Electrophysiological evidence for formation of new corticorubral synapses associated with classical conditioning in the cat

Abstract

The present study was performed to clarify whether or not structural plasticity of synaptic connections underlies classical conditioning mediated by the red nucleus (RN) in the cat. Conditioned forelimb flexion is established by pairing electrical conditioned stimuli (CS), applied to corticorubral fibers at the cerebral peduncle (CP), with a forelimb skin shock (the unconditioned stimulus, US), but not by applying the CS alone or by pairing the CS and US at random intervals. In our previous study, it was shown that the firing probability of rubrospinal neurons (RN neurons) in response to the CS was well correlated with acquisition of the conditioned forelimb flexion and that the primary site of neural change underlying establishment of the conditioned forelimb flexion was suggested to be at corticorubral synapses. In the present study, we investigated corticorubral excitatory postsynaptic potentials evoked by CP stimulation (CP-EPSPs), in order to identify the neuronal mechanism underlying establishment of classical conditioning. In normal cats, CP-EPSPs had a typical slow-rising phase, which has been attributed to the distal location of corticorubral synapses on the dendrites of RN neurons. In contrast, in animals that received paired conditioning, subsequent CP stimulation evoked potentials with a fast-rising time course. In control groups of cats that received CS alone, CS randomly paired with the US, or only the same surgical operations as the conditioned animals, most of the CP-EPSPs displayed slow-rising EPSPs that similar to those observed in normal cats. The mean time from onset to peak of the potentials in the conditioned animals was significantly shorter than that seen in other groups. Therefore, the appearance of a fast-rising potential correlates well with acquisition of the conditioned forelimb flexion. The amplitude of the fast-rising potential was gradually changed with stimulus intensity. It had a short onset latency following CP stimulation (0.9 ms), which was similar to that of the slow-rising EPSP in normal cats. It followed high-frequency stimulation up to 100 Hz. These results suggest that the newly appearing, fast-rising potential was a monosynaptically evoked EPSP. Fast-rising EPSPs were also induced by stimulation of the sensorimotor cortex (SM). Since the SM-EPSP was occluded by the CP-EPSP, the SM cortex is, at least in part, a likely source of fast-rising EPSPs. Fast-rising SM-EPSPs were also observed at the unitary level. The SM-EPSPs in the conditioned animals exhibited somatotopical representation in their cortical origin, as has been described in normal cats. The electrotonic length was calculated from the voltage transient responses to current steps injected into the RN neurons. There was no concomitant change in the electrotonic length following the classical conditioning. Furthermore, the fastrising EPSPs were often observed as if they were superposed on the slow-rising EPSPs that were observed in normal animals. These observations suggest that the appearance of fast-rising EPSPs is due to the formation of new corticorubral synapses on the somata or the proximal dendrites of the RN neurons, and not as a result of a reduction in the electrotonic length of the RN neurons. The present study provides further evidence that this type of structural plasticity of synaptic connections underlies establishment of the classically conditioned forelimb flexion.