Determinants of synaptic integration and heterogeneity in rebound firing explored with data-driven models of deep cerebellar nucleus cells

Significant inroads have been made to understand cerebellar cortical processing but neural coding at the output stage of the cerebellum in the deep cerebellar nuclei (DCN) remains poorly understood. The DCN are unlikely to just present a relay nucleus because Purkinje cell inhibition has to be turned into an excitatory output signal, and DCN neurons exhibit complex intrinsic properties. In particular, DCN neurons exhibit a range of rebound spiking properties following hyperpolarizing current injection, raising the question how this could contribute to signal processing in behaving animals. Computer modeling presents an ideal tool to investigate how intrinsic voltage-gated conductances in DCN neurons could generate the heterogeneous firing behavior observed, and what input conditions could result in rebound responses. To enable such an investigation we built a compartmental DCN neuron model with a full dendritic morphology and appropriate active conductances. We generated a good match of our simulations with DCN current clamp data we recorded in acute slices, including the heterogeneity in the rebound responses. We then examined how inhibitory and excitatory synaptic input interacted with these intrinsic conductances to control DCN firing. We found that the output spiking of the model reflected the ongoing balance of excitatory and inhibitory input rates and that changing the level of inhibition performed an additive operation. Rebound firing following strong Purkinje cell input bursts was also possible, but only if the chloride reversal potential was more negative than −70 mV to allow de-inactivation of rebound currents. Fast rebound bursts due to T-type calcium current and slow rebounds due to persistent sodium current could be differentially regulated by synaptic input, and the pattern of these rebounds was further influenced by HCN current. Our findings suggest that active properties of DCN neurons could play a crucial role for signal processing in the cerebellum.     

Development and expression of cytoplasmic antigens in Purkinje cells recognized by monoclonal antibodies

Summary Five monoclonal antibodies reacting with intracellular constituents of Purkinje cells were investigated by means of indirect immunofluorescence on fresh-frozen sections of the cerebellum and retina from developing and adult normal and mutant mice. Antibodies PC1, PC2 and PC3, which recognize Purkinje cells, but no other cerebellar neuron type, label these cells from day 4 onward. PC4 antigen is expressed in addition to Purkinje cells also in granule cells and neurons of deep cerebellar nuclei and appears in Purkinje cells at day 4. M1 antigen (Lagenaur et al. 1980) is first detectable in Purkinje cell bodies by day 5; it is also detectable in deep cerebellar neurons. In the adult retina, only PC4 antigen is detectably expressed and is localized in the inner segments of photoreceptor cells.The neurological mutants weaver, reeler,jimpy and wobbler show detectable levels of these antigens in Purkinje cells. However, the mutants staggerer and Purkinje cell degeneration are abnormal in expression PC1, PC2, PC3, and M1 antigens. Staggerer never starts to express the antigens during development, whereas Purkinje cell degeneration first expresses the antigens, but then loses antigen expression after day 23. PC4 antigen is detectable in the remaining Purkinje cells in staggerer and Purkinje cell degeneration mice at all ages tested in this study. Deep cerebellar neurons are positive for both antigens, PC4 and M1, in all mutants and at all ages studied. In retinas of staggerer and Purkinje cell degeneration mutants, PC4 antigen is normally detectable in the inner segments of photoreceptor cells, even when these have started to degenerate in the case of Purkinje cell degeneration.     

Electrophysiological analysis of the functional organization of cortico-cerebellar connections

The effect of stimulation of cortical association (orbito-frontal, parietal) and projection (auditory, sensomotor) areas on the activity of Purkinje neurons of the cerebellar cortex was studied in adult cats anesthetized with pentobarbital, with or without chloralose. These responses were compared with those to peripheral stimuli. Definite similarity was found between the responses of Purkinje cells to different cortical (association and projection) stimuli as regards both the types of responses of the neurons and their ability to respond. No similarity was observed in the responses of Purkinje cells to peripheral (visual, auditory, electrodermal) stimulation. Whereas almost identical numbers of neurons (over 50%) were excited in response to the different forms of cortical stimulation, the ability of the neurons to respond to peripheral stimuli differed considerably: 44.6% of neurons responded to electrodermal stimulation, 34.2% to auditory, and 18.8% to visual.Medical Institute, Kemerovo. Translated from Neirofiziologiya, Vol. 8, No. 5, pp. 483–489, September–October, 1976.     

A computational mechanism for unified gain and timing control in the cerebellum

Precise gain and timing control is the goal of cerebellar motor learning. Because the basic neural circuitry of the cerebellum is homogeneous throughout the cerebellar cortex, a single computational mechanism may be used for simultaneous gain and timing control. Although many computational models of the cerebellum have been proposed for either gain or timing control, few models have aimed to unify them. In this paper, we hypothesize that gain and timing control can be unified by learning of the complete waveform of the desired movement profile instructed by climbing fiber signals. To justify our hypothesis, we adopted a large-scale spiking network model of the cerebellum, which was originally developed for cerebellar timing mechanisms to explain the experimental data of Pavlovian delay eyeblink conditioning, to the gain adaptation of optokinetic response (OKR) eye movements. By conducting large-scale computer simulations, we could reproduce some features of OKR adaptation, such as the learning-related change of simple spike firing of model Purkinje cells and vestibular nuclear neurons, simulated gain increase, and frequency-dependent gain increase. These results suggest that the cerebellum may use a single computational mechanism to control gain and timing simultaneously.     

Aftereffect phenomena in neurons of the cerebellar cortex

The reactions of Purkinje cells (PC) of the cerebellar cortex during electrocutaneous stimulation of one of the extremities with different frequencies (from one stimulus in 10 sec to one to five stimuli per sec) were studied in experiments on cats. It was shown that the reactions to the first and subsequent stimuli were different. This indicates the presence of an aftereffect from the first stimulus. It is assumed that the variability of the responses of PC to infrequent stimuli is connected with changes in their functional state which develop in response to "spontaneous" cerebellopetal impulses, as well as to circulation of excitation in intracerebellar circuits. With an increase in the frequency of the stimuli, the changes in excitability induced by previous peripheral stimuli, not only in the reacting PC, but also in the whole neuronal network of the corresponding cerebellopetal pathway, evidently acquire paramount importance. The absence of a direct relationship between strong peripheral stimulation and the degree of the reactions of the PC may be due to the involvement of intermediate neurons both of the exciting and inhibitory type in the transmission of impulses at the level of the cerebellar cortex.N. I. Pirogov Vinnitsa Medical Institute. Translated from Neirofiziologiya, Vol. 2, No. 6, pp. 573–580, November–December, 1970.     

Electrophysiological analysis of the circuitry and of the corticonuclear relationships in the agranular cerebellum of irradiated rats.

The cerebellar circuitry and the corticonuclear relationships were studied in the cerebellum of adult rats rendered agranular through 7 successive exposures to X-ray radiations during infancy. Data were obtained through examination of electrical responses induced in Purkinje cells (PC) and in neurons of the lateral vestibular nucleus (LVN) by cerebellar and spinal stimulations. In irradiated rats, PC exhibited antidromic activation with a high axonal threshold and 70% of them also presented typical climbing fiber responses (CFRs). By contrast, they exceptionnally exhibited responses via the mossy fiber (MF)-granule cell pathway, but two other classes of responses were identified: i) short latency single spike responses attributed to a direct excitatory impingement of MF onto PC; ii) atypical CFRs formed of high frequency bursts of simple spikes which were seen in 76% of PC tested. Furthermore, 53% of these cells also presented typical CFRs, strongly suggesting these PC were innervated by more than one CF, thus confirming previous data on the same type of agranular cerebellum. In the LVN neurons of control and irradiated rats, spinal and cerebellar stimulations evoked clear cut IPSPs. On the basis of their shape, latency, and occurrence in animals with or without cerebellum and with or without lesion of the CF pathway, they were interpreted as mediated through direct or synaptic activation of PC or through an extracerebellar pathway. In irradiated rats, the quantitative study of these IPSPs gave further arguments in favor of a multiinnervation of PC by CF and of an important reafferentation of MF onto PC. However, the functional efficiency of this reafferentation appeared very low, as tested by activation of MF originating in the spinal cord. Finally, the intracellular recording of LVN neurons showed that a large majority of PC axons retained normal synaptic connections with nuclear cells in treated animals, indicating that corticonuclear relationships do not markedly depend upon granule cells and normal CF input.     

Development and evolution of cerebellar neural circuits

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.     

Increased Density of Dystrophin Protein in the Lateral Versus the Vermal Mouse Cerebellum

Dystrophin, present in muscle, also resides in the brain, including cerebellar Purkinje neurons. The cerebellum, although historically associated with motor abilities, is also implicated in cognition. An absence of brain dystrophin in Duchenne muscular dystrophy (DMD) and in the mdx mouse model results in cognitive impairments. Localization studies of cerebellar dystrophin, however, have focused on the vermal cerebellum, associated with motor function, and have not investigated dystrophin distribution in the lateral cerebellum, considered to mediate cognitive function. The present study examined dystrophin localization in vermal and lateral cerebellar regions and across subcellular areas of Purkinje neurons in the mouse using immunohistochemistry. In both vermal and lateral cerebellum, dystrophin was restricted to puncta on somatic and dendritic membranes of Purkinje neurons. The density of dystrophin puncta was greater in the lateral than the vermal region. Neither the size of puncta nor the area of Purkinje neuron somata differed between regions. Results support the view that cognitive deficits in the DMD and the mdx model may be mediated by the loss of dystrophin, particularly in the lateral cerebellum. Findings have important implications for future studies examining the neurophysiological sequelae of neuronal dystrophin deficiency and the role of the lateral cerebellum in cognition.     

Specific Relationship between Excitatory Inputs and Climbing Fiber Receptive Fields in Deep Cerebellar Nuclear Neurons

Many mossy fiber pathways to the neurons of the deep cerebellar nucleus (DCN) originate from the spinal motor circuitry. For cutaneously activated spinal neurons, the receptive field is a tag indicating the specific motor function the spinal neuron has. Similarly, the climbing fiber receptive field of the DCN neuron reflects the specific motor output function of the DCN neuron. To explore the relationship between the motor information the DCN neuron receives and the output it issues, we made patch clamp recordings of DCN cell responses to tactile skin stimulation in the forelimb region of the anterior interposed nucleus in vivo. The excitatory responses were organized according to a general principle, in which the DCN cell responses became stronger the closer the skin site was located to its climbing fiber receptive field. The findings represent a novel functional principle of cerebellar connectivity, with crucial importance for our understanding of the function of the cerebellum in movement coordination.     

In vivo analysis of inhibitory synaptic inputs and rebounds in deep cerebellar nuclear neurons

Neuronal function depends on the properties of the synaptic inputs the neuron receive and on its intrinsic responsive properties. However, the conditions for synaptic integration and activation of intrinsic responses may to a large extent depend on the level of background synaptic input. In this respect, the deep cerebellar nuclear (DCN) neurons are of particular interest: they feature a massive background synaptic input and an intrinsic, postinhibitory rebound depolarization with profound effects on the synaptic integration. Using in vivo whole cell patch clamp recordings from DCN cells in the cat, we find that the background of Purkinje cell input provides a tonic inhibitory synaptic noise in the DCN cell. Under these conditions, individual Purkinje cells appear to have a near negligible influence on the DCN cell and clear-cut rebounds are difficult to induce. Peripheral input that drives the simple spike output of the afferent PCs to the DCN cell generates a relatively strong DCN cell inhibition, but do not induce rebounds. In contrast, synchronized climbing fiber activation, which leads to a synchronized input from a large number of Purkinje cells, can induce profound rebound responses. In light of what is known about climbing fiber activation under behaviour, the present findings suggest that DCN cell rebound responses may be an unusual event. Our results also suggest that cortical modulation of DCN cell output require a substantial co-modulation of a large proportion of the PCs that innervate the cell, which is a possible rationale for the existence of the cerebellar microcomplex.     

Histone Deacetylase 3 Is Necessary for Proper Brain Development

The functional role of histone deacetylase 3 (HDAC3) in the developing brain has yet to be elucidated. We show that mice lacking HDAC3 in neurons and glia of the central nervous system, Nes-Cre/HDAC3 conditional KO mice, show major abnormalities in the cytoarchitecture of the neocortex and cerebellum and die within 24 h of birth. Later-born neurons do not localize properly in the cortex. A similar mislocalization is observed with cerebellar Purkinje neurons. Although the proportion of astrocytes is higher than normal, the numbers of oligodendrocytes are reduced. In contrast, conditional knockout of HDAC3 in neurons of the forebrain and certain other brain regions, using Thy1-Cre and calcium/calmodulin dependent protein kinase II α-Cre for ablation, produces no overt abnormalities in the organization of cells within the cortex or of cerebellar Purkinje neurons at birth. However, both lines of conditional knockout mice suffer from progressive hind limb paralysis and ataxia and die around 6 weeks after birth. The mice display an increase in overall numbers of cells, higher numbers of astrocytes, and Purkinje neuron degeneration. Taken together, our results demonstrate that HDAC3 plays an essential role in regulating brain development, with effects on both neurons and glia in different brain regions.     

Cerebellar localization and colocalization of GABA and calcium binding protein-D28K

Immunocytochemical studies using antibodies raised against the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) and against the 28 Kd vitamin D dependent calcium binding protein (calbindin) in the cerebellum, are reviewed. The GABA immunoreactive neurones found in the cerebellar cortex were the Purkinje cell (PC), the three classes of intrinsic inhibitory interneurones, stellate, basket and Golgi cells and the cells of Lugaro. Some of the neurons of the cerebellar nuclei were also found to be GABA immunoreactive. A part of these could be identified as extrinsic neurones projecting either back to the cerebellar cortex, or to the inferior olive, both these pathways being topographically highly organized but arising from independent parent neurons. The presumed inhibitory function of these two pathways are discussed. Calbindin immunoreactivity in the cerebellum was confined to the PCs, staining concerned the whole cell including soma, branching dendrites, axons and axons terminals. The antibody, which appears to be tightly bound to the PC in vivo, failed to stain some of the PC when cerebellar slices maintained in vitro were studied. The stability of the antigen-antibody binding and the use of calbindin as a marker specific for the PC in the cerebellum, is discussed. Co-localization of GABA with calbindin as well as with other calcium binding proteins are reported to be found in the PCs. While these co-localizations have led to much speculation, conclusive functional roles for them have not been identified at present.     

Cerebellar Nuclear Neurons Use Time and Rate Coding to Transmit Purkinje Neuron Pauses

Neurons of the cerebellar nuclei convey the final output of the cerebellum to their targets in various parts of the brain. Within the cerebellum their direct upstream connections originate from inhibitory Purkinje neurons. Purkinje neurons have a complex firing pattern of regular spikes interrupted by intermittent pauses of variable length. How can the cerebellar nucleus process this complex input pattern? In this modeling study, we investigate different forms of Purkinje neuron simple spike pause synchrony and its influence on candidate coding strategies in the cerebellar nuclei. That is, we investigate how different alignments of synchronous pauses in synthetic Purkinje neuron spike trains affect either time-locking or rate-changes in the downstream nuclei. We find that Purkinje neuron synchrony is mainly represented by changes in the firing rate of cerebellar nuclei neurons. Pause beginning synchronization produced a unique effect on nuclei neuron firing, while the effect of pause ending and pause overlapping synchronization could not be distinguished from each other. Pause beginning synchronization produced better time-locking of nuclear neurons for short length pauses. We also characterize the effect of pause length and spike jitter on the nuclear neuron firing. Additionally, we find that the rate of rebound responses in nuclear neurons after a synchronous pause is controlled by the firing rate of Purkinje neurons preceding it.     

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO₂. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.     

Immunohistochemical characterization of the orphan nuclear receptor ROR alpha in the mouse nervous system.

ROR alpha is an orphan nuclear receptor. A deletion mutation in the ROR alpha gene leads to severe cerebellar defects, known as the staggerer mutant mouse. Although previous in situ hybridization (ISH) studies have shown that ROR alpha is highly expressed in the cerebellum, especially in Purkinje cells, and in the thalamus, sufficient immunohistochemical (IHC) study has not yet been presented. I demonstrate here the IHC analysis of ROR alpha using a specific anti-ROR alpha antibody, in adult and developing mouse nervous system. ROR alpha immunoreactivity was observed in the Purkinje cell and molecular layers of the cerebellum. The co-localization of ROR alpha with calbindin D(28K) (CaBP) and parvalbumin indicates that ROR alpha-positive cells were Purkinje cells, stellate cells, and basket cells. In addition to the cerebellum, strong to medium ROR alpha immunoreactivity was found in the thalamus, cerebral cortex (mainly in the layer IV), dorsal cochlear nucleus (DCN), suprachiasmatic nucleus (SCN), superior colliculus, spinal trigeminal nucleus, and retina. The immunostaining was restricted in nuclei of neurons. Developmentally, ROR alpha immunoreactivity was observed in the cerebellum and thalamus from embryonal day 16 (E16). The distribution of ROR alpha immunoreactivity and ROR alpha mRNA hybridization signal was almost coincident. However, the intensity of hybridization signal was not always parallel to that of immunoreactivity.     

The role of the cerebellum in modulating voluntary limb movement commands

We recorded the activity of cerebellar Purkinje cells (PCs), primary motor cortical (M1) neurons, and limb EMG signals while monkeys executed a sequential reaching and button pressing task. PC simple spike discharge generally correlated well with the activity of one or more forelimb muscles. Surprisingly, given the inhibitory projection of PCs, only about one quarter of the correlations were negative. The largest group of neurons burst during movement and were positively correlated with EMG signals, while another significant group burst and were negatively correlated. Among the PCs that paused during movement most were negatively correlated with EMG. The strength of these various correlations was somewhat weaker, on average, than equivalent correlations between M1 neurons and EMG signals. On the other hand, there were no significant differences in the timing of the onset of movement related discharge among these groups of PCs, or between the PCs and M1 neurons. PC discharge was modulated largely in phase, or directly out of phase, with muscle activity. The nearly synchronous activation of PCs and muscles yielded positive correlations, despite the fact that the synaptic effect of the PC discharge is inhibitory. The apparent function of this inhibition is to restrain activity in the limb premotor network, shaping it into a spatiotemporal pattern that is appropriate for controlling the many muscles that participate in this task. The observed timing suggests that the cerebellar cortex learns to modulate PC discharge predictively. Through the cerebellar nucleus, this PC signal is combined with an underlying cerebral cortical signal. In this manner the cerebellum refines the descending command as compared with the relatively crude version generated when the cerebellum is damaged.     

A real-time spiking cerebellum model for learning robot control

We describe a neural network model of the cerebellum based on integrate-and-fire spiking neurons with conductance-based synapses. The neuron characteristics are derived from our earlier detailed models of the different cerebellar neurons. We tested the cerebellum model in a real-time control application with a robotic platform. Delays were introduced in the different sensorimotor pathways according to the biological system. The main plasticity in the cerebellar model is a spike-timing dependent plasticity (STDP) at the parallel fiber to Purkinje cell connections. This STDP is driven by the inferior olive (IO) activity, which encodes an error signal using a novel probabilistic low frequency model. We demonstrate the cerebellar model in a robot control system using a target-reaching task. We test whether the system learns to reach different target positions in a non-destructive way, therefore abstracting a general dynamics model. To test the system's ability to self-adapt to different dynamical situations, we present results obtained after changing the dynamics of the robotic platform significantly (its friction and load). The experimental results show that the cerebellar-based system is able to adapt dynamically to different contexts.     

A novel inhibitor rescues cerebellar defects in a zebrafish model of Down syndrome–associated kinase Dyrk1A overexpression

The highly conserved dual-specificity tyrosine phosphorylation–regulated kinase 1A (Dyrk1A) plays crucial roles during central nervous system development and homeostasis. Furthermore, its hyperactivity is considered responsible for some neurological defects in individuals with Down syndrome. We set out to establish a zebrafish model expressing human Dyrk1A that could be further used to characterize the interaction between Dyrk1A and neurological phenotypes. First, we revealed the prominent expression of dyrk1a homologs in cerebellar neurons in the zebrafish larval and adult brains. Overexpression of human dyrk1a in postmitotic cerebellar Purkinje neurons resulted in a structural misorganization of the Purkinje cells in cerebellar hemispheres and a compaction of this cell population. This impaired Purkinje cell organization was progressive, leading to an age-dependent dispersal of Purkinje neurons throughout the cerebellar molecular layer with larval swim deficits resulting in miscoordination of swimming and reduced exploratory behavior in aged adults. We also found that the structural misorganization of the larval Purkinje cell layer could be rescued by pharmacological treatment with Dyrk1A inhibitors. We further reveal the in vivo efficiency of a novel selective Dyrk1A inhibitor, KuFal194. These findings demonstrate that the zebrafish is a well-suited vertebrate organism to genetically model severe neurological diseases with single cell type specificity. Such models can be used to relate molecular malfunction to cellular deficits, impaired tissue formation, and organismal behavior and can also be used for pharmacological compound testing and validation.     

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca²⁺-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca²⁺-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.