SUMMARY1. In high decerebrate rabbits, cells were sampled extracellularly from the rostral flocculus. Purkinje cells were identified by their characteristic responses to stimulation of the contralateral inferior olive. Identification of basket cells was based on the absence of olivary responses and also on their location in the molecular layer adjacent to identified Purkinje cells. Mass field potentials in the flocculus were also studied.2. Single pulse stimulation of a vestibular nerve, either ipsilateral or contralateral, at a rate of 2/sec excited Purkinje cells with a latency of 3-6 msec. This early excitation represents activation through vestibular mossy fibres, granule cells and their axons (parallel fibres). Similar early excitation also occurred in putative basket cells.3. Conjunctive stimulation of a vestibular nerve at 20/sec and the inferior olive at 4/sec, for 25 sec per trial, effectively depressed the early excitation of Purkinje cells by that nerve, without an associated change in spontaneous discharge. The depression recovered in about ten minutes. This recovery was followed by the onset of a slow depression lasting for an hour.4. Conjunctive vestibular-olivary stimulation produced no such depression in the following responses: early excitation in Purkinje cells induced from the vestibular nerve not involved in the conjunctive stimulation; early excitation in putative basket cells from either vestibular nerve; inhibition or rebound facilitation in Purkinje cells following the early excitation; vestibular-evoked field potentials in the granular layer and white matter of the flocculus. These observations lead to the conclusion that the depression occurs specifically at parallel fibre-Purkinje cell synapses involved in conjunctive stimulation.5. Ionophoretic application of glutamate to Purkinje cells in conjunction with 4/sec olivary stimulation depressed the glutamate sensitivity of Purkinje cells; aspartate sensitivity was depressed to a much lesser degree. The depression diminished in about 10 min, but this recovery was succeeded by a slow depression lasting

SUMMARY1. Synaptic transmission from parallel fibres to Purkinje cells and its modification by paired stimulation of parallel fibres and climbing fibres were studied in in vitro slices of the cerebellum obtained from guinea-pigs.2. Intracellular recordings were made from Purkinje cells, mainly from dendrites in the middle third of the molecular layer, but also, in a few cases, from somata.Climbing fibres were activated by stimulation of the white matter, while parallel fibres were stimulated with an electrode placed near the pial surface of the molecular layer.3. Stimulation of the white matter elicited antidromic spikes, all-or-none climbing fibre responses, disynaptic responses through mossy fibres and parallel fibres, and trisynaptic responses through inhibitory interneurones. Climbing fibre responses were often followed by a small plateau potential, usually less than 2-3 mV in amplitude and less than 100 ms in duration, followed by a slow hyperpolarization which reached its peak in several seconds. Inhibitory inputs to Purkinje cells were blocked with picrotoxin for the experiments described below.4. Stimulation of the superficial molecular layer with currents less than 50 ,uA produced graded parallel fibre-mediated excitatory postsynaptic potentials (e.p.s.p.s) ranging from 4 to 8 mV in peak amplitude.5. Conjunctive stimulation of climbing fibres and parallel fibres at 4 Hz for 25 s induced depression of parallel fibre-mediated e.p.s.p.s in Purkinje cells, both in the peak amplitudes and in the slopes. The depression was about 30 % on average and lasted for more than 50 min.6. No such depression occurred when the intensity of the white matter stimulation was set just subthreshold for the climbing fibre innervating the Purkinje cell under study. Instead, the parallel fibre-mediated e.p.s.p.s were moderately potentiated for a period ranging from 10 to 50 min. Repetitive stimulation of the climbing fibre alone did not affect parallel fibre-mediated e.p.s.p.s.7. Immediately after the conjunctive stimulation or the repetitive stimulation of climbing fibres alone, a transient hyperpolarization which lasted for several minutes was seen. Its time course was similar to that of the hyperpolarization following a climbing fibre response. Except for this, there were no associated changes in the membrane potential, the input resistance, or the magnitudes of climbing fibre responses in any of the cases mentioned in 5 and 6 above.

In cerebellar Purkinje cells, conjunctive stimulation of parallel fibers and the climbing fiber causes longterm depression of parallel fiber-Purkinje cell transmission. It has been postulated that calcium is an intracellular mediator of the climbing fiber to induce this synaptic modification. To directly test the hypothesis, a calcium-chelating agent, EGTA, was intracellularly injected into Purkinle cells. In these injected cells, conjunctive stimulation failed to induce depression. Instead, it caused potentiation similar to that observed after repetitive stimulation of parallel fibers alone.A unique and characteristic feature of the cerebellar cortical network structure is that the final output neuron, the Purkinje cell (PC), receives two distinct types of excitatory afferents: numerous parallel fibers (PFs), which are axons of cerebellar granule cells; and a single but powerful climbing fiber (CF), which derives from the inferior olive (1, 2). Synapses supplied by PFs to PCs exhibit prominent activity-dependent modifiability. A long-term depression (LTD) is induced by conjunctive stimulation of PFs with a CF (3-5), whereas a potentiation is produced by repetitive stimulation of PFs alone (5). This modifiability of PF-PC synapses would explain the role of the cerebellum in motor learning (1,6).Involvement of calcium in induction of LTD has been postulated (1,(3)(4)(5) from the findings that the CF response is associated with a large amount of calcium influx to PCs (7-9) and that strong postsynaptic inhibition of PC dendrites, which presumably depresses the calcium influx, prevented the induction of LTD (4). However, there has been no direct evidence for this hypothesis. Here, I report that when EGTA was injected into PCs, conjunctive CF-PF activation no longer produced LTD but, rather, elicited a potentiation. This observation suggests that calcium chelation by EGTA cancels the action ofCF impulses in inducing LTD, consequently leaving the same potentiating effect as with PF stimulation alone (5). The present results provide direct evidence for a role of calcium in induction of LTD. MATERIALS AND METHODSParasagittal slices (each 300-330 ,um thick) were prepared from guinea pig cerebellar vermis and maintained in vitro as described (5). Slices were prepared using a Microslicer (Dosaka EM, Kyoto) and preincubated for >1 hr in a standard medium containing 124 mM NaCl, 5 mM KCl, 1.24 mM KH2PO4, 1.3 mM MgSO4, 1.24 mM CaC12, 26 mM NaHCO3, and 10 mM glucose and equilibrated with a 95% 02/5% CO2 gas mixture. The temperature was kept constant at 360C.Intracellular recordings were made from PC somata or proximal dendrites. PCs were identified by their characteristic CF responses and/or antidromic spike responses (2). A stimulating electrode was placed at the superficial molecular layer for stimulating PFs with positive-negative current pulses. Another stimulating electrode was inserted into white matter for stimulating CUs. Since stimulation of the white matter often elicited inhibitory postsynaptic potentials...

The corticospinal (CS) tract is essential for voluntary movement, but what we know about the organization and development of the CS tract remains limited. To determine the total cortical area innervating the seventh cervical spinal cord segment (C7), which controls forelimb movement, we injected a retrograde tracer (fluorescent microspheres) into C7 such that it would spread widely within the unilateral gray matter (to Ͼ80%), but not to the CS tract. Subsequent detection of the tracer showed that, in both juvenile and adult mice, neurons distributed over an unexpectedly broad portion of the rostral two-thirds of the cerebral cortex converge to C7. This even included cortical areas controlling the hindlimbs (the fourth lumbar segment, L4). With aging, cell densities greatly declined, mainly due to axon branch elimination. Whole-cell recordings from spinal cord cells upon selective optogenetic stimulation of CS axons, and labeling of axons (DsRed) and presynaptic structures (synaptophysin) through cotransfection using exo utero electroporation, showed that overgrowing CS axons make synaptic connections with spinal cells in juveniles. This suggests that neuronal circuits involved in the CS tract to C7 are largely reorganized during development. By contrast, the cortical areas innervating L4 are limited to the conventional hindlimb area, and the cell distribution and density do not change during development. These findings call for an update of the traditional notion of somatotopic CS projection and imply that there are substantial developmental differences in the cortical control of forelimb and hindlimb movements, at least in rodents.

Two opposite types of synaptic plasticity in the CA1 hippocampus, long-term potentiation (LTP) and long-term depression (LTD), require postsynaptic Ca2+ elevation. To explain these apparently contradictory phenomena, the current view assumes that a moderate postsynaptic increase in Ca2+ leads to LTD, whereas a large increase leads to LTP. No detailed study has so far been attempted to investigate whether the instantaneous Ca2+ elevation level differentially induces LTP or LTD. We therefore used low-frequency (1 Hz) stimulation of Schaffer collateral/commissural fibers in rat hippocampal slices, during a Mg2+-free period, as the conditioning stimulus to investigate this. This allowed low-frequency afferent stimulation to cause a postsynaptic Ca2+ influx because the voltage-dependent block of N-methyl-D-aspartate (NMDA) receptor-channels by Mg2+ was removed. When delivered during the Mg2+-free period, a single pulse, as well as 2-600 pulses, induced LTP that was occluded with tetanus-induced LTP. To decrease the Ca2+ influx, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors were completely blocked by the addition of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the conditioning medium, in which 1 Hz afferent stimuli (1-600 pulses) induced less LTP and never induced LTD. To further reduce the Ca2+ influx, NMDA receptors were partially blocked with D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5). A small number of 1 Hz stimuli, however, never induced LTD. Only when the conditioning stimuli exceeded 200 pulses was LTD induced. The present findings provide definitive evidence that protracted conditioning is a prerequisite for the induction of LTD. Thus, not only the amplitude but also the duration of postsynaptic Ca2+ elevation could be essential factors for differentially inducing LTP or LTD.

Purpose To evaluate choroidal circulatory changes in diabetic patients with and without background diabetic retinopathy (BDR) by

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Objectives-To investigate whether interstitial collagenase (MMP-1) concentration in synovial fluid can be useful as a marker for disease activity in rheumatoid arthritis (RA), to determine the main route by which collagenase degrades the matrix of articular cartlage, and to investigate if an imbalance between metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP) is responsible for the activity ofMMPs in RA. Methods-Collagenase concentrations were measured in synovial fluid and paired serum samples using a specific sandwich enzyme linked immunosorbent assay. Coliagenase activities were also assayed in synovial fluid samples. Synovial tissues obtained from the same patient were examined by inmunohistochemical staining and the numbers of cells expressing collagenase were counted.