Cerebellar Cortex Granular Layer Interneurons in the Macaque Monkey Are Functionally Driven by Mossy Fiber Pathways through Net Excitation or Inhibition

The granular layer is the input layer of the cerebellar cortex. It receives information through mossy fibers, which contact local granular layer interneurons (GLIs) and granular layer output neurons (granule cells). GLIs provide one of the first signal processing stages in the cerebellar cortex by exciting or inhibiting granule cells. Despite the importance of this early processing stage for later cerebellar computations, the responses of GLIs and the functional connections of mossy fibers with GLIs in awake animals are poorly understood. Here, we recorded GLIs and mossy fibers in the macaque ventral-paraflocculus (VPFL) during oculomotor tasks, providing the first full inventory of GLI responses in the VPFL of awake primates. We found that while mossy fiber responses are characterized by a linear monotonic relationship between firing rate and eye position, GLIs show complex response profiles characterized by “eye position fields” and single or double directional tunings. For the majority of GLIs, prominent features of their responses can be explained by assuming that a single GLI receives inputs from mossy fibers with similar or opposite directional preferences, and that these mossy fiber inputs influence GLI discharge through net excitatory or inhibitory pathways. Importantly, GLIs receiving mossy fiber inputs through these putative excitatory and inhibitory pathways show different firing properties, suggesting that they indeed correspond to two distinct classes of interneurons. We propose a new interpretation of the information flow through the cerebellar cortex granular layer, in which mossy fiber input patterns drive the responses of GLIs not only through excitatory but also through net inhibitory pathways, and that excited and inhibited GLIs can be identified based on their responses and their intrinsic properties.     

Cerebellar cortex: its simulation and the relevance of Marr's theory.

Marr's theory of the cerebellar cortex as an associative learning device is one of the best examples of a theory that directly relates the function of a neural system to its neural structure. However, although he assigned a precise function to each of the identified cell types of the cerebellar cortex, many of the crucial aspects of the implementation of his theory remained unspecified. We attempted to resolve these difficulties by constructing a computer simulation which contained a direct representation of the 13,000 mossy fibres and the 200,000 granule cells associated with a single Purkinje cell of the cerebellar cortex, together with the supporting Golgi, basket and stellate cells. In this paper we present a detailed explanation of Marr's theory based upon an analogy between Marr's cerebellar model and an abstract model called the associative net. Although some of Marr's assumptions contravene neuroanatomical findings, we found that in general terms his conclusion that each Purkinje cell can learn to respond to a large number of different patterns of activity in the mossy fibres is substantially correct. However, we found that this system has a lower capacity and acts more stochastically than he envisaged. The biologically realistic simulated structure that we designed can be used to assess the computational capabilities of other network theories of the cerebellum.     

Formation of new synaptic contacts by Purkinje axon collaterals in the granular layer of deafferented cerebellar cortex of adult rat

In addition to (i) mossy terminals, (ii) Golgi axons, (iii) granule cell dendrites and (iv), occasionally, Golgi cell dendrites, a third axonal profile identified by morphological criteria as the collateral of Purkinje axons, has been found in 2% of all cerebellar glomeruli. These infrequent components of a few glomeruli, however, were never seen in normal cerebellar cortex to establish specialized synaptic contact with glomerular dendrites. Two to four weeks after surgical isolation of the cerebellar cortex, i.e. following the destruction of both efferent and afferent fibres, the number of glomeruli containing (hypertrophic) axonal branches of Purkinje cells has increased to 13% of all surveyed glomeruli. In addition, the Purkinje axon terminals in the mossy fibre-deprived glomeruli were observed to establish numerous Gray II-type synaptic contacts with surrounding granule cell dendrites. It is suggested that the development of heterologous synapses between hypertrophic, or even intact, Purkinje axon collaterals on the one hand and the mossy fibre-vacated granule cell dendrites on the other, is a compensatory, reactive process to the synaptic "desaturation" of granule neurons, which demonstrate a dormant potential of Purkinje cells to form new synaptic contacts in the adult cerebellum.     

Electron microscopical study of synaptic glomeruli in cerebellum transplanted to the anterior eye chamber

Fetal cerebellar anlage from rat fetuses of 15-16 operational days were grafted into the anterior chamber of the eye of adult female albino rat recipients. Survival time of the transplants--containing both cerebellar cortex and cerebellar nuclei--was 2 to 2 1/2 months. Electron microscopical (EM) studies of the thin, under-developed granular layer of the laminated cerebellar cortex revealed the presence of well differentiated cerebellar glomeruli, surrounded by granule cell perikarya. As in the normal cerebellar cortex, the central profile of the glomerular complex was the large mossy terminal, containing spheroid synaptic vesicles, and forming synaptic contacts with dendrites and dendritic digits of the granule cells. Golgi cell axonal varicosities, containing ovoid or pleomorphic synaptic vesicles were found also on the periphery of the glomeruli. In addition, in several synaptic glomeruli, a third neuronal element was also observed, containing flat, discoidal vesicles and receiving synaptic contacts from mossy and Golgi axons, but being also presynaptic to granule cell dendrites. It is suggested that all mossy terminals in the cerebellar transplant originate from the cerebellar nucleus. Morphological evidence is also provided that the presynaptic dendrite-like processes--never found in normal cerebellar cortex--are also processes of nuclear neurons.     

Identification of an inhibitory circuit that regulates cerebellar Golgi cell activity

Here we provide evidence that revises the inhibitory circuit diagram of the cerebellar cortex. It was previously thought that Golgi cells, interneurons that are the sole source of inhibition onto granule cells, were exclusively coupled via gap junctions. Moreover, Golgi cells were believed to receive GABAergic inhibition from molecular layer interneurons (MLIs). Here we challenge these views by optogenetically activating the cerebellar circuitry to determine the timing and pharmacology of inhibition onto Golgi cells and by performing paired recordings to directly assess synaptic connectivity. In contrast to current thought, we find that Golgi cells, not MLIs, make inhibitory GABAergic synapses onto other Golgi cells. As a result, MLI feedback does not regulate the Golgi cell network, and Golgi cells are inhibited approximately 2?ms before Purkinje cells, following a mossy fiber input. Hence, Golgi cells and Purkinje cells receive unique sources of inhibition and can differentially process shared granule cell inputs.     

Stimulus-dependent state transition between synchronized oscillation and randomly repetitive burst in a model cerebellar granular layer

Information processing of the cerebellar granular layer composed of granule and Golgi cells is regarded as an important first step toward the cerebellar computation. Our previous theoretical studies have shown that granule cells can exhibit random alternation between burst and silent modes, which provides a basis of population representation of the passage-of-time (POT) from the onset of external input stimuli. On the other hand, another computational study has reported that granule cells can exhibit synchronized oscillation of activity, as consistent with observed oscillation in local field potential recorded from the granular layer while animals keep still. Here we have a question of whether an identical network model can explain these distinct dynamics. In the present study, we carried out computer simulations based on a spiking network model of the granular layer varying two parameters: the strength of a current injected to granule cells and the concentration of Mg²⁺ which controls the conductance of NMDA channels assumed on the Golgi cell dendrites. The simulations showed that cells in the granular layer can switch activity states between synchronized oscillation and random burst-silent alternation depending on the two parameters. For higher Mg²⁺ concentration and a weaker injected current, granule and Golgi cells elicited spikes synchronously (synchronized oscillation state). In contrast, for lower Mg²⁺ concentration and a stronger injected current, those cells showed the random burst-silent alternation (POT-representing state). It is suggested that NMDA channels on the Golgi cell dendrites play an important role for determining how the granular layer works in response to external input.     

Reliability of pattern separation by the cerebellar mossy fiber — granule cell system

As a simple example of a neuronal network in which synaptic connectivity among neurons is probabilistic, Marr's model for the granular layer of cat cerebellar cortex is examined. The mean and variance are computed for the fraction of granule cells activated, and for the extent of pattern separation by granule cells, for various mossy fiber inputs and various values of connectivity and electrical parameters of the network structure. Results suggest different functions for the network, and different optimal ranges for its parameters, depending on whether Golgi cells are present or absent. The model network does not perform the functions originally prescribed for it with high reliability.     

Golgi Cell-Mediated Activation of Postsynaptic GABA(B) Receptors Induces Disinhibition of the Golgi Cell-Granule Cell Synapse in Rat Cerebellum

In the cerebellar glomerulus, GABAergic synapses formed by Golgi cells regulate excitatory transmission from mossy fibers to granule cells through feed-forward and feedback mechanisms. In acute cerebellar slices, we found that stimulating Golgi cell axons with a train of 10 impulses at 100 Hz transiently inhibited both the phasic and the tonic components of inhibitory responses recorded in granule cells. This effect was blocked by the GABA(B) receptor blocker CGP35348, and could be mimicked by bath-application of baclofen (30 μM). This depression of IPSCs was prevented when granule cells were dialyzed with GDPβS. Furthermore, when synaptic transmission was blocked, GABA(A) currents induced in granule cells by localized muscimol application were inhibited by the GABA(B) receptor agonist baclofen. These findings indicate that postsynaptic GABA(B) receptors are primarily responsible for the depression of IPSCs. This inhibition of inhibitory events results in an unexpected excitatory action by Golgi cells on granule cell targets. The reduction of Golgi cell-mediated inhibition in the cerebellar glomerulus may represent a regulatory mechanism to shift the balance between excitation and inhibition in the glomerulus during cerebellar information processing.     

Cerebellar globular cells receive monoaminergic excitation and monosynaptic inhibition from Purkinje cells

Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control.     

mGluR2 postsynaptically senses granule cell inputs at Golgi cell synapses

In the cerebellar circuit, Golgi cells are thought to contribute to information processing and integration via feedback mechanisms. In these mechanisms, dynamic modulation of Golgi cell excitability is necessary because GABA from Golgi cells causes tonic inhibition on granule cells. We studied the role and synaptic mechanisms of postsynaptic metabotropic glutamate receptor subtype 2 (mGluR2) at granule cell-Golgi cell synapses, using whole-cell recording of green fluorescent protein-positive Golgi cells of wild-type and mGluR2-deficient mice. Postsynaptic mGluR2 was activated by glutamate from granule cells and hyperpolarized Golgi cells via G protein-coupled inwardly rectifying K+ channels (GIRKs). This hyperpolarization conferred long-lasting silencing of Golgi cells, the duration and extents of which were dependent on stimulus strengths. Postsynaptic mGluR2 thus senses inputs from granule cells and is most likely important for spatiotemporal modulation of mossy fiber-granule cell transmission before distributing inputs to Purkinje cells.     

Interrelated modification of excitatory and inhibitory synapses in three-layer olivary-cerebellar neural network

The model of three-layer olivary-cerebellar neural network with modifiable excitatory and inhibitory connections between diverse elements is suggested. The same Hebbian modification rules are proposed for Purkinje cells, granule (input) cells, and deep cerebellar nuclei (output) cells. The inverse calcium-dependent modification rules for these cells and hippocampal/neocortical neurones or Golgi cells are conceivably the result of the involvement of cGMP and cAMP in postsynaptic processes. The sign of simultaneous modification of excitatory and inhibitory inputs to a cell is opposite and determined by the variations in pre- and/or postsynaptic cell activity. Modification of excitatory transmission between parallel fibers and Purkinje cells, mossy fibers and granule cells, and mossy fibers and deep cerebellar nuclei cells essentially depends on inhibition effected by stellate/basket cells, Golgi cells and Purkinje cells, respectively. The character of interrelated modifications of diverse synapses in all three layers of the network is influenced by olivary cell activity. In the absence (presence) of a signal from inferior olive, the long-term potentiation (depression) in the efficacy of a synapse between input mossy fiber and output cell can be induced. The results of the suggested model are in accordance with known experimental data.     

The enzymehistochemical maturation of the cerebellar cortex of rats after early postnatal X-irradiation

Summary The enzymehistochemical maturation of the cerebellar cortex was studied at 8, 16 and 30 days in rats whose cerebellar area was irradiated with 1,000 r at 2 days of age, and was compared with that in normal animals.After irradiation, the granule cells and basket and stellate neurones are absent due to lethal damage to their precursors. Only large neurones, glial cells and bloodvessels are present in a disorderly arrangement; the laminar pattern of the cortex and specialized structures such as the glomeruli do not develop.Enzymehistochemically the absence of specialized structures such as the molecular layer and the glomeruli is reflected in a uniformly strong diffuse activity of most of the dehydrogenases studied. AMPase-activity shows a strong positive correlation with the presence of granule cells. High subcortical activity of AChE in the posterior half of the vermis is presumed to be located within mossy fibres which lack their normal goal: the granule cells. Capillaries in the irradiated cortex show a close spatial relation to Purkinje cells. The development of the cytoplasmic organization of the Purkinje cells as demonstrated by TPPase and AcPase activities proceeds in a normal way. Some enzymes can be used as markers of certain cell-types in the irradiated cortex: ICDH and G6PDH of Bergmann glial cells, AcPase of Golgi cells.It is suggested that enzymehistochemical studies of experimentally disturbed development may lead to better understanding of normal maturation.     

A Reinforcing Circuit Action of Extrasynaptic GABAA Receptor Modulators on Cerebellar Granule Cell Inhibition

GABA_A receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.     

Further consideration on pattern separability in a random neural net with inhibitory connections

A two-layer random neural net with inhibitory connections composing of threshold elements has been regarded as a model of the cerebellar cortex. Many properties of pattern separation with the model have been disclosed through consideration on the degree of pattern separation. However, we have not shown yet that the degree of pattern separation is given by some different functions which are decided by the relation between the firing rates of input patterns. The present study is intended to reveal that the functions of the degree of pattern separation are synthesized with some different partial functions, and they are differently given on the relation between the firing rates of input patterns. Simultaneously, it is proved that the number of the functions also depend on the number of connections between two layers in the model. We also disclose the properties of the degree of pattern separation, and give some suggestions on the sizes of the firing rates of mossy fibers and granule cells under the knowledge about them.     

At the Edge of Chaos: How Cerebellar Granular Layer Network Dynamics Can Provide the Basis for Temporal Filters

Models of the cerebellar microcircuit often assume that input signals from the mossy-fibers are expanded and recoded to provide a foundation from which the Purkinje cells can synthesize output filters to implement specific input-signal transformations. Details of this process are however unclear. While previous work has shown that recurrent granule cell inhibition could in principle generate a wide variety of random outputs suitable for coding signal onsets, the more general application for temporally varying signals has yet to be demonstrated. Here we show for the first time that using a mechanism very similar to reservoir computing enables random neuronal networks in the granule cell layer to provide the necessary signal separation and extension from which Purkinje cells could construct basis filters of various time-constants. The main requirement for this is that the network operates in a state of criticality close to the edge of random chaotic behavior. We further show that the lack of recurrent excitation in the granular layer as commonly required in traditional reservoir networks can be circumvented by considering other inherent granular layer features such as inverted input signals or mGluR2 inhibition of Golgi cells. Other properties that facilitate filter construction are direct mossy fiber excitation of Golgi cells, variability of synaptic weights or input signals and output-feedback via the nucleocortical pathway. Our findings are well supported by previous experimental and theoretical work and will help to bridge the gap between system-level models and detailed models of the granular layer network.     

Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex

We show that information flow through the adult cerebellar cortex, from the mossy fiber input to the Purkinje cell output, is controlled by furosemide-sensitive, diazepam- and neurosteroid-insensitive GABA(A) receptors on granule cells, which are activated both tonically and by GABA spillover from synaptic release sites. Tonic activation of these receptors contributes a 3-fold larger mean inhibitory conductance than GABA released synaptically by high-frequency stimulation. Tonic and spillover inhibition reduce the fraction of granule cells activated by mossy fiber input, generating an increase of coding sparseness, which is predicted to improve the information storage capacity of the cerebellum.     

Delayed reverberation through time windows as a key to cerebellar function

We present a functional model of the cerebellum comprising cerebellar cortex, inferior olive, deep cerebellar nuclei, and brain stem nuclei. The discerning feature of the model being time coding, we consistently describe the system in terms of postsynaptic potentials, synchronous action potentials, and propagation delays. We show by means of detailed single-neuron modeling that (i) Golgi cells can fulfill a gating task in that they form short and well-defined time windows within which granule cells can reach firing threshold, thus organizing neuronal activity in discrete `time slices', and that (ii) rebound firing in cerebellar nuclei cells is a robust mechanism leading to a delayed reverberation of Purkinje cell activity through cerebellar-reticular projections back to the cerebellar cortex. Computer simulations of the whole cerebellar network consisting of several thousand neurons reveal that reverberation in conjunction with long-term plasticity at the parallel fiber-Purkinje cell synapses enables the system to learn, store, and recall spatio-temporal patterns of neuronal activity. Climbing fiber spikes act both as a synchronization and as a teacher signal, not as an error signal. They are due to intrinsic oscillatory properties of inferior olivary neurons and to delayed reverberation within the network. In addition to clear experimental predictions the present theory sheds new light on a number of experimental observation such as the synchronicity of climbing fiber spikes and provides a novel explanation of how the cerebellum solves timing tasks on a time scale of several hundreds of milliseconds. Received: 23 July 1999 / Accepted in revised form: 31 August 1999     

人体小脑神经元发育的定量观察

目的:研究人体小脑神经元的发育过程。方法:应用体视学方法,对18例不同时期人体小脑组织Golgi染色后进行观察,观测小脑皮质分层出现的时间,观测并计算神经元的数密度、体密度和表面积密度。结果:6月龄时,小脑皮质出现较明显的分子层、蒲肯野细胞层和颗粒层;星形细胞、篮状细胞、蒲肯野细胞、颗粒细胞和高尔基细胞的的数密度随月龄/年龄的增长而减少,体密度和表面积密度随月龄/年龄的增长而增加,但这些减小和增大是不等速的,6-8月龄变化最明显。结论:人体小脑神经元的发育呈现快慢交替、不均速发展,6～8月是小脑神经元发育的重要时期。     

Detection of sequences in the cerebellar cortex: numerical estimate of the possible number of tidal-wave inducing sequences represented

The two major cortices of the brain--the cerebral and cerebellar cortex--are massively connected through intercalated nuclei (pontine, cerebellar and thalamic nuclei). We suggest that the two cortices co-operate by generating precise temporal patterns in the cerebral cortex that are detected in the cerebellar cortex as temporal patterns assembled spatially in the mossy fibers. We will begin by showing that the tidal-wave mechanism works in the cerebellar cortex as a read-out mechanism for such spatio-temporal patterns due to the synchronous activity they generate in the parallel fiber system which drives the Purkinje cells--the output neurons of the cerebellar cortex--to fire action potentials. We will review the anatomy of the mossy fibers and show that within a "beam", or "row" of cerebellar cortex the mossy fibers in principle could embed a vast number of tidal-wave generating sequences. Based on anatomical data we will argue that the cerebellar mossy fiber-granule cell-Purkinje cell system can potentially detect and--through learning--select from an enormous number of spatio-temporal patterns.