SUMMARY1. The mechanism of reciprocal inhibition between antagonistic motor centres during swimming in the paralysed Xenopus embryo has been investigated further. Paired intracellular recordings have been made from interneurones and motoneurones in an attempt to identify neurones which make direct inhibitory synapses onto motoneurones on the opposite side of the spinal cord.2. A physiological class of inhibitory interneurones is described which, when stimulated by intracellular current passage, evoke short-latency, probably monosynaptic, strychnine-sensitive inhibitory potentials in contralateral motoneurones.3. These inhibitory interneurones fire once per swimming cycle in phase with the ipsilateral motor root discharge. They therefore have a pattern of activity which would cause them to inhibit motoneurones of the antagonistic motor centre at an appropriate part of the swimming cycle.4. The intracellular injection of horseradish peroxidase (HRP) has allowed the morphology ofthese inhibitory interneurones to be characterized. They have unipolar cell bodies with a thick proximal process with short dendrites which crosses the spinal cord ventrally and then bifurcates with one axonal branch ascending into the hind brain and the other descending the spinal cord. These anatomical features are

1. Simulations of Xenopus embryo spinal neurons were endowed with Hodgkin-Huxley-style models of voltage-dependent Na+, Ca2+, slow K+ and fast K+ currents together with a Naedependent K+ current. The parameters describing the activation, inactivation and relaxation of these currents were derived from previous voltage-clamp studies of Xenopus embryo spinal neurons. Each of the currents was present at realistic densities.2. The model neurons fired repetitively in response to current injection. The Ca!+ current was essential for repetitive firing in response to current injection. The fast K+ current appeared mainly to control spike width, whereas the slow K+ current exerted a powerful influence on the repetitive firing properties of the neurons without markedly affecting spike width. 3. The properties of the model neurons could be made more consistent with those previously reported for Xenopus embryo neurons during intracellular recordings in vivo, if the shunting effect of the sharp microelectrode was incorporated into the model. 4. The model neurons were then used to create a simplified version of the spinal network that controls swimming in the frog embryo. This model network could generate the motor pattern for swimming: the activity between the left and right sides alternated with a cycle period that varied from 50 to 120 ms. This is very similar to the range of cycle periods observed in the real embryo. The shunting effect of the microelectrode was once again taken into account.5. Reductions of the K+ currents perturbed the motor pattern and gave three forms of aberrant motor activity very similar to those previously seen during the application of K+ channel blockers to the real embryo. The ability to generate the correct motor pattern for swimming in the model depended on the balance between the K+ currents and the inward Na+ and Ca2+ currents rather than their absolute values.6. The model network could generate a motor pattern for swimming over a very wide range of excitatory (2-10 nS) and inhibitory (2-400 nS) synaptic strengths. Rough estimates of the physiological synaptic strengths in the real circuit (around 20-60 nS for inhibition and 2-5 nS for excitation) fall within the range of synaptic strengths that gave simulation of the swimming motor pattern in the model. 7. The cycle period of the motor activity in the model shortened either as the excitatory synapses were strengthened or as the inhibitory synapses were weakened. 8. The prediction that the strength of the mid-cycle inhibition determines cycle period has been tested by using low levels of strychnine to reduce glycinergic reciprocal inhibition in a graded manner in the real embryo. As the inhibition was reduced, the cycle period of fictive swimming in the embryo shortened by amounts very close to those predicted by the model. 9. This new experimentally derived model can replicate many of the known features of fictive swimming in the real embryo and may be of value as an analytical tool in attempting to understand how the spinal circuitry of th...

SUMMARY1. The monoamine transmitter 5-hydroxytryptamine (5-HT) and the peptide Phe-Met-Arg-Phe-amide (FMRFa), which appear to contribute to presynaptic facilitation and inhibition of the sensorimotor synapse in the abdominal ganglion of Aplysia, can modulate the frequency of spontaneous transmitter release at synapses formed between dissociated Aplysia sensory neurones and motoneurones in vitro.2. 5-HT caused a decrease in the mean time interval between consecutive miniature EPSPs (mEPSPs), while FMRFa, applied either by itself or together with 5-HT, caused an increase in the mean time interval between consecutive mEPSPs.3. Depolarization of the presynaptic neurone caused a decrease in the mean time interval between consecutive mEPSPs. This modulation required external Cai±.4. 5-HT and FMRFa were able to modulate spontaneous release when applied in saline solutions lacking Ca2± and containing Ca2+-chelating agents, suggesting that the modulation of spontaneous release by 5-HT and FMRFa did not require a Ca2+ influx. Similarly, spontaneous release could still be modulated by 5-HT and FMRFa in saline solutions containing 1 mM-Cd2 , which blocked both the voltage-gated Ca2, channels and the evoked transmitter release.5. To prevent a rise in intracellular Ca +, we buffered the concentration of Cai+ in the presynaptic terminals by injecting into the sensory neurone the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The injection of BAPTA blocked evoked transmitter release, suggesting that it acted as an effective buffer of Ca2+ in the terminals. However, spontaneous release could still be observed and was still modulated by 5-HT and FMRFa. This suggests that the modulation of spontaneous release does not require an elevation of intracellular Cai±.6. We propose that 5-HT and FMRFa can modulate the rate of spontaneous release directly by mechanisms that do not require changes in the intracellular concentration of Ca2+. These mechanisms might contribute an additional component to the presynaptic inhibition and facilitation of evoked transmitter release.

When isolated and grown in cell culture, the sensory and motor neurons of the gill withdrawal reflex of Aplysia readily form synaptic connections. Repeated exposures to 5-HT cause facilitation of the synaptic connections between co-cultured sensory and motor neurons lasting at least 24 hr. As a first step toward understanding the locus and the mechanisms underlying this long-term synaptic facilitation, we have examined the membrane excitability of the isolated presynaptic sensory neurons grown alone in dissociated cell culture. Four repeated applications of 1 microM 5-HT caused a significant increase in the excitability of sensory neurons, lasting at least 24 hr. This resembles the short-term changes in excitability seen in response to a single application of 5-HT. Unlike the short-term effect, this long-lasting change was blocked by exposure of the cells during the 5-HT treatment to 10 microM anisomycin, an inhibitor of protein synthesis. Thus, like the synaptic facilitation, the long-term change in excitability of the isolated presynaptic neurons differs from the short-term in requiring the synthesis of new protein. This finding suggests that the sensory neuron uses gene products to modulate membrane currents in its long-term response to repeated external stimuli that are not required in its short-term response to a single stimulus.

1. Using the whole-cell patch clamp technique, the voltage-gated currents of neurons acutely isolated from the Xenopus embryo spinal cord were studied. 2. The spinal neurons possessed a very fast Na+ current, which activated with time constants that ranged from 0.1 to 0.25 ms. It was also subject to rapid inactivation with time constants ranging from 0.3 to 8 ms. This current could only be fitted with Hodgkin-Huxley equations once the rapid inactivation that occurs by the time of the peak current had been taken into account. 3. Xenopus embryo neurons also possessed a mixture of kinetically similar Ca2+ currents, which activated with time constants that ranged from 0.3 to 0.8 ms. Sometimes the Ca2+ currents showed very slow inactivation at more positive voltages (> 20 mV). The Ca2+ current was modelled as a single non-inactivating current. 4. As might be expected, the embryonic neurons possessed a mixture of outward currents that were hard to separate either pharmacologically or through differences in voltage dependence. The delayed rectifier seemed to consist of varying proportions of two currents: a fast-activating K+ current (with time constants of activation ranging from 0.6 to 2 ms) and a slow K+ current (with time constants of activation ranging from 5 to 25 ms). The slow current was occasionally seen in isolation. 5. For the Ca2+, fast K+ and slow K+ currents the rate of deactivation was faster than would be predicted from the kinetics of activation. This was modelled by allowing the closing rate constant of the channels to be described by one of two different functions of voltage that between them covered the whole range of transmembrane voltage. Although this was done for empirical reasons, it could be interpreted to suggest that the channels have more than one open state and predominantly close from a state that is distinct from the one to which they originally opened.