Diversity of cellular physiology and morphology of Purkinje cells in the adult zebrafish cerebellum.

This study was designed to explore the functional circuitry of the adult zebrafish cerebellum, focusing on its Purkinje cells and using whole-cell patch recordings and single cell labeling in slice preparations. Following physiological characterizations, the recorded single cells were labeled for morphological identification. It was found that the zebrafish Purkinje cells are surprisingly diverse. Based on their physiology and morphology, they can be classified into at least three subtypes: Type I, a narrow spike cell, which fires only narrow Na+ spikes (<3 ms in duration), and has a single primary dendrite with an arbor restricted to the distal molecular layer; Type II, a broad spike cell, which fires broad Ca2+ spikes (5-7 ms in duration) and has a primary dendrite with limited branching in the inner molecular layer and then further radiates throughout the molecular layer; and Type III, a very broad spike cell, which fires very broad Ca2+ spikes (≥10 ms in duration) and has a dense proximal dendritic arbor that is either restricted to the inner molecular layer (Type IIIa), or radiates throughout the entire molecular layer (Type IIIb). The graded paired-pulse facilitation of these Purkinje cells' responses to parallel fiber activations and the all-or-none, paired-pulse depression of climbing fiber activation are largely similar to those reported for mammals. The labeled axon terminals of these Purkinje cells end locally, as reported for larval zebrafish. The present study provides evidence that the corresponding functional circuitry and information processing differ from what has been well-established in the mammalian cerebellum.

Non-synaptic Cell-Autonomous Mechanisms Underlie Neuronal Hyperactivity in a Genetic Model of PIK3CA-Driven Intractable Epilepsy.

Patients harboring mutations in the PI3K-AKT-MTOR pathway-encoding genes often develop a spectrum of neurodevelopmental disorders including epilepsy. A significant proportion remains unresponsive to conventional anti-seizure medications. Understanding mutation-specific pathophysiology is thus critical for molecularly targeted therapies. We previously determined that mouse models expressing a patient-related activating mutation in PIK3CA, encoding the p110α catalytic subunit of phosphoinositide-3-kinase (PI3K), are epileptic and acutely treatable by PI3K inhibition, irrespective of dysmorphology. Here we report the physiological mechanisms underlying this dysregulated neuronal excitability. In vivo, we demonstrate epileptiform events in the Pik3ca mutant hippocampus. By ex vivo analyses, we show that Pik3ca-driven hyperactivation of hippocampal pyramidal neurons is mediated by changes in multiple non-synaptic, cell-intrinsic properties. Finally, we report that acute inhibition of PI3K or AKT, but not MTOR activity, suppresses the intrinsic hyperactivity of the mutant neurons. These acute mechanisms are distinct from those causing neuronal hyperactivity in other AKT-MTOR epileptic models and define parameters to facilitate the development of new molecularly rational therapeutic interventions for intractable epilepsy.

[Neurobiological mechanisms of autistic savant and acquired savant].

The autism spectrum is a pervasive developmental disorder characterized by profound social and verbal communication deficits, stereotypical motor behaviors, restricted interests, and cognitive abnormalities. It affects approximately 1% of children in most of the reported nations and regions. One of the most fascinating and mysterious features of autism, however, is the remarkable talent frequently found in people affected by it, namely autistic savant. A parallel and equally mysterious phenomenon is that some otherwise normal and ordinary individuals develop similarly remarkable talent after brain injuries, a disorder known as acquired savant. After decades of intensive investigation, significant progress has been made in these fields. Current studies indicate that autistic savant and acquired savant are neuropathologically related, and these disorders share many neurobiological mechanisms. This review summarizes current knowledge of autism and both two savant types, and how it may aid our understanding of higher brain functionalities.

Cell type-specific plasticity at parallel fiber synapses onto Purkinje cells in the posterior caudal lobe of the mormyrid fish cerebellum.

It has been demonstrated that there are two morphological subtypes of Purkinje cells (PCs)-fan-shaped Purkinje cells (fPCs) and multipolar Purkinje cells (mPCs)-in the posterior caudal lobe of the mormyrid fish cerebellum, but whether these cell types are also functionally distinct is unknown. Here, we have used electrophysiological and pharmacological tools in a slice preparation to demonstrate that pairing parallel fiber (PF) and climbing fiber (CF) inputs at a low frequency induces long-term depression (LTD) in fPCs but long-term potentiation (LTP) in mPCs. The induction of plasticity in both cell types required postsynaptic Ca2+ and type 1α metabotropic glutamate receptors. However, the LTD in fPCs was inducted via a calcium/calmodulin-dependent protein kinase II cascade, whereas LTP induction in mPCs required calcineurin. Moreover, the LTD in fPCs and LTP in mPCs were accompanied by changes to the corresponding paired-pulse ratios and their coefficients of variation, suggesting presynaptic modes of expression for the plasticity at PF terminals for both cell types. Hence, the synaptic plasticity at PF synapses onto PCs in the posterior caudal lobe of the mormyrid cerebellum is cell type specific, with both pre- and postsynaptic mechanisms contributing to its induction and expression. NEW & NOTEWORTHY Much has been learnt about the cerebellar long-term depression (LTD) in the cortex. More recent work has shown that long-term potentiation (LTP) is equally important for cerebellar motor learning. Here we report for the first time that plasticity in the mormyrid cerebellum is cell type specific, e.g., following the conventional pairing of parallel and climbing fiber inputs in an in vitro preparation leads to LTD in one Purkinje cell subtype and LTP in another.

Electrical Coupling and Synchronized Subthreshold Oscillations in the Inferior Olive of the Rhesus Macaque.

UNLABELLED: Inferior olive (IO) neurons are critical for motor coordination and exhibit oscillations in membrane potential that are subthreshold for spiking. The prevalence, coherence, and continuity of those subthreshold oscillations (STOs) depend upon resonant interactions between neighboring neurons supported by electrical coupling. Many studies of the olivocerebellar system in rodents, in which STOs were related to tremor, whisking, and licking, fueled a debate over whether IO STOs were relevant for primates whose repertoire of movement is generally less periodic. The debate was never well informed due to the lack of a direct examination of the physiological properties of primate IO neurons. Here, we obtained dual patch-clamp recordings of neighboring IO neurons from young adult macaques in brainstem slices and compared them to identical recordings from rats. Macaque IO neurons exhibited an equivalent prevalence of continuous STOs as rats (45 vs 54%, respectively). However, macaque STOs were slower (1-4 Hz) and did not overlap with the dominant 4-9 Hz frequency of rats. The slower STO frequency of macaques was at least partially due to a prolonged membrane time constant and increased membrane capacitance that could be attributed to stronger electrical coupling and greater total dendritic length. The presence of synchronized STOs in the IO of adult macaques, coincident with strong and prevalent electrical coupling, answers a fundamental outstanding question in cerebellar neuroscience and is consistent with a prominent role for synchronized oscillation in primate sensory-motor control. SIGNIFICANCE STATEMENT: It was debated whether inferior olive (IO) neurons of primates behave as synchronized oscillators as was found for rodents using intracellular, optical, and multielectrode recordings. An inability to resolve this issue using single-Purkinje cell extracellular recordings in monkeys limited our understanding of timing mechanisms in the primate brain. Using dual whole-cell recordings from the IO of young adult rhesus macaques in acutely prepared brainstem slices, our work demonstrates that pairs of primate IO neurons show synchronized oscillations in membrane potential. The findings have strong mechanistic and translational relevance, as IO activation has been implicated in humans' perceptual timing of sensory events and motricity.

RNA interference of GluN1 inhibits neuronal rhythmogenesis in the adult inferior olive.

RNA interference (RNAi) to knockdown N-methyl-D-aspartate receptor (NMDAR) function is being investigated to address disorders associated with pathological brain rhythms. A motivating finding has been that pharmacological block of NMDARs inhibited oscillations in neuronal membrane potential that entrain rhythmic bursts of action potentials. To determine whether transient effects of NMDAR antagonist drugs to inhibit neuronal rhythmicity can be stably induced with genetic specificity, we examined the effects of RNAi of GluN1 protein on the subthreshold oscillations (STOs) of neurons in the inferior olive (IO), a pacemaking nucleus necessary for motor and cognitive timing. Western blot of dissociated neurons demonstrated 90% knockdown of GluN1 after a strong in vivo transduction by a dual-microRNA lentiviral vector. GluN1 RNAi in whole-cell-patched IO neurons blocked both membrane depolarization and STOs typically induced by NMDAR activation for up to 54 days without affecting input resistance, membrane capacitance, action potential firing, high-threshold Ca(2+) spikes, the hyperpolarization-activated current Ih, or the activation of the low-threshold Ca(2+) current I(T). Although an off-target effect on Cav3 expression was ruled out also by BlastN query, we found that GluN1 RNAi chronically eliminated I(T)-dependent STOs at resting membrane potential, well below the activation threshold of the NMDAR channel. In the context of a recent report showing that NMDAR activation induces STOs as it strengthens electrical coupling, the long-term block of STOs by GluN1 RNAi may relate to the loss of an essential support mechanism. Lentivector-mediated RNAi of GluN1 provides a novel technique for future investigations of NMDAR involvement in electrical oscillations and behavior.

NMDA receptor activation strengthens weak electrical coupling in mammalian brain.

Electrical synapses are formed by gap junctions and permit electrical coupling, which shapes the synchrony of neuronal ensembles. Here, we provide a direct demonstration of receptor-mediated strengthening of electrical coupling in mammalian brain. Electrical coupling in the inferior olive of rats was strengthened by activation of NMDA-type glutamate receptors (NMDARs), which were found at synaptic loci and at extrasynaptic loci 20-100 nm proximal to gap junctions. Electrical coupling was strengthened by pharmacological and synaptic activation of NMDARs, whereas costimulation of ionotropic non-NMDAR glutamate receptors transiently antagonized the effect of NMDAR activation. NMDAR-dependent strengthening (1) occurred despite increased input conductance, (2) induced Ca(2+)-influx microdomains near dendritic spines, (3) required activation of the Ca(2+)/calmodulin-dependent protein-kinase II, (4) was restricted to neurons that were weakly coupled, and (5) thus strengthened coupling, mainly between nonadjacent neurons. This provided a mechanism to expand the synchronization of rhythmic membrane potential oscillations by chemical neurotransmitter input.

[Current understanding of sleep, dreaming and related memory consolidation].

Sleep is a naturally recurring state found throughout the animal kingdom and characterized by a reversible loss of consciousness. Although in humans the daily amount of sleep decreases with aging, the total amount of time spent for sleep is estimated as up to one-third of one's lifetime. In mammals, sleep shows a clear daily rhythmicity as well as nightly phases, which are strongly controlled by the circadian clock located in the hypothalamic suprachiasmatic nuclei and are also regulated by ambient light. While it is certain that sleep is critical for survival in general, the functional significance of sleep is still under investigation. Dreaming is a common psychological phenomenon occurring during human sleep, yet its content and natural function, if any, are still a matter of debate. In recent years, accumulated evidence strongly supports the notion that new information acquired during the day time is processed and transformed into long-term memory in a complicated and sophisticated way during sleeping. Such information processing is commonly referred to as memory consolidation.

Bidirectional plasticity in the primate inferior olive induced by chronic ethanol intoxication and sustained abstinence.

The brain adapts to chronic ethanol intoxication by altering synaptic and ion-channel function to increase excitability, a homeostatic counterbalance to inhibition by alcohol. Delirium tremens occurs when those adaptations are unmasked during withdrawal, but little is known about whether the primate brain returns to normal with repeated bouts of ethanol abuse and abstinence. Here, we show a form of bidirectional plasticity of pacemaking currents induced by chronic heavy drinking within the inferior olive of cynomolgus monkeys. Intracellular recordings of inferior olive neurons demonstrated that ethanol inhibited the tail current triggered by release from hyperpolarization (I(tail)). Both the slow deactivation of hyperpolarization-activated cyclic nucleotide-gated channels conducting the hyperpolarization-activated inward current and the activation of Ca(v)3.1 channels conducting the T-type calcium current (I(T)) contributed to I(tail), but ethanol inhibited only the I(T) component of I(tail). Recordings of inferior olive neurons obtained from chronically intoxicated monkeys revealed a significant up-regulation in I(tail) that was induced by 1 y of daily ethanol self-administration. The up-regulation was caused by a specific increase in I(T) which (i) greatly increased neurons' susceptibility for rebound excitation following hyperpolarization and (ii) may have accounted for intention tremors observed during ethanol withdrawal. In another set of monkeys, sustained abstinence produced the opposite effects: (i) a reduction in rebound excitability and (ii) a down-regulation of I(tail) caused by the down-regulation of both the hyperpolarization-activated inward current and I(T). Bidirectional plasticity of two hyperpolarization-sensitive currents following chronic ethanol abuse and abstinence may underlie persistent brain dysfunction in primates and be a target for therapy.

Morphological analysis of the mormyrid cerebellum using immunohistochemistry, with emphasis on the unusual neuronal organization of the valvula.

This study used immunohistochemistry, Golgi impregnation, and electron microscopy to examine the circuitry of the cerebellum of mormyrid fish. We used antibodies against the following antigens: the neurotransmitters glutamate and gamma-aminobutyric acid (GABA); the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD); GABA transporter 1; the anchoring protein for GABA and glycine receptors, gephyrin; the calcium binding proteins calbindin and calretinin; the NR1 subunit of the N-methyl-D-aspartate glutamate receptor; the metabotropic glutamate receptors mGluR1alpha and mGluR2/3; the intracellular signaling molecules calcineurin and calcium calmodulin kinase IIalpha (CAMKIIalpha); and the receptor for inositol triphosphate (IP3RIalpha). Purkinje cells are immunoreactive to anti-IP3R1alpha, anticalcineurin, and anti-mGluR1alpha. Cerebellar efferent cells (eurydendroid cells) are anticalretinin and anti-NR1 positive in the valvula but not in the corpus and caudal lobe. In contrast, climbing fibers are anticalretinin and anti-NR1 immunopositive in the corpus and caudal lobe but not in the valvula. Purkinje cells, Golgi cells, and stellate cells are GABA positive, whereas efferent cells are glutamate positive. Unipolar brush cells are immunoreactive to anti-mGluR2/3, anticalretinin, and anticalbindin. We describe a "new" cell type in the mormyrid valvula, the deep stellate cell. These cells are GABA, calretinin, and calbindin positive. They are different from superficial stellate cells in having myelinated axons that terminate massively with GAD- and gephyrin-positive terminals on the cell bodies and proximal dendrites of efferent cells. We discuss how the valvula specializations described here may act in concert with the palisade pattern of Purkinje cell dendrites for analyzing spatiotemporal patterns of parallel fiber activity.

The neuronal organization of a unique cerebellar specialization: the valvula cerebelli of a mormyrid fish.

The distal valvula cerebelli is the most prominent part of the mormyrid cerebellum. It is organized in ridges of ganglionic and molecular layers, oriented perpendicular to the granular layer. We have combined intracellular recording and labeling techniques to reveal the cellular morphology of the valvula ridges in slice preparations. We have also locally ejected tracer in slices and in intact animals to examine its input fibers. The palisade dendrites and fine axon arbors of Purkinje cells are oriented in the horizontal plane of the ridge. The dendrites of basal efferent cells and large central cells are confined to the molecular layer but are not planar. Basal efferent cell axons are thick and join the basal bundle leaving the cerebellum. Large central cell axons are also thick, and they traverse long distances in the transverse plane, with local collaterals in the ganglionic layer. Vertical cells and small central cells also have thick axons with local collaterals. The dendrites of Golgi cells are confined to the molecular layer, but their axon arbors are either confined to the granular layer or proliferate in both the granular and ganglionic layers. Dendrites of deep stellate cells are distributed in the molecular layer, with fine axon arbors in the ganglionic layer. Granule cell axons enter the molecular layer as parallel fibers without bifurcating. Climbing fibers run in the horizontal plane and terminate exclusively in the ganglionic layer. Our results confirm and extend previous studies and suggest a new concept of the circuitry of the mormyrid valvula cerebelli.

Local circuitry in the anterior caudal lobe of the mormyrid cerebellum: a study of intracellular recording and labeling.

The caudal lobe of the mormyrid cerebellum includes the anterior portion, which is associated with the lateral line and eighth nerve senses, and the posterior portion, which is associated with the electrosense. This study examines the physiology and morphology of cells in the anterior portion in slice preparations. Two subtypes of Purkinje cells, efferent cells and stellate cells, are described. Multipolar Purkinje cells are located in the central region of the lobe, with large, multipolar, spiny dendrites and locally ending axons. Small Purkinje cells are located along its anterior border with the eminentia granularis anterior (EGa), with spiny dendrites in the molecular region. Axons of some small Purkinje cells end locally, whereas axons of other such cells are cut at the surface of the slices, suggesting that they project outside the lobe. Efferent cells are also distributed along the border with EGa. These cells have thin, smooth dendrites in the molecular region, and their axons are cut at the sliced surface. Stellate cells have thin, smooth dendrites and locally terminating axons. Physiologically, all types of cells respond to parallel fiber activation, but only multipolar Purkinje cells showed characteristic all-or-none climbing fiber responses. Although the majority of Purkinje cells fire a single type of spikes at resting level, a subset of small Purkinje cells fire small, narrow and large, broad spikes. Thus, the anterior caudal lobe of the mormyrid cerebellum is different from the mammalian cerebellum in having different subtypes of Purkinje cells and local termination of many Purkinje cell axons.

Synaptic plasticity and calcium signaling in Purkinje cells of the central cerebellar lobes of mormyrid fish.

Climbing fiber (CF)-evoked calcium transients play a key role in plasticity at parallel fiber (PF) to Purkinje cell synapses in the mammalian cerebellum. Whereas PF activation alone causes long-term potentiation (LTP), coactivation of the heterosynaptic CF input, which evokes large dendritic calcium transients, induces long-term depression (LTD). This unique type of heterosynaptic interaction is a hallmark feature of synaptic plasticity in mammalian Purkinje cells. Purkinje cells in the cerebellum of mormyrid electric fish are characterized by a different architecture of their dendritic trees and by a more pronounced separation of CF and PF synaptic contact sites. We therefore examined the conditions for bidirectional plasticity at PF synapses onto Purkinje cells in the mormyrid cerebellum in vitro. PF stimulation at elevated frequencies induces LTP, whereas LTD results from PF stimulation at enhanced intensities and depends on dendritic calcium influx and metabotropic glutamate receptor type 1 activation. LTD can also be observed after pairing of low intensity PF stimulation with CF stimulation. Using a combination of whole-cell patch-clamp recordings and fluorometric calcium imaging, we characterized calcium transients in Purkinje cell dendrites. CF activation elicits calcium transients not only within the CF input territory (smooth proximal dendrites) but also within the PF input territory (spiny palisade dendrites). Paired PF and CF activation elicits larger calcium transients than stimulation of either input alone. A major source for dendritic calcium signaling is provided by P/Q-type calcium channels. Our data show that despite the spatial separation between the two inputs CF activity facilitates LTD induction at PF synapses.

Physiology of morphologically identified cells in the posterior caudal lobe of the mormyrid cerebellum.

The cerebellum of the mormyrid fish consists of three major divisions: the valvula, the central lobes, and the caudal lobes. Several studies have focused on the central lobes and the valvula, but little is known about the caudal lobes. The mormyrid caudal lobe includes anterior and posterior components. The anterior caudal lobe is associated with the lateral line and eighth nerve end organs, whereas the posterior caudal lobe is associated with the electrosensory system. The present study examines the physiology and pharmacology of morphologically identified Purkinje cells and efferent cells in an in vitro slice preparation of the posterior caudal lobe. We found that the Purkinje cells in the posterior caudal lobe can be classified into three subtypes based on both their morphology and on their physiological responses to intracellular current injection and to synaptic inputs from parallel fibers and climbing fibers. Similarities and differences between the physiology of the caudal lobe and that of other regions of the mormyrid cerebellum and the mammalian cerebellum are discussed.

Granular cells of the mormyrid electrosensory lobe and postsynaptic control over presynaptic spike occurrence and amplitude through an electrical synapse.

Primary afferent fibers from the electroreceptors of mormyrid electric fish use a latency code to signal the intensity of electrical current evoked by the fish's own electric organ discharge (EOD). The afferent fibers terminate centrally in the deep and superficial granular layers of the electrosensory lobe with morphologically mixed chemical-electrical synapses. The granular cells in these layers seem to decode afferent latency through an interaction between primary afferent input and a corollary discharge input associated with the EOD motor command. We studied the physiology of deep and superficial granular cells in a slice preparation with whole cell patch recording and electrical stimulation of afferent fibers. Afferent stimulation evoked large all-or-none electrical excitatory postsynaptic potentials (EPSPs) and large all or none GABAergic inhibitory postsynaptic potentials (IPSPs) in both superficial and deep granular cells. The amplitudes of the electrical EPSPs depended on postsynaptic membrane potential, with maximum amplitudes at membrane potentials between -65 and -110 mV. Hyperpolarization beyond this level resulted in either the abrupt disappearance of EPSPs, a step-like reduction to a smaller EPSP, or a graded reduction in EPSP amplitude. Depolarization to membrane potentials lower than that yielding a maximum caused a linear decrease in EPSP amplitude, with EPSP amplitude reaching 0 mV at potentials between -55 and -40 mV. We suggest that the dependence of EPSP size on postsynaptic membrane potential is caused by close linkage of pre- and postsynaptic membrane potentials through a high-conductance gap junction. We also suggest that this dependence may result in functionally important nonlinear interactions between synaptic inputs.

Cell morphology and circuitry in the central lobes of the mormyrid cerebellum.

The cerebellum of mormyrid electric fish is large and unusually regular in its histological structure. We have examined the morphology of cellular elements in the central lobes of the mormyrid cerebellum. We have used intracellular injection of biocytin to determine the morphology of cells with somas in the cortex, and we have used extracellular placement of anterograde tracers in the inferior olive to label climbing fibers. Our results confirm previous Golgi studies and extend them by providing a more complete description of axonal trajectories. Most Purkinje cells in mormyrids and other actinopterygian fishes are interneurons that terminate locally in the cortex on efferent neurons that are equivalent to cerebellar nucleus cells in mammals. We confirm the markedly sagittal distribution of the fan-like dendrites of Purkinje cells, efferent cells, and molecular layer interneurons. We show that Purkinje cell axons extend further than was previously thought in the sagittal plane. We show that climbing fibers are distributed in narrow sagittal strips and that these fibers terminate exclusively in the ganglionic layer below the molecular layer where parallel fibers terminate. Our results together with those of others show that the central lobes of the mormyrid cerebellum, similar to the mammalian cerebellum, are composed of sagittally oriented modules made up of Purkinje cells, climbing fibers, molecular layer interneurons, and cerebellar efferent cells (cerebellar nucleus cells in mammals) that Purkinje cells inhibit. This modular organization is more apparent and more sharply defined in the mormyrid than in the mammal.

Physiology of cells in the central lobes of the mormyrid cerebellum.

The cerebellum of mormyrid electric fish is unusual for its size and for the regularity of its histology. The circuitry of the mormyrid cerebellum is also different from that of the mammalian cerebellum in that mormyrid Purkinje cell axons terminate locally within the cortex on efferent cells, and the cellular regions of termination for climbing fibers and parallel fibers are well separated. These and other features suggest that the mormyrid cerebellum may be a useful site for addressing some functional issues regarding cerebellar circuitry. We have therefore begun to examine the physiology of the mormyrid cerebellum by recording intracellularly from morphologically identified Purkinje cells, efferent cells, Golgi cells, and stellate cells in in vitro slices. Mormyrid Purkinje cells respond to parallel fiber input with an AMPA-mediated EPSP that shows paired pulse facilitation and to climbing fiber input with a large all-or-none AMPA-mediated EPSP that shows paired pulse depression. Recordings from the somas of Purkinje cells show three types of spikes in response to injected current: a small, narrow sodium spike; a large, broad sodium spike; and a large broad calcium spike. Efferent cells, Golgi cells, and stellate cells respond to parallel fiber input with an EPSP or EPSP-IPSP sequence and show only large, narrow spikes in response to intracellular current injection. We conclude that the physiology of the mormyrid cerebellum is similar in many ways to the mammalian cerebellum but is also different in ways that may prove instructive concerning the functional circuitry of the cerebellum.