Faculty Bibliography

Morphological methods were used to study the plasticity of target-deprived mammalian dopaminergic (DA) neurons. Slices of substantia nigra (SN) were taken from the midbrain of rats aged one to twelve days, and cultured for one to two weeks. Localization of tyrosine hydroxylase (TH) was used to examine the distribution and shapes of DA neurons. Histochemical staining for acetylcholinesterase (AChE) was carried out to estimate both survival and biosynthesis of SN neurons. We found that some DA neurons can survive in vitro without their usual target neurons. This was demonstrated by injecting rhodamine-conjugated microspheres (RD) into the caudate putamen, a SN target area, at 6 to 8 days prior to culturing. RD-labeled cells survived in SN cultures and some of them were doubly labeled with AChE. TH neurons had different shapes and their axon terminals formed close contacts with adjacent nondopaminergic neurons. These findings suggest that a subset of DA neurons may switch targets, but the majority of them require target interactions with the caudate putamen for survival in vitro

Intracellular recording in the guinea-pig brainstem slice has demonstrated that high molecular weight alcohols block the low threshold calcium channel (LTCC) in the inferior olive (IO). These alcohols thus provide a tool for understanding the function of the pacemaking cellular networks of the olivo-cerebellar system, since the LTCC has been implicated in the oscillatory behavior of these neurons. Aspects of normal and pathological tremor are also believed to be mediated by these circuits, and thus development of effective ways of blocking the LTCC in vivo may eventually lead to novel treatments for essential tremor. The present experiments evaluated the effectiveness of the isomers of octanol in decreasing harmaline-induced tremor in vivo in the rat. Harmaline was used in this study because its tremorgenic action is mediated at the level of IO; octanol was found to be a potent antagonist of harmaline-induced tremor. Significant differences between the isomers further suggested conformational differences. This, taken in conjunction with the lack of effect of octanol in both IO lesioned rats and oxotremorine-induced tremor, implied that the action of the alcohol may be mediated at a specific binding site. These findings thus support the conclusions that the antagonism of harmaline-induced tremor by octanol occurs in the IO, and, in view of the previously reported in vitro data, that octanol may be an effective blocker of the LTCC in vivo

A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the 'P' channel, as it was first described in Purkinje cells

Multiple recordings from Purkinje cells in the rat cerebellum allowed the mechanism responsible for the activation of rows of synchronous complex spikes to be investigated. By determining the spatial distribution of the climbing fibre reflex that follows electrical microstimulation of the cerebellar cortex, it was shown that the mechanism for the simultaneity of firing was the electrotonic interactions between neurons in the inferior olive (IO). The spatial organization of the complex spike activity was shown to be regulated by GABAergic inhibitory input into the IO, probably arising from the cerebellar nuclear neurons. The rostro-caudal organizion of the complex spike activity following physiological stimulation (tactile stimulation of the upper and lower lip) demonstrated the same spatial distribution of synchronous activity in the cerebellar cortex as did the spontaneous activity and this was also disrupted by GABA blockers. Finally, complex spike responses to physiological stimulation indicate that the IO is capable of gating sensory inputs in accordance with its intrinsic autorhythmicity and that strong peripheral stimuli reset the oscillatory properties of the IO. The functional implications of the synchronicity and of the temporo-spatial organizion of complex spikes in the cerebellar cortex are discussed in the context of motor coordination and timing

The spatial and temporal organization of climbing fibre activation of Purkinje cells, the so-called complex spikes, were studied in the rat cerebellar Crus II folium utilizing a multiple microeletrode recording technique. As many as 32 Purkinje cells could be simultaneously recorded by using a custom-built electronic amplifier system and a special data storage device. Analysis of the auto-correlation activity of complex spikes in any given group of Purkinje cells indicated that activation occurs with a particular rhythmicity having a base firing of 10 Hz. Cross-correlation of spontaneous complex spikes demonstrated, in addition to a particular rhythmicity, an extraordinarily high degree of synchronicity within a particular spatial distribution of Purkinje cells. Thus, Purkinje cells organized in rostro-caudal rows tend to fire within 1 ms of each other for distances as far as 800 microm (the width of a folium) from the 'master' neuron. By contrast, Purkinje cells located medial or lateral to the master neuron showed almost no cross-correlation. Administration of harmaline to the animal increased the degree of auto- and cross-correlation but did not change the spatial order of the distribution of the cross-correlation. The results indicate that the olivo-cerebellar system is organized in such a way that climbing fibre afferents may be activated in a close-to-synchronous and rhythmic fashion. The spatial distribution of these afferents over the cortex is such as to activate rostro-caudal bands of Purkinje cells which tend to fire in a close-to-synchronous manner