The Golgi cells have been recently shown to beat regularly in vitro (Forti et al., 2006. J. Physiol. 574, 711-729). Four main currents were shown to be involved, namely a persistent sodium current (I Na-p ), an h current (I h ), an SK-type calcium-dependent potassium current (I K-AHP ), and a slow M-like potassium current (I K-slow ). These ionic currents could take part, together with others, also to different aspects of neuronal excitability like responses to depolarizing and hyperpolarizing current injection. However, the ionic mechanisms and their interactions remained largely hypothetical. In this work, we have investigated the mechanisms of Golgi cell excitability by developing a computational model. The model predicts that pacemaking is sustained by subthreshold oscillations tightly coupled to spikes. I Na-p and I K-slow emerged as the critical determinants of oscillations. I h also played a role by setting the oscillatory mechanism into the appropriate membrane potential range. I K-AHP , though taking part to the oscillation, appeared primarily involved in regulating the ISI following spikes. The combination with other currents, in particular a resurgent sodium current (I Na-r ) and an A-current (I K-A ), allowed a precise regulation of response frequency and delay. These results provide a coherent reconstruction of the ionic mechanisms determining Golgi cell intrinsic electroresponsiveness and suggests important implications for cerebellar signal processing, which will be fully developed in a companion paper (Solinas et al., .

The Golgi cells are inhibitory interneurons of the cerebellar granular layer, which respond to afferent stimulation in vivo with a burst-pause sequence interrupting their irregular background low-frequency firing (Vos et al., 1999a. Eur. J. Neurosci. 11, 2621-2634. However, Golgi cells in vitro are regular pacemakers (Forti et al., 2006. J. Physiol. 574, 711-729), raising the question how their ionic mechanisms could impact on responses during physiological activity. Using patch-clamp recordings in cerebellar slices we show that the pacemaker cycle can be suddenly reset by spikes, making the cell highly sensitive to input variations. Moreover, the neuron resonates around the pacemaker frequency, making it specifically sensitive to patterned stimulation in the theta-frequency band. Computational analysis based spike-triggered activation of SK channels and that resonance was sustained by a slow voltage-dependent potassium current and amplified by a persistent sodium current. Adding balanced synaptic noise to mimic the irregular discharge observed in vivo, we found that pacemaking converts into spontaneous irregular discharge, that phase-reset plays an important role in generating the burst-pause pattern evoked by sensory stimulation, and that repetitive stimulation at theta-frequency enhances the time-precision of spike coding in the burst. These results suggest that Golgi cell intrinsic properties exert a profound impact on time-dependent signal processing in the cerebellar granular layer.

Synapses in the central nervous system are typically studied by recording electrical responses from the cell body of the postsynaptic cell. Because neurons are normally connected by multiple synaptic contacts, these postsynaptic responses reflect the combined activity of many thousands synapses, and it remains unclear to what extent the properties of individual synapses can be deduced from the population response. We have therefore developed a method for recording the activity of individual hippocampal synapses. By capturing an isolated presynaptic bouton inside a loose-patch pipette and recording from the associated patch of postsynaptic membrane, we were able to detect miniature excitatory postsynaptic currents ('minis') arising from spontaneous vesicle exocytosis at a single synaptic site, and to compare these with minis recorded simultaneously from the cell body. The average peak conductance at a single synapse was about 900 pS, corresponding roughly to the opening of 90 AMPA-type glutamate-receptor channels. The variability in this conductance was about 30%, matching the value reported for the neuromuscular junction. Given that our synapses displayed single postsynaptic densities (PSDs), this variability is larger than would be predicted from the random opening of receptor channels, suggesting that they are not saturated by the content of a single vesicle. Therefore the response to a quantum of neurotransmitter at these synapses is not limited by the number of available postsynaptic receptors.

Variations in intracellular calcium concentration ([Ca2ϩ

No abstract

Although Golgi cells (GoCs), the main type of inhibitory interneuron in the cerebellar granular layer (GL), are thought to play a central role in cerebellar network function, their excitable properties have remained unexplored. GoCs fire rhythmically in vivo and in slices, but it was unclear whether this activity originated from pacemaker ionic mechanisms. We explored this issue in acute cerebellar slices from 3-week-old rats by combining loose cell-attached (LCA) and whole-cell (WC) recordings. GoCs displayed spontaneous firing at 1-10 Hz (room temperature) and 2-20 Hz (35-37• C), which persisted in the presence of blockers of fast synaptic receptors and mGluR and GABA B receptors, thus behaving, in our conditions, as pacemaker neurons. ZD 7288 (20 μM), a potent hyperpolarization-activated current (I h ) blocker, slowed down pacemaker frequency. The role of subthreshold Na + currents (I Na,sub ) could not be tested directly, but we observed a robust TTX-sensitive, non-inactivating Na + current in the subthreshold voltage range. When studying repolarizing currents, we found that retigabine (5 μM), an activator of KCNQ K + channels generating neuronal M-type K + (I M ) currents, reduced GoC excitability in the threshold region. The KCNQ channel antagonist XE991 (5 μM) did not modify firing, suggesting that GoC I M has low XE991 sensitivity. Spike repolarization was followed by an after-hyperpolarization (AHP) supported by apamin-sensitive Ca 2+ -dependent K + currents (I apa ). Block of I apa decreased pacemaker precision without altering average frequency. We propose that feed-forward depolarization is sustained by I h and I Na,sub , and that delayed repolarizing feedback involves an I M -like current whose properties remain to be characterized. The multiple ionic mechanisms shown here to contribute to GoC pacemaking should provide the substrate for fine regulation of firing frequency and precision, thus influencing the cyclic inhibition exerted by GoCs onto the cerebellar GL.

The function of inhibitory interneurons within brain microcircuits depends critically on the nature and properties of their excitatory synaptic drive. Golgi cells (GoCs) of the cerebellum inhibit cerebellar granule cells (GrCs) and are driven both by feedforward mossy fiber (mf) and feedback GrC excitation. Here, we have characterized GrC inputs to GoCs in rats and mice. We show that, during sustained mf discharge, synapses from local GrCs contribute equivalent charge to GoCs as mf synapses, arguing for the importance of the feedback inhibition. Previous studies predicted that GrC-GoC synapses occur predominantly between parallel fibers (pfs) and apical GoC dendrites in the molecular layer (ML). By combining EM and Ca 2ϩ imaging, we now demonstrate the presence of functional synaptic contacts between ascending axons (aa) of GrCs and basolateral dendrites of GoCs in the granular layer (GL). Immunohistochemical quantification estimates these contacts to be ϳ400 per GoC. Using Ca 2ϩ imaging to identify synaptic inputs, we show that EPSCs from aa and mf contacts in basolateral dendrites display similarly fast kinetics, whereas pf inputs in the ML exhibit markedly slower kinetics as they undergo strong filtering by apical dendrites. We estimate that approximately half of the local GrC contacts generate fast EPSCs, indicating their basolateral location in the GL. We conclude that GrCs, through their aa contacts onto proximal GoC dendrites, define a powerful feedback inhibitory circuit in the GL.

Axonal [Ca2+] transients evoked by action potential (AP) propagation were studied by monitoring the fluorescence of the high‐affinity calcium‐sensitive dye Oregon Green 488 BAPTA‐1, introduced through whole‐cell recording pipettes in the molecular layer of interneurones from cerebellar slices of young rats. The spatiotemporal profile of Ca2+‐dependent fluorescence changes was analysed in well‐focused axonal stretches a few tens of micrometres long. AP‐evoked Ca2+ signals were heterogeneously distributed along axons, with the largest and fastest responses appearing in hot spots on average ∼5 μm apart. The spatial distribution of fluorescence responses was independent of the position of the focal plane, uncorrelated to basal dye fluorescence, and independent of dye concentration. Recordings using the low‐affinity dye mag‐fura‐2 and a Cs+‐based intracellular solution revealed a similar pattern of hot spots in response to depolarisation, ruling out measurement artefacts or possible effects of inhomogeneous dye distribution in the generation of hot spots. Fluorescence responses to a short train of APs in hot spots decreased by 41–76 % after bath perfusion of ω‐conotoxin MVIIC (5–6 μM), and by 17–65 % after application of ω‐agatoxin IVA (500 nM). ω‐Conotoxin GVIA (1 μM) had a variable, small effect (0–31 % inhibition), and nimodipine (5 μM) had none. Somatically recorded voltage‐gated currents during depolarising pulses were unaffected in all cases. These data indicate that P/Q‐type Ca2+ channels, and to a lesser extent N‐type channels, are responsible for a large fraction of the [Ca2+] rise in axonalhot spots. [Ca2+] responses never failed during low‐frequency (≤ 0.5 Hz) stimulation, indicating reliable AP propagation to the imaged sites. Axonal branching points coincided with a hot spot in ∼50 % of the cases. The spacing of presynaptic varicosities, as determined by a morphological analysis of Neurobiotin‐filled axons, was ∼10 times larger than the one measured for hot spots. The latter is comparable to the spacing reported for varicosities in mature animals. We discuss the nature of hot spots, considering as the most parsimonious explanation that they represent functional clusters of voltage‐dependent Ca2+ channels, and possibly other [Ca2+] sources, marking the position of developing presynaptic terminals before the formation of en passant varicosities.