Extracellularly applied steady electric fields of 0.1 to 10 V/cm were found to have marked effects on the neurite growth of single dissociated Xenopus neurons in culture: (1) neurites facing the cathode showed accelerated growth, while the growth of those facing the anode was reduced. Neurites growing relatively perpendicular to the field axis were prompted to curve toward the cathode. (2) More neurites appeared to be initiate from the cathodal side of the cell. (3) The number of neurite-bearing neurons per culture and the average neurite length were increased. These effects are absent in cultures treated with electric fields of similar strength but alternating polarity and cannot be attributed either to a gradient of extracellular diffusible substances or to the flow of culture medium produced by the field. The field effects are reversible: (1) removal of the electric field resulted in the loss of neurite orientation in a few hours and (2) reversal of the polarity of the electric field led to a rapid reversal in the neurite orientation. To determine the cellular loci of these field effects, we treated the neurons with a number of pharmacological agents or altered their ionic environments. Incubation with concanavalin A (Con A) was found to abolish these filed effects completely. Since the binding of Con A to the neuronal surface was shown to prevent field-induced accumulation of the Con A receptors toward the cathodal side of these neurons, our finding is accumulation of the Con A receptors toward the cathodal side of these neurons, our finding is consistent with the notion that cathodal accumulation of growth-controlling surface glycoproteins by the field is the underlying mechanism of the field-induced orientation of neurite growth toward the cathode.

We have studied the orientation of neurite growth in the culture of embryonic Xenopus neurons in response to three types of extracellular electric fields: spatially uniform pulsed fields, focally applied steady (DC) fields, and focally applied pulsed fields. Under uniform pulsed fields, neurites showed a preferential orientation toward the cathode pole of the field in a manner similar to that previously found for DC fields. The extent of neurite orientation depended upon the duration, amplitude, and frequency of the pulse but appeared to be similar to that produced by a uniform DC field of an equivalent time-averaged field intensity. For square pulses of 5 msec duration, the minimal amplitude and frequency required to produce a detectable orientation of neurite growth over a period of 24 hr were 2.5 V/cm and 10 Hz, which correspond to a time-averaged field intensity of 125 mV/cm. Steady or pulsed focal fields were applied by passing a current through a micropipette placed near the growth cone of the neurite. Fields of negative polarity (current sink) were found to attract the growth cone, whereas fields of positive polarity (current source) were found to deflect the growth cone away from the pipette. The threshold DC current density needed at the growth cone to perturb its direction of growth within 15 min was 0.2 to 2 pA/micron2 (or 3 to 30 mV/cm); and for focal pulsed currents (pulse duration 5 msec), a typical combination of minimal pulse amplitude and frequency was 4 pA/micron2 and 10 Hz. This threshold focal current is similar to that which occurs at the synaptic cleft during active synaptic activity.

Monopolar electric current pulses were focally applied through a micropipette to the growth cone of Xenopus embryonic neurons in culture. Application of the current directly in front of the growth cone modulated the rate of growth cone extension: Negative (sink) currents increased the growth rate, while positive (source) currents reduced the growth rate. When the currents were applied in a direction perpendicular to the direction of the neurite growth, both negative and positive currents produced inhibitory effects. Application of a negative focal current at a 45 degree angle with respect to the direction of neurite growth resulted in an oriented growth of the neurite toward the current sink. However, after the growth cone had been attracted to the vicinity of a current sink, further extension of the neurite was inhibited. These current effects occur rapidly after the onset of the current application, and are at least partially reversible within 1 hr after the termination of the current. The magnitude of current density required to induce a growth cone response was found to be in the order of a few pA per micron2. Such current density is close to that which may be generated at the muscle cell surface by the acetylcholine molecules released from the growth cone during the early phase of nerve-muscle contact.