there individual differences of a materialsort? Can we essay some guess about the generative events from noting similaritiesbetween fibers ?Our preparation was, again, the curarized frog with optic nerve exposed intracra-nially through retraction rather than ablation of the brain (to preserve a strong circula-tion). Our electrodes were, as before, our modification of the Dowben-Rose probes. Our100I0z- 100z0o10DURATION OF 2n d INHIBITION( TIME OF RECOVERY MINUS TIME OF OFF-EXCITATION)( 12 sec UNITS )Fig. XIX-1. Time of recovery vs time of illumination.QPR No. 78 284(XIX. NEUROPHYSIOLOGY)only new apparatus was the CLOOGE (Continuous Leg Of On-Going Events). With thisinstrument we could record the logarithm of pulse interval over 4 decades (1 kc-0. 1 cycle) and, using a sample-hold circuit and voltage comparator, generate the recordsshown in Fig. XIX-1. When we had isolated a single off-unit in the optic nerve by pulse-height pick-off, and were assured of its stability, we let the eye dark adapt for 35minutes in very close to absolute darkness. At the end of this time we would administera flash of light of 1-sec duration, wait for full recovery, then give a light of 2-sec dura-tion, wait for recovery, and so on with 4, 8, 16, 32, and 64 sec flashes. Then we wouldleave the light on (it had always the same intensity in our initial experiments) until, asoften happened, the fiber would begin firing again, then turn the light off to absolutedarkness and watch the patterns of firing afterward. The CLOOGE yields a very easilyread record of these patterns.The most salient feature of our study is that over a wide range the time of return toresting activity in the dark is a linear function of the total amount of pigment bleached.These values were taken well below the asymptotic times of recovery for a light of thisintensity left on indefinitely long and then switched off. We have not studied the asymp-totes sufficiently to plot them as a function of light intensity. The data strongly suggestthat the generating event is governed by a bottleneck and, whatever sign of recovery weare measuring, it changes at a fixed rate. Recently such processes have been observedin pigment changes of the frog's eye.J. Y. Lettvin, S. Frenk, H. MaturanaQPR No. 78 285

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