The spatial organization of long-term synaptic plasticity [long-term potentiation (LTP) and long-term depression (LTD)] is supposed to play a critical role for distributed signal processing in neuronal networks, but its nature remains undetermined in most central circuits. By using multielectrode array recordings, we have reconstructed activation maps of the granular layer in cerebellar slices. LTP and LTD induced by theta-burst stimulation appeared in patches organized in such a way that, on average, LTP was surrounded by LTD. The sign of long-term synaptic plasticity in a given granular layer region was directly correlated with excitation and inversely correlated with inhibition: the most active areas tended to generate LTP, whereas the least active areas tended to generate LTD. Plasticity was almost entirely prevented by application of the NMDA receptor blocker, APV. This suggests that synaptic inhibition, through a control of membrane depolarization, effectively regulates NMDA channel unblock, postsynaptic calcium entry, and the induction of bidirectional synaptic plasticity at the mossy fiber-granule cell relay (Gall et al., 2005). By this mechanism, LTP and LTD could regulate the geometry and contrast of network computations, preprocessing the mossy fiber input to be conveyed to Purkinje cells and molecular layer interneurons.

Long-term potentiation (LTP) is a synaptic change supposed to provide the cellular basis for learning and memory in brain neuronal circuits. Although specific LTP expression mechanisms could be critical to determine the dynamics of repetitive neurotransmission, this important issue remained largely unexplored. In this paper, we have performed whole cell patch-clamp recordings of mossy fiber-granule cell LTP in acute rat cerebellar slices and studied its computational implications with a mathematical model. During LTP, stimulation with short impulse trains at 100 Hz revealed earlier initiation of granule cell spike bursts and a smaller nonsignificant spike frequency increase. In voltage-clamp recordings, short AMPA excitatory postsynaptic current (EPSC) trains showed short-term facilitation and depression and a sustained component probably generated by spillover. During LTP, facilitation disappeared, depression accelerated, and the sustained current increased. The N-methyl-d-aspartate (NMDA) current also increased. In agreement with a presynaptic expression caused by increased release probability, similar changes were observed by raising extracellular [Ca(2+)]. A mathematical model of mossy fiber-granule cell neurotransmission showed that increasing release probability efficiently modulated the first-spike delay. Glutamate spillover, by causing tonic NMDA and AMPA receptor activation, accelerated excitatory postsynaptic potential (EPSP) temporal summation and maintained a sustained spike discharge. The effect of increasing neurotransmitter release could not be replicated by increasing receptor conductance, which, like postsynaptic manipulations enhancing intrinsic excitability, proved very effective in raising granule cell output frequency. Independent regulation of spike burst initiation and frequency during LTP may provide mechanisms for temporal recoding and gain control of afferent signals at the input stage of cerebellar cortex.

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.

Signal elaboration in the cerebellum mossy fiber input pathway presents controversial aspects, especially concerning gain regulation and the spot-like (rather than beam-like) appearance of granular to molecular layer transmission. By using voltage-sensitive dye imaging in rat cerebellar slices (Mapelli et al., 2010), we found that mossy fiber bursts optimally excited the granular layer above ∼50 Hz and the overlaying molecular layer above ∼100 Hz, thus generating a cascade of high-pass filters. NMDA receptors enhanced transmission in the granular, while GABA-A receptors depressed transmission in both the granular and molecular layer. Burst transmission gain was controlled through a dynamic frequency-dependent involvement of these receptors. Moreover, while high-frequency transmission was enhanced along vertical lines connecting the granular to molecular layer, no high-frequency enhancement was observed along the parallel fiber axis in the molecular layer. This was probably due to the stronger effect of Purkinje cell GABA-A receptor-mediated inhibition occurring along the parallel fibers than along the granule cell axon ascending branch. The consequent amplification of burst responses along vertical transmission lines could explain the spot-like activation of Purkinje cells observed following punctuate stimulation in vivo.

The granular layer of cerebellum has been long hypothesized to perform combinatorial operations on incoming signals. Although this assumption is at the basis of main computational theories of cerebellum, it has never been assessed experimentally. Here, by applying high-resolution voltage-sensitive dye imaging techniques, we show that simultaneous activation of two partially overlapping mossy fiber bundles (either with single pulses or high-frequency bursts) can cause combined excitation and combined inhibition, which are compatible with the concepts of coincidence detection and spatial pattern separation predicted by theory. Combined excitation appeared as an area in which the combination of two inputs is greater than the arithmetic sum of the individual inputs and was enhanced by gamma-aminobutyric acid type A (GABA(A)) receptor blockers. Combined inhibition was manifest as an area where two inputs combined resulted in a reduction to less than half of the activity evoked from either one of the two inputs alone and was prevented by GABA(A) receptor blockers. The combinatorial responses occupied small granular layer regions (approximately 30 microm diameter), with combined inhibition being interspersed among extended areas of combined excitation. Moreover, the combinatorial effects lasted for tens of milliseconds and combined inhibition occurred only after termination of the stimuli. These combinatorial operations, if engaged by natural input patterns in vivo, may be important to influence incoming impulses organizing spatiotemporal spike sequences to be relayed to Purkinje cells.

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.

Human dental pulp is considered an interesting source of adult stem cells, due to the low-invasive isolation procedures, high content of stem cells and its peculiar embryological origin from neural crest. Based on our previous findings, a dental pulp stem cells sub-population, enriched for the expression of STRO-1, c-Kit, and CD34, showed a higher neural commitment. However, their biological properties were compromised when cells were cultured in adherent standard conditions. The aim of this study was to evaluate the ability of three dimensional floating spheres to preserve embryological and biological properties of this sub-population. In addition, the expression of the inwardly rectifying potassium channel Kir4.1, Fas and FasL was investigated in 3D-sphere derived hDPSCs. Our data showed that 3D sphere-derived hDPSCs maintained their fibroblast-like morphology, preserved stemness markers expression and proliferative capability. The expression of neural crest markers and Kir4.1 was observed in undifferentiated hDPSCs, furthermore this culture system also preserved hDPSCs differentiation potential. The expression of Fas and FasL was observed in undifferentiated hDPSCs derived from sphere culture and, noteworthy, FasL was maintained even after the neurogenic commitment was reached, with a significantly higher expression compared to osteogenic and myogenic commitments. These data demonstrate that 3D sphere culture provides a favorable micro-environment for neural crest-derived hDPSCs to preserve their biological properties.