Contains reports on three research projects.Bell Telephone Laboratories, IncorporatedTeagle FoundationNational Science FoundationOffner Electronics, Incorporate

Duchane, E. M.

Gesteland, R. C.

Lettvin, Jerome Y.

McCulloch, Warren S.

Pitts, Walter H.

Wall, P. D.

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XV. NEUROPHYSIOLOGY*W. S. McCulloch R. C. Gesteland W. H. PittsE. M. Duchane J. Y. Lettvin P. D. WallA. MEMBRANE PROPERTIESResting squid nerve membrane actively extrudes Na+ against a large concentrationgradient, while another cation, K + , is concentrated passively within the cell by theDonnan equilibrium. During the passage of an impulse, large transient current flowsacross the membrane; some K + leaves, some Na + enters, but which ions carry thecurrent at which instant, and how it is turned on and off cannot be told from simpleexternal measurements. Thus Marmont, and later, exhaustively, Hodgkin and Huxley,applied a voltage clamp between the inside and outside of a squid giant axon, i. e., theyapplied step functions of voltage across the membrane and measured the currentrequired to maintain the steps. By systematically varying the external concentrationof different ions they were finally brought to a description of the nonlinear behavior ofthe membrane in terms of specific ion conductances, each conductance being dependenton the concentration potential of its species and certain rate constants. With this verycareful job they accurately predicted the shape of traveling nerve spikes and after-potentials. However, that tour de force was meant to apply only to squid axon - thesubject of their experiments.Very soon other physiologists began using as laws interpretations which Hodgkinand Huxley had only diffidently suggested, and these laws were applied to nerve-nerveand nerve-muscle transmission with great ingenuity. While such guessing is fair inthe absence of data, it is clear that one must in the event test, say, frog nerve, forwhat nonlinear conductances govern its behavior. Too many differences in chemicalcomposition, speed of transmission, shape of spike, and so forth, exist between squidand frog nerve to allow the voltage-current relations for one to be used on the other.Some workers even suspect that certain quaternary amines rather than Na + are impor-tant in frog-nerve conduction.The physical difficulty of examining other than giant fibers is very great. One caninsert down a squid nerve of some 0. 5 mm in diameter two reasonably large wires,one to measure voltage against some external probe, the other to supply current tosome outer electrode. This technique is not possible with fibers that are only 10 . indiameter. Cell bodies some 50 1 across could, in principle, admit two microelectrodes,one acting as a point source of current, but such a measure would be valid only if thecell were spherical. (Eccles' results with bipolar microelectrodes are not relevant* This work was supported in part by Bell Telephone Laboratories, Incorporated; inpart by Teagle Foundation; in part by National Science Foundation; and in part by OffnerElectronics, Incorporated.142(XV. NEUROPHYSIOLOGY)Fig. XV-1. Voltage clamp with one electrode.here, since he impaled such things as motoneurons which have large irregularprocesses.)The trouble in making a bipolar microelectrode (two compartments, fluid-filled,with a total outer diameter of less than 2 1) is great, the problem of filling it properlyis worse, and the patience required in handling it in tissue must be staggering. Thuswe honor Eccles but shall not imitate him except as driven to it. Instead, we havedevised two methods whereby vertebrate nervous tissue can be submitted to a voltageclamp; both are also trying to the patience. The first method involves examiningspherical ganglion cells such as those of the dorsal root. Here we use the circuitshown in Fig. XV-1. A is a differential amplifier (gain = 250) so arranged that theleft-hand loop through R 1 is degenerative and the right-hand loop through R2 is regen-erative. RN represents the variable membrane conductance, C N the constant membranecapacitance, EN the membrane potential which is interdependent with RN, and R theimpedance to ground from the outside of the cell. Although this is not a true bridge itis operated as one in this fashion: Suppose that R N remains constant for small pertur-bations of the voltage across CN and that Ro is zero. Then, with X disconnected fromA and connected to some external generator of square waves, if the shuntcapacitance to ground C at the junction of R 1 and RE is compensated by -C, thenR1/RE = R 2 /(R 3 + R 4 ) when the sharp front of the square wave is balanced out in theoutput of A (so that only the charging and discharging current of C is visible). If,with this configuration, X is reattached to A, the effect is that of a negative resistance,-RE, in series between RE and E S , and Y follows E S but not EN . Thus, without accessto Y except through RE it is possible to keep Y at any constant voltage independent ofwide changes in RN and EN.143(XV. NEUROPHYSIOLOGY)Fig. XV-2. Voltage clamp for node of Ranvier.In this way it is possible to enter a cell with a resistive microelectrode, and throughthat electrode maintain the potential to ground constant across the cell membrane,measuring the necessary current at the output of A.The second method involves applying a voltage clamp (Fig. XV-2) to a single nodeof Ranvier. Consider three sequential nodes, N I , N 2 , N 3 , lying in three troughs, themyelin between troughs dried off to isolate them, the two end nodes lying in KC1 orK2SO 4 , and the center node lying in Ringer's solution. The membrane potential of N 2is given by E l - E 2 , and the output of the amplifier is fed back degeneratively throughE3. Then E l - E 2 will follow G independent of change in membrane potential and imped-ance at N 2 . With the visit of del Castillo we are using this method to measure the"quantal" nonlinear elements in the nodal membrane.B. OTHER RECENT WORKDuring the past quarter, we have completed work on two studies. First, on thequestion of the effects of a high-frequency burst of nerve impulses, we have examinedthe failure of the immediate appearance of the full potentiation. We found that, duringthe height of the hyperpolarization of the nerve fibers immediately after the end of thetetanus, there are signs of an interruption in the conduction of nerve impulses. Thus,while the individual nerve impulses are greatly increased in height, some are not con-ducted to the synaptic region. Next, we have examined the relation between the slow"synaptic potentials" and the excitability changes of the afferent fibers that end on thecells. There is no doubt that the changes seen in the skin sensory afferent fibers thatend on internuncials are exactly correlated with the slow fifth-dorsal-root potential. Inthe case of the sensory fibers that originate in muscle and end on the motor cells, there144(XV. NEUROPHYSIOLOGY)is a strong suspicion that the changes restricted to the active fibers are correlated withthe slow ventral-root potential. However, a new series of experiments will be requiredto clarify this very important relation.As a result of our experience in measuring the prolonged effect of a single nervevolley on internuncial cells, we can postulate that change in the speed of reaction of thenervous system in a very old animal might be caused by a change in the reaction of thistype of cell. We have available a small number of ancient rats. We hope to establishthe normal behavior of the internuncial cells in adult rats and then to compare theirbehavior in the senile rat.Finally, we are starting a study of the behavior of spinal sensory internuncial cellsin response to a touch on the skin. Our aim here is to extract the mechanism by whichthe localization of the peripheral stimulus is coded in the nervous system. Individualcells can be shown to respond to stimulation of huge areas of the skin. The pattern ofresponse varies as the stimulus moves within the area. This is made particularly clearif two cells responding to the same area are examined simultaneously. We are makinga catalogue of cells and it is already clear that they fall into a number of categories.In some the first response is at a very definite time interval after the stimulus, pro-viding a time marker. Others fire off in a definite pattern which, however, is notexactly repeated at each stimulus. Others with a high spontaneous rate of firing do notrespond by the addition of new impulses, but only by a temporary variation in the fre-quency of their spontaneous firing. We hope to extract various classes of cells fromthis plethora of types and to determine methods of coding, since it is clear that no onecell transfers all the information on the nature, intensity, and localization of a smalltouch on the skin.145

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