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Purkinje cells are the sole output neurons of the cerebellar cortex that project to cerebellar deep nuclei (such as the interpositus nucleus) [M. Ito, The Cerebellum and Neural Control (Raven Press, New York, 1985)], and these neurons demonstrate synaptic plasticity (for example, long-term depression) hypothesized to be involved in eyeblink conditioning (5).
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Ito, M.1
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A circular craniotomy (6 mm diameter) was performed over the left cerebellum, centered over Larsell's HVI (4.5 mm lateral and 1.0 mm rostral to the lambda skull suture). A stainless steel recording base for a hydraulic microdrive (Kopf 650) was cemented over the craniotomy with dental acrylic and stainless steel skull screws. Electrode penetrations could be precisely located anywhere within the 6-mm opening. All surgeries were performed under ketamine HCI (60 mg per kilogram body weight), xylazine (6 mg/kg), and 1 to 2% halothane gas anesthesia. Rabbits (n = 14) received 7 days of postoperative recovery.
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For recording, a single-unit electrode (Z 64 5 megohm at 1 kHz) was lowered to the surface of the cerebellum and then further lowered in steps of <5 μm to isolate the complex spike activity of a Purkinje cell. The signal was fed into a window discriminator that allowed the number of complex spikes above the noise envelope to be sampled and stored in the computer. Once a unit was isolated, the rabbit was presented with either unpaired tones and airpuffs or paired tone-airpuff trials. Trials were presented until the cell was lost or enough trials were collected to characterize the cell's activity. Small marking lesions were placed at interesting recording sites or at the bottom of electrode tracts to histologically reconstruct the path of electrode penetrations. Single units were recorded from several regions within cerebellar cortex likely to be involved in eyeblink conditioning, including lobule HVI, HV (anterior lobe), HVIIA (crus I), and paramedian lobule. The following criteria were used to identify Purkinje cells: (i) the presence of both simple and complex spikes in the same unit recording; (ii) characteristic activity through different cortical layers (molecular, Purkinje cell, granule cell); and (iii) histological reconstructions - recording sites located within the Purkinje cell layer were identified by Nissl staining. Only the cells that reliably fit each of the above criteria were classified as Purkinje cells.
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For PTX infusions (1 μl of 10 μM at a rate of 0.1 μl/min), a guide cannulae was implanted into the contralateral dorsal accessory portion of the inferior olive (n = 2 animals). The placement of the cannulae was aided by monitoring characteristic inferior olive cell activity (groups of two to three spikes, low spontaneous rates) with a recording electrode protruding from the cannulae. Interestingly, by removing the GABA-mediated inhibition in the inferior olive, PTX seems to increase the overall frequency of complex spike activity recorded in the cerebellum.
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2 and 100 ms. In our preparation, the external eyelids were held open and the corneal airpuff US was delivered to the temporal region of the cornea, a region not covered by the nictitating membrane at full extension. Both CSs preceded the US by 400 ms and the stimuli coterminated.
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We thank A. R. Wagner and M. M. Chun for their helpful comments on the manuscript. Supported by grants (R.F.T.) from the U.S. National Institute on Aging (AF05142), NSF (IBN9215069), and the U.S. Office of Naval Research (N00014-95-1-1152), and also the Sankyo Corporation.
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