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.     

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.     

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.     

Plausible function of Golgi cells in the cerebellar cortex

The function of Golgi cells in the cerebellar cortex is quantitatively examined in consideration of the nonlinear input-output characteristics and convergence and divergence numbers of cells. It is strongly suggested that the two signal paths to Golgi cells have different function. The feed-forward path will have the same function as assumed in the previous theories of the cerebellar cortex, that is, to keep the firing rate of granule cells approximately constant over considerable variation in the firing rate of mossy fibers. The feedback path will, on the other hand, have a new function which has not been assumed in the previous theories. The function is to cause oscillation of the firing rate of granule cells for stationary mossy fiber inputs. The assumption of the new function enables us to explain cerebellar function to keep stationary posture.     

Light and scanning electron microscopic study of cerebellar cortex of teleost fishes

Summary The teleostean cerebellar cortex has been studied with respect to its cytoarchitectonic arrangement and intracortical neuronal circuits. Samples of fish cerebellum were fixed either by immersion or vascular perfusion in 5% glutaraldehyde solution and processed for light and scanning electron microscopy. The cerebellar cortex shows four distinct layers: granular; fibrous stratum; Purkinje cell; and molecular layers. In the granular layer, mossy and climbing fiber glomeruli were characterized. The mossy glomerular region appeared as polygonal, round or ovoid clews formed by the convergence of up to 17 dendritic profiles upon a thick mossy fiber branch. The en passant nature of mossy fiber-granule cell dendrite synaptic relationship was clearly appreciated. The climbing fibers showed tendril and glomerular collaterals. The latter form thin, elongated glomeruli. Remnants of a neuroglial envelope were observed in the mossy fiber glomeruli but are apparently absent from the climbing fiber glomeruli. The beaded-shape Golgi cell axonal ramifications were observed participating in the formation of both glomerular types. Velate protoplasmic astrocytes and oligodendrocytes were also identified. The fibrous stratum appeared to be formed by compact bundles of thick and thin myelinated axons, running horizontally beneath the Purkinje cell layer and apparently belonging to ascending climbing fibers and descending Purkinje cell axons. At the Purkinje cell layer a selective removal of Bergmann glial cells was observed allowing the visualization of the pericellular basket and the pinceaux. Climbing fiber stems and their tendril collaterals were seen on their way to the molecular layer ascending parallel to the Purkinje dendritic ramifications. Stellate neuron processes were found passing through the fan-like arborescence of Purkinje cell dendrites.     

Reliability of Computation in the Cerebellum

The mossy fiber-granule cell-parallel fiber-Purkinje cell system of the cerebellar cortex is investigated from the viewpoint of reliability of computation. It is shown that the effects of variability in the inputs to a Purkinje cell can be reduced by having a large number of parallel fibers whose activities are statistically independent. The mossy fiber-granule cell relay is shown to be capable of performing the required function of transforming the activity in a small number of mossy fibers into activity in a much larger number of parallel fibers, while ensuring that there is little correlation between the activities of individual parallel fibers. The effects of variability in the outputs of Purkinje cells may be reduced by redundancy and convergence schemes, as evidenced by the geometrical pattern of parallel fibers and Purkinje cells and the convergence of these cells onto their target neurons.     

Production of an antiserum against cyclic nucleotide phosphodiesterase and its use for the immunocytochemical demonstration of this enzyme in rat cerebellum

A procedure for the separation of cyclic AMP phosphodiesterase from a commercially available preparation and for raising antibodies against this enzyme in rabbits is described. An antiserum thus obtained was used for the immunocytochemical detection of cyclic nucleotide phosphodiesterase in rat cerebellum. The molecular layer, the granular layer and the cerebellar white matter exhibited different degrees of immunoreactivity. Only a few cell bodies (possibly glial cells) were stained. Most of the antigenic sites were present in the neuropil of the molecular layer and around Purkinje cells. Cerebellar glomeruli, sites of synaptic interactions between mossy fibres, Golgi cells and granule cells, were also stained by this antiserum. Control reactions using preimmune serum were consistently negative.     

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.     

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.     

Histochemical and cytochemical localization of acetylcholinesterase in the cerebellar cortex of the chicken

The localization of acetylcholinesterase (AChE) was studied in the cerebellar cortex of the crossbred trembler chickens by means of histo- and cytochemical methods. No essential differences between the crossbred normal and the crossbred trembler chickens were observed. The common results were as follows: Under a light microscope AChE activity was predominantly evident in the molecular layer, and secondly in the granular layer. AChE was ultrastructurally distributed principally in the cisternae of rough endoplasmic reticulum (ER) and in a part of nuclear envelope of the Purkinje, the Golgi and some of the basket and granule cells, and in a portion of the sacculus of the Golgi apparatus of the Purkinje cell only. In dendrites and the initial axon of the Purkinje cells the smooth ER also showed AChE activity. Although dendritic terminals of the Golgi cells contained AChE reaction products, the axon terminal did not. Some of the afferent terminal fibers forming the cerebellar glomerulus exhibited weakly a positive AChE reaction, while others in the vicinity did not show any AChE activity at all. However, the enzyme reaction product was localized in the intercellular spaces between a presynaptic afferent terminal and the postsynaptic granule cell dendritic terminals in the glomerulus. In addition, AChE activity was found in the form of spots in the intercellular spaces of both molecular and granular layers.     

Cumulative effects of alcohol (ethanol) on the activity of Purkinje cells in the cat cerebellum

The effects of three consecutive injections of 2 ml/kg 30% alcohol (ethanol, i.v.) on the activity of identified cerebellar Purkinje cells were investigated during experiments on adult cats anesthetized by a Nembutal-chloralose mixture. It was found that repeated alcohol injections exert a cumulative effect on the firing rate of these cells. The alcohol-induced rise in Purkinje cell firing rate, produced by excitation of the mossy fibers, was generally accompanied by a reduction in that of cells responding to excitation of climbing fibers. The inhibitory pause occurring after complex discharges in cerebellar Purkinje cells grew shorter with successive alcohol injections.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 74–80, January–February, 1987.     

Production of an antiserum against cyclic nucleotide phosphodiesterase and its use for the immunocytochemical demonstration of this enzyme in rat cerebellum

Summary A procedure for the separation of cyclic AMP phosphodiesterase from a commercially available preparation and for raising antibodies against this enzyme in rabbits is described. An antiserum thus obtained was used for the immunocytochemical detection of cyclic nucleotide phosphodiesterase in rat cerebellum. The molecular layer, the granular layer and the cerebellar white matter exhibited different degrees of immunoreactivity. Only a few cell bodies (possibly glial cells) were stained. Most of the antigenic sites were present in the neuropil of the molecular layer and around Purkinje cells. Cerebellar glomeruli, sites of synaptic interactions between mossy fibres, Golgi cells and granule cells, were also stained by this antiserum. Control reactions using preimmune serum were consistently negative.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday.     

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。     

Distribution and morphology of ghrelin-immunopositive cells in the cerebellum of the African ostrich

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, has been found in the cerebellum of many vertebrates and in the gastrointestinal tract of African ostrich chicks, but little is known about its distribution in the cerebellum of the African ostrich. In the present study, the distribution and morphological characteristics of ghrelin-producing cells in the cerebellum of the African ostrich were investigated using immunohistochemistry. The results indicate that the cerebellum is divided into two sections: the outer cerebellar cortex and the inner medulla of cerebellum. The cerebellar cortex comprises a molecular layer, a Purkinje cell layer and a granular layer; ghrelin-immunopositive (ghrelin-ip) cells were localized throughout the entire cerebellum, but sparsely in the medulla. The greatest number of ghrelin-ip cells was found in the stratum granulosum, and the density decreased gradually from the molecular layer to the Purkinje cell layer in the cerebellar cortex. The ghrelin-ip cells were fusiform or irregular polygons and their cytoplasm was stained intensely. These results clearly demonstrate the presence of ghrelin-ip cells in the cerebellum of the African ostrich. It is speculated that ghrelin may have a physiological function in 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.     

Two types of granular cells in the cerebellar cortex

We recorded the activity of two types of granular cells in the rostral folia of the paramedial lobe (the projection region of the front legs) of the cerebellar cortex in cats immobilized by administration of ditiline; these cells differed in their receptive fields, the characteristics of their reaction to single stimulation of somatic nerves, and the character of their background activity. The granular cells of the first type were excited only when the nerves of the front legs were stimulated (reacting with 1–3 impulses with a latent period of 8–20 msec) and were inhibited between 20–50 and 70–180 msec after stimulation of the nerves of any leg. The cells of the second type responded with volleys of 3–6 impulses with a latent period of 20–40 msec to stimulation of the nerves of all four legs. Comparison of the reactions of the granular cells and other neurons of the cerebellar cortex showed that the cells of the first type cause excitation of the Purkinje and Golgi cells and the neurons of the molecular layer. The granular cells of the second type have an excitatory effect on the Golgi cells. The differences in the reactions of the two types of granular cells result from the fact that they are selectively innervated by the mossy fibers of different afferent pathways. Comparison with the data in the literature enables us to surmise that the fibers of the cuneocerebellar tract terminate at granular cells of the first type, while the reticular fibers terminate at cells of the second type.Institute of Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 1, No. 2, pp. 167–176, September–October, 1969.     

Localization of Hexokinase in Neural Tissue: Electron Microscopic Studies of Rat Cerebellar Cortex

: The distribution of hexokinase (ATP:d -hexose 6-phosphotransferase, EC 2.7.1.1) in the rat cerebellar cortex has been studied at the electron microscopic level using the peroxidase-antiperoxidase procedure. Extensive staining of cytoplasmic regions, with some increased staining at mitochondrial profiles, was seen in the cell bodies of both neurons (basket, stellate, Lugaro, Golgi, and granule cells) and astrocytes. Oligodendrocytes showed little or no detectable staining. Purkinje cell perikarya were much less intensely stained than were the perikarya of other neurons. The initial portion of the Purkinje dendrite was, like the perikaryon from which it emerged, lightly stained. More intense staining was seen in the secondary and tertiary branches of the Purkinje dendrite, but the terminal branches were devoid of stain. Granule cell dendrites were well stained in their initial portions but devoid of stain in their terminal dendritic digits which form part of the cerebellar glomeruli. In contrast to the unstained granule cell dendritic digits, the central mossy fiber nerve terminal of the glomerulus exhibited intense staining of the mitochondrial profiles and of synaptic vesicles adjacent to the mitochondria. Axons of basket cells showed intense staining in the segments adjacent to the Purkinje cell soma, while terminal twigs of the basket axons in the pinceau surrounding the (unstained) initial segment of the Purkinje axon showed markedly decreased staining intensity. These results indicate that there may be substantial variation in hexokinase levels between the various regions of neuronal processes. Hexokinase was seen at both cytoplasmic and mitochondrial locations in a variety of cells. It does not appear likely that location of hexokinase can be directly correlated with cell type, i.e., with neurons versus glia.     

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.     

Potentiation of mossy fiber EPSCs in the cerebellar nuclei by NMDA receptor activation followed by postinhibitory rebound current

Behavioral and computational studies predict that synaptic plasticity of excitatory mossy fiber inputs to cerebellar nuclear neurons is required for associative learning, but standard tetanization protocols fail to potentiate nuclear cell EPSCs in mouse cerebellar slices. Nuclear neurons fire action potentials spontaneously unless strongly inhibited by Purkinje neurons, raising the possibility that plasticity-triggering signals in these cells differ from those at classical Hebbian synapses. Based on predictions of neuronal activity during delay eyelid conditioning, we developed quasi-physiological induction protocols consisting of high-frequency mossy fiber stimulation and postsynaptic hyperpolarization. Robust, NMDA receptor-dependent potentiation of nuclear cell EPSCs occurred with protocols including a 150-250 ms hyperpolarization in which mossy fiber stimulation preceded a postinhibitory rebound depolarization. Mossy fiber stimulation potentiated EPSCs even when postsynaptic spiking was prevented by voltage-clamp, as long as rebound current was evoked. These data suggest that Purkinje cell inhibition guides the strengthening of excitatory synapses in the cerebellar nuclei.     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.