Egidio D'Angelo

Synaptic excitation of individual rat cerebellar granule cells in situ: evidence for the role of NMDA receptors.

1. Current‐clamp recordings were made in whole‐cell patch‐clamp configuration from ninety‐one granule cells in parasagittal cerebellar slices obtained from 21‐ to 31‐day‐old rats. Recordings were performed at 30 degrees C. 2. Resting membrane potential was ‐58 +/‐ 6 mV (n = 43). The membrane voltage response to step current injection showed inward rectification consistent with increasing input resistance during membrane depolarization. Over ‐35 +/‐ 7 mV (n = 14) repetitive firing with little or no adaptation was activated. Spike frequency increased nearly linearly with injected current. 3. Unitary EPSPs obtained by stimulating the mossy fibre bundle had an amplitude of 11.4 +/‐ 2.1 mV (n = 22, holding potential = ‐75 mV). Synchronous activation of greater than one to two mossy fibres was needed to elicit action potentials. Antidromic stimulation elicited antidromic spikes and also EPSPs, presumably through a mossy fibre ‘axon reflex’. 4. EPSPs were brought about by NMDA and non‐NMDA receptor activation, accounting for about 70 and 30%, respectively, of peak amplitude at the holding potential of ‐70 mV. The EPSP decay conformed to passive membrane discharge after blocking the NMDA receptors. 5. No appreciable correlation was found between the time‐to‐peak and decay time constant of the EPSPs, consistent with the compact electrotonic structure of these neurons. 6. During membrane depolarization EPSP amplitude increased transiently, due to both a voltage‐dependent increase of the NMDA component and inward rectification. In addition, EPSPs slowed down due to a slowdown of the NMDA component. 7. Temporal summation during high‐frequency stimulation was sustained by NMDA receptors, whose contribution to depolarization tended to prevail over that of non‐NMDA receptors during the trains. A block of the NMDA receptors resulted in reduced depolarization and output spike frequency. 8. This study, as well as extending previous knowledge to the intracellular level in vivo, provides evidence for a primary role of NMDA receptors in determining mossy fibre excitation of granule cells. It is suggested that the marked voltage dependence of the EPSP time course, which was mainly caused by voltage dependence in NMDA conductance, promotes the NMDA receptor‐dependent enhancement of granule cell coding observed during repetitive mossy fibre activity.

Seeking a unified framework for cerebellar function and dysfunction: from circuit operations to cognition

Following the fundamental recognition of its involvement in sensory-motor coordination and learning, the cerebellum is now also believed to take part in the processing of cognition and emotion. This hypothesis is recurrent in numerous papers reporting anatomical and functional observations, and it requires an explanation. We argue that a similar circuit structure in all cerebellar areas may carry out various operations using a common computational scheme. On the basis of a broad review of anatomical data, it is conceivable that the different roles of the cerebellum lie in the specific connectivity of the cerebellar modules, with motor, cognitive, and emotional functions (at least partially) segregated into different cerebro-cerebellar loops. We here develop a conceptual and operational framework based on multiple interconnected levels (a meta-levels hypothesis): from cellular/molecular to network mechanisms leading to generation of computational primitives, thence to high-level cognitive/emotional processing, and finally to the sphere of mental function and dysfunction. The main concept explored is that of intimate interplay between timing and learning (reminiscent of the “timing and learning machine” capabilities long attributed to the cerebellum), which reverberates from cellular to circuit mechanisms. Subsequently, integration within large-scale brain loops could generate the disparate cognitive/emotional and mental functions in which the cerebellum has been implicated. We propose, therefore, that the cerebellum operates as a general-purpose co-processor, whose effects depend on the specific brain centers to which individual modules are connected. Abnormal functioning in these loops could eventually contribute to the pathogenesis of major brain pathologies including not just ataxia but also dyslexia, autism, schizophrenia, and depression.

Increased neurotransmitter release during long-term potentiation at mossy fibre–granule cell synapses in rat cerebellum

During long‐term potentiation (LTP) at mossy fibre–granule cell synapses in rat cerebellum synaptic transmission and granule cell intrinsic excitability are enhanced. Although it is clear that changes in granule cell excitability are mediated postsynaptically, there is as yet no direct evidence for the site and mechanism of changes in transmission. To approach this problem, evoked postsynaptic currents (EPSCs) and miniature synaptic currents (mEPSCs) were recorded by patch‐clamp in cerebellar slices obtained from P17–P23 rats. LTP was induced by theta‐burst stimulation paired with depolarization. During LTP, the EPSCs showed a significant decrease in the coefficient of variation (CV; 28.9 ± 5.2%, n= 8; P < 0.002), the number of failures (87.1 ± 41.9%, n= 8; P < 0.04), and the paired‐pulse ratio (PPR; 25.5 ± 4.1%n= 5; P < 0.02). Similar changes were observed by increasing neurotransmitter release (extracellular solutions with high Ca2+/Mg2+ ratio), whereas increases in CV, numbers of failures and PPR occurred when release was decreased (extracellular solutions with low Ca2+/Mg2+ ratio; 10 μm Cl‐adenosine). No changes followed modifications of postsynaptic holding potentials, while CV and failures were reduced when the number of active synapses was increased. LTP was prevented by use of solutions with high Ca2+/Mg2+ ratio. Moreover, LTP and the associated CV decrease were observed in the spillover‐mediated component of AMPA EPSCs and in NMDA EPSCs. During LTP, mEPSCs did not change in amplitude or variability but significantly increased in frequency (47.6 ± 16%, n= 4; P < 0.03). By binomial analysis changes in EPSCs were shown to be due to increased release probability (from 0.6 ± 0.08 to 0.73 ± 0.06, n= 7; P < 0.02) with a constant number of three to four releasing sites. These observations provide evidence for increased neurotransmitter release during LTP at mossy fibre–granule cell synapses.

The spatial organization of long-term synaptic plasticity at the input stage of cerebellum.

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.

Different proportions of N-methyl-d-aspartate and non-N-methyl-d-aspartate receptor currents at the mossy fibre-granule cell synapse of developing rat cerebellum

The mossy fibre-granule cell synapse undergoes major developmental changes during the second and third weeks after birth. We investigated synaptic transmission during postnatal days 10-22 by means of whole-cell patch-clamp recordings from granule cells in situ. Parasagittal slices were cut from rat cerebellar vermis, and excitatory postsynaptic currents were evoked in granule cells by mossy fibre stimulation with 1.2 mM Mg++ in the extracellular solution. In the majority of granule cells recorded at postnatal days 16-22, excitatory currents were characterized by a fast initial peak followed by a slower component, while in many of the cells recorded at more immature stages, the fast peak was virtually absent. Pharmacological and kinetic data indicated that the fast and slow components were mediated by non-N-methyl-D-aspartate and N-methyl-D-aspartate receptor activation, respectively. The magnitude of the non-N-methyl-D-aspartate current increased with developmental age, while the magnitude of the NMDA current did not change markedly. The age-dependent change of the non-N-methyl-D-aspartate currents could not be accounted for by changes in recording conditions or granule cell electrotonic properties. Furthermore, from postnatal day 11 to 16 the extent of Mg++ block on the N-methyl-D-aspartate receptor did not change, and could not explain the increasing non-N-methyl-D-aspartate/N-methyl-D-aspartate current ratio. We concluded therefore that the age-dependent increase of the non-N-methyl-D-aspartate current was the main cause of the different postsynaptic current waveforms observed at different ages. The developmental change in the proportion of N-methyl-D-aspartate and non-N-methyl-D-aspartate currents may be relevant to the processes regulating granule cell maturation and excitability.

Ionic mechanisms of autorhythmic firing in rat cerebellar Golgi 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 GABAB receptors, thus behaving, in our conditions, as pacemaker neurons. ZD 7288 (20 μm), a potent hyperpolarization‐activated current (Ih) blocker, slowed down pacemaker frequency. The role of subthreshold Na+ currents (INa,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+ (IM) currents, reduced GoC excitability in the threshold region. The KCNQ channel antagonist XE991 (5 μm) did not modify firing, suggesting that GoC IM has low XE991 sensitivity. Spike repolarization was followed by an after‐hyperpolarization (AHP) supported by apamin‐sensitive Ca2+‐dependent K+ currents (Iapa). Block of Iapa decreased pacemaker precision without altering average frequency. We propose that feed‐forward depolarization is sustained by Ih and INa,sub, and that delayed repolarizing feedback involves an IM‐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.

Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.

A realistic large-scale model of the cerebellum granular layer predicts circuit spatio-temporal filtering properties

The way the cerebellar granular layer transforms incoming mossy fiber signals into new spike patterns to be related to Purkinje cells is not yet clear. Here, a realistic computational model of the granular layer was developed and used to address four main functional hypotheses: center-surround organization, time-windowing, high-pass filtering in responses to spike bursts and coherent oscillations in response to diffuse random activity. The model network was activated using patterns inspired by those recorded in vivo. Burst stimulation of a small mossy fiber bundle resulted in granule cell bursts delimited in time (time windowing) and space (center-surround) by network inhibition. This burst–burst transmission showed marked frequency-dependence configuring a high-pass filter with cut-off frequency around 100 Hz. The contrast between center and surround properties was regulated by the excitatory–inhibitory balance. The stronger excitation made the center more responsive to 10–50 Hz input frequencies and enhanced the granule cell output (with spikes occurring earlier and with higher frequency and number) compared to the surround. Finally, over a certain level of mossy fiber background activity, the circuit generated coherent oscillations in the theta-frequency band. All these processes were fine-tuned by NMDA and GABA-A receptor activation and neurotransmitter vesicle cycling in the cerebellar glomeruli. This model shows that available knowledge on cellular mechanisms is sufficient to unify the main functional hypotheses on the cerebellum granular layer and suggests that this network can behave as an adaptable spatio-temporal filter coordinated by theta-frequency oscillations.

Intracellular calcium regulation by burst discharge determines bidirectional long-term synaptic plasticity at the cerebellum input stage.

Variations in intracellular calcium concentration ([Ca2+]i) provide a critical signal for synaptic plasticity. In accordance with Hebbs postulate (Hebb, 1949), an increase in postsynaptic [Ca2+]i can induce bidirectional changes in synaptic strength depending on activation of specific biochemical pathways (Bienenstock et al., 1982; Lisman, 1989; Stanton and Sejnowski, 1989). Despite its strategic location for signal processing, spatiotemporal dynamics of [Ca2+]i changes and their relationship with synaptic plasticity at the cerebellar mossy fiber (mf)-granule cell (GrC) relay were unknown. In this paper, we report the plasticity/[Ca2+]i relationship for GrCs, which are typically activated by mf bursts (Chadderton et al., 2004). Mf bursts caused a remarkable [Ca2+]i increase in GrC dendritic terminals through the activation of NMDA receptors, metabotropic glutamate receptors (probably acting through IP3-sensitive stores), voltage-dependent calcium channels, and Ca2+-induced Ca2+ release. Although [Ca2+]i increased with the duration of mf bursts, long-term depression was found with a small [Ca2+]i increase (bursts <250 ms), and long-term potentiation (LTP) was found with a large [Ca2+]i increase (bursts >250 ms). LTP and [Ca2+]i saturated for bursts >500 ms and with theta-burst stimulation. Thus, bursting enabled a Ca2+-dependent bidirectional Bienenstock-Cooper-Munro-like learning mechanism providing the cellular basis for effective learning of burst patterns at the input stage of the cerebellum.

Timing in the cerebellum: oscillations and resonance in the granular layer.

The brain generates many rhythmic activities, and the olivo-cerebellar system is not an exception. In recent years, the cerebellum has revealed activities ranging from low frequency to very high-frequency oscillations. These rhythms depend on the brain functional state and are typical of certain circuit sections or specific neurons. Interestingly, the granular layer, which gates sensorimotor and cognitive signals to the cerebellar cortex, can also sustain low frequency (7-25 Hz) and perhaps higher-frequency oscillations. In this review we have considered (i) how these oscillations are generated in the granular layer network depending on intrinsic electroresponsiveness and circuit connections, (ii) how these oscillations are correlated with those in other cerebellar circuit sections, and (iii) how the oscillating cerebellum communicates with extracerebellar structures. It is suggested that the granular layer can generate oscillations that integrate well with those generated in the inferior olive, in deep-cerebellar nuclei and in Purkinje cells. These rhythms, in turn, might play a role in cognition and memory consolidation by interacting with the mechanisms of long-term synaptic plasticity.