The optogenetic revolution in cerebellar investigations

The cerebellum is most renowned for its role in sensorimotor control and coordination, but a growing number of anatomical and physiological studies are demonstrating its deep involvement in cognitive and emotional functions. Recently, the development and refinement of optogenetic techniques boosted research in the cerebellar field and, impressively, revolutionized the methodological approach and endowed the investigations with entirely new capabilities. This translated into a significant improvement in the data acquired for sensorimotor tests, allowing one to correlate single-cell activity with motor behavior to the extent of determining the role of single neuronal types and single connection pathways in controlling precise aspects of movement kinematics. These levels of specificity in correlating neuronal activity to behavior could not be achieved in the past, when electrical and pharmacological stimulations were the only available experimental tools. The application of optogenetics to the investigation of the cerebellar role in higher-order and cognitive functions, which involves a high degree of connectivity with multiple brain areas, has been even more significant. It is possible that, in this field, optogenetics has changed the game, and the number of investigations using optogenetics to study the cerebellar role in non-sensorimotor functions in awake animals is growing. The main issues addressed by these studies are the cerebellar role in epilepsy (through connections to the hippocampus and the temporal lobe), schizophrenia and cognition, working memory for decision making, and social behavior. It is also worth noting that optogenetics opened a new perspective for cerebellar neurostimulation in patients (e.g., for epilepsy treatment and stroke rehabilitation), promising unprecedented specificity in the targeted pathways that could be either activated or inhibited

Gating of Long-Term Potentiation by Nicotinic Acetylcholine Receptors at the Cerebellum Input Stage

The brain needs mechanisms able to correlate plastic changes with local circuit activity and internal functional states. At the cerebellum input stage, uncontrolled induction of long-term potentiation or depression (LTP or LTD) between mossy fibres and granule cells can saturate synaptic capacity and impair cerebellar functioning, which suggests that neuromodulators are required to gate plasticity processes. Cholinergic systems innervating the cerebellum are thought to enhance procedural learning and memory. Here we show that a specific subtype of acetylcholine receptors, the α7-nAChRs, are distributed both in cerebellar mossy fibre terminals and granule cell dendrites and contribute substantially to synaptic regulation. Selective α7-nAChR activation enhances the postsynaptic calcium increase, allowing weak mossy fibre bursts, which would otherwise cause LTD, to generate robust LTP. The local microperfusion of α7-nAChR agonists could also lead to in vivo switching of LTD to LTP following sensory stimulation of the whisker pad. In the cerebellar flocculus, α7-nAChR pharmacological activation impaired vestibulo-ocular-reflex adaptation, probably because LTP was saturated, preventing the fine adjustment of synaptic weights. These results show that gating mechanisms mediated by specific subtypes of nicotinic receptors are required to control the LTD/LTP balance at the mossy fibre-granule cell relay in order to regulate cerebellar plasticity and behavioural adaptation

Exacerbation of experimental autoimmune encephalomyelitis in prion protein (PrPc)-null mice: evidence for a critical role of the central nervous system

<p>Abstract</p> <p>Background</p> <p>The cellular prion protein (PrPc) is a host-encoded glycoprotein whose transconformation into PrP scrapie (PrPSc) initiates prion diseases. The role of PrPc in health is still obscure, but many candidate functions have been attributed to the protein, both in the immune and the nervous systems. Recent data show that experimental autoimmune encephalomyelitis (EAE) is worsened in mice lacking PrPc. Disease exacerbation has been attributed to T cells that would differentiate into more aggressive effectors when deprived of PrPc. However, alternative interpretations such as reduced resistance of neurons to autoimmune insult and exacerbated gliosis leading to neuronal deficits were not considered.</p> <p>Method</p> <p>To better discriminate the contribution of immune cells versus neural cells, reciprocal bone marrow chimeras with differential expression of PrPc in the lymphoid or in the central nervous system (CNS) were generated. Mice were subsequently challenged with MOG_35-55peptide and clinical disease as well as histopathology were compared in both groups. Furthermore, to test directly the T cell hypothesis, we compared the encephalitogenicity of adoptively transferred PrPc-deficient versus PrPc-sufficient, anti-MOG T cells.</p> <p>Results</p> <p>First, EAE exacerbation in PrPc-deficient mice was confirmed. Irradiation exacerbated EAE in all the chimeras and controls, but disease was more severe in mice with a PrPc-deleted CNS and a normal immune system than in the reciprocal construction. Moreover, there was no indication that anti-MOG responses were different in PrPc-sufficient and PrPc-deficient mice. Paradoxically, PrPc-deficient anti-MOG 2D2 T cells were less pathogenic than PrPc-expressing 2D2 T cells.</p> <p>Conclusions</p> <p>In view of the present data, it can be concluded that the origin of EAE exacerbation in PrPc-ablated mice resides in the absence of the prion protein in the CNS. Furthermore, the absence of PrPc on both neural and immune cells does not synergize for disease worsening. These conclusions highlight the critical role of PrPc in maintaining the integrity of the CNS in situations of stress, especially during a neuroinflammatory insult.</p

Role of the Cellular Prion Protein in Oligodendrocyte Precursor Cell Proliferation and Differentiation in the Developing and Adult Mouse CNS

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrPc) to this process remains unclear. PrPc is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrPc influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrPc proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrPc knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells

Disrupted Calcium Signaling in Animal Models of Human Spinocerebellar Ataxia (SCA)

Spinocerebellar ataxias (SCAs) constitute a heterogeneous group of more than 40 autosomal-dominant genetic and neurodegenerative diseases characterized by loss of balance and motor coordination due to dysfunction of the cerebellum and its efferent connections. Despite a well-described clinical and pathological phenotype, the molecular and cellular events that underlie neurodegeneration are still poorly undaerstood. Emerging research suggests that mutations in SCA genes cause disruptions in multiple cellular pathways but the characteristic SCA pathogenesis does not begin until calcium signaling pathways are disrupted in cerebellar Purkinje cells. Ca2+ signaling in Purkinje cells is important for normal cellular function as these neurons express a variety of Ca2+ channels, Ca2+-dependent kinases and phosphatases, and Ca2+-binding proteins to tightly maintain Ca2+ homeostasis and regulate physiological Ca2+-dependent processes. Abnormal Ca2+ levels can activate toxic cascades leading to characteristic death of Purkinje cells, cerebellar atrophy, and ataxia that occur in many SCAs. The output of the cerebellar cortex is conveyed to the deep cerebellar nuclei (DCN) by Purkinje cells via inhibitory signals; thus, Purkinje cell dysfunction or degeneration would partially or completely impair the cerebellar output in SCAs. In the absence of the inhibitory signal emanating from Purkinje cells, DCN will become more excitable, thereby affecting the motor areas receiving DCN input and resulting in uncoordinated movements. An outstanding advantage in studying the pathogenesis of SCAs is represented by the availability of a large number of animal models which mimic the phenotype observed in humans. By mainly focusing on mouse models displaying mutations or deletions in genes which encode for Ca2+ signaling-related proteins, in this review we will discuss the several pathogenic mechanisms related to deranged Ca2+ homeostasis that leads to significant Purkinje cell degeneration and dysfunctio

Nicotinic receptor activation increases glutamatergic transmission and plasticity in the rat cerebellum

Neuromodulatory systems of the brain have been suggested to profoundly impact on neurotransmission and long-term synaptic plasticity, the cellular correlate for learning and memory. The cerebellum, involved in procedural memory, receives abundant cholinergic innervation and shows a dense nicotinic acetylcholine receptor (nAChRs) expression. However, the functional effects of nAChRs in the cerebellum are still largely unknown. To address this issue we have performed voltage-clamp recordings in whole cell configuration in the granular layer of acute slices obtained from the cerebellar vermis of P18-P22 rats. A 100-sec application of nicotine (1M) significantly enhanced glutamatergic EPSCs. The effect was transient, suggesting that nAChR were progressively desensitizing.AsnAChRsareoftenlocatedinthepresynaptic terminals where they modulate other neurotransmitter release we have therefore investigated whether a similar mechanism could operate in the cerebellum. EPSCs mediated by AMPA receptors were elicited in pairs with an interpulse interval of 20ms. Nicotine exposure readily caused a reduction of the pair pulse ratio (PPR). Moreover, a high calcium buffer concentration in the intracellular solution was still accompanied by a significant PPR decrease during nicotine application supporting its presynaptic origin. EPSCs mediated by NMDA receptors were not influenced by nicotine. Interestingly, when a high calcium buffer concentration was added to the intracellular solution, the effect of nicotine was restored and NMDA EPSCs increased. Therefore, nicotine could act both pre- and postsynaptically. The enhancement of neurotransmission caused by nicotine suggested that nicotine could also enhance the induction of LTP. We therefore tested whether a single100ms/100Hzburst,whichdeterminesalong-termdepression of EPSC peak could turn into LTP induction in the presence of nicotine. Exposure to 1M nicotine led the development of LTP of the EPSCs following the 100ms/100Hz burst. To explore which nAChR subtype mediated the facilitating effect of nicotine on LTP, recordings were performed in the presence of 7 nAChR agonist and antagonist: choline (10 mM) and MLA (100 nM), respectively. The application of choline (100 s) increased the EPSC and then a single 100 ms/100 Hz burst led to LTP. The co-application of nicotine with MLA (100 s) prevented switching form LTD to LTP. These results suggest that cholinergic stimulation mediated by nAChRs markedly potentiates synaptic transmission and long term synaptic plasticity along the mossy fibre pathway of the cerebellum

Diverse Neuron Properties and Complex Network Dynamics in the Cerebellar Cortical Inhibitory Circuit

Neuronal inhibition can be defined as a spatiotemporal restriction or suppression of local microcircuit activity. The importance of inhibition relies in its fundamental role in shaping signal processing in single neurons and neuronal circuits. In this context, the activity of inhibitory interneurons proved the key to endow networks with complex computational and dynamic properties. In the last 50 years, the prevailing view on the functional role of cerebellar cortical inhibitory circuits was that excitatory and inhibitory inputs sum spatially and temporally in order to determine the motor output through Purkinje cells (PCs). Consequently, cerebellar inhibition has traditionally been conceived in terms of restricting or blocking excitation. This assumption has been challenged, in particular in the cerebellar cortex where all neurons except granule cells (and unipolar brush cells in specific lobules) are inhibitory and fire spontaneously at high rates. Recently, a combination of electrophysiological recordings in vitro and in vivo, imaging, optogenetics and computational modeling, has revealed that inhibitory interneurons play a much more complex role in regulating cerebellar microcircuit functions: inhibition shapes neuronal response dynamics in the whole circuit and eventually regulate the PC output. This review elaborates current knowledge on cerebellar inhibitory interneurons [Golgi cells, Lugaro cells (LCs), basket cells (BCs) and stellate cells (SCs)], starting from their ontogenesis and moving up to their morphological, physiological and plastic properties, and integrates this knowledge with that on the more renown granule cells and PCs. We will focus on the circuit loops in which these interneurons are involved and on the way they generate feed-forward, feedback and lateral inhibition along with complex spatio-temporal response dynamics. In this perspective, inhibitory interneurons emerge as the real controllers of cerebellar functioning

Evidence for long-term synaptic plasticity at the mossy fiber - Golgi cell synapse of cerebellum

Programme and Abstracts of the 66th National Congress of the Italian Physiological Society (Società Italiana di Fisiologia

Late-onset bursts evoked by mossy fiber bundle stimulation in unipolar brush cells: evidence for the involvement of H- and TRP-currents

Synaptic transmission at central synapses has usually short latency and graded amplitude, thereby regulating threshold crossing and the probability of action potential generation. In the granular layer of vestibulo-cerebellum, the unipolar brush cells (UBCs) receive a giant synapse generating a stereotyped EPSP-burst complex with early-onset (~ 2 ms) and high reliability. By using patch-clamp recordings in cerebellar slices of the rat vestibulo-cerebellum, we found that mossy fiber bundle stimulation also evoked (in ~80% of cases) a late-onset burst (after tens to hundreds milliseconds) independent from EPSP generation. Different from the early-onset, the late-onset burst delay decreased and its duration increased by raising stimulation intensity or the number of impulses. Though depending on synaptic activity, the late-onset response was insensitive to APV, NBQX and MCPG perfusion and did not therefore depend on conventional glutamatergic transmission mechanisms. The late-onset response was initiated by a slow depolarizing ramp driven by activation of an H-current (sensitive to ZD7288- and Cs+) and of a TRP-current (sensitive to SKF96365), while the HVA and LVA Ca2+-currents (sensitive to nimodipine and mibefradil) played a negligible role. The late-onset burst was occluded by intracellular cAMP. These results indicate that afferent activity can regulate H- and TRP-current gating in UBCs generating synaptically-driven EPSP-independent responses, in which the delay rather than amplitude is graded with the intensity of the input pattern. This modality of synaptic transmission may play an important role for regulating UBC activation and granular layer functions in the vestibulo-cerebellu

Presynaptic current changes at the mossy fiber–granule cell synapse of cerebellum during LTP

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