Mutant mice as a model for cerebellar ataxia.

Not later than two synapses after their arrival in the cerebellar cortex all excitatory afferent signals are subsequently transformed into inhibitory ones. Guaranteed by the exceedingly ordered and stereotyped synaptic arrangement of its cellular elements, the cerebellar cortex transmits this inhibitory result of cerebellar integration exclusively via Purkinje cells (PCs) in a precise temporal succession directly onto the target neurons of the deep cerebellar and vestibular nuclei. Thus the cerebellar cortex seems to produce a temporal pattern of inhibitory influence on these target neurons that modifies their excitatory action in such a way that an activation of muscle fibers occurs which progressively integrates the intended motion into the actual condition of the motoric inventory. In consequence, disturbances that affect this cerebellar inhibition will cause uncoordinated, decomposed and ataxic movements, commonly referred to as cerebellar ataxia. Electrophysiological investigations using different cerebellar mouse mutants have shown that alterations in the cerebellar inhibitory input in the target nuclei lead to diverse neuronal responses and to different consequences for the behavioural phenotype. A dependence between the reconstitution of inhibition and the behavioural outcome seems to exist. Obviously two different basic mechanisms are responsible for these observations: (1) ineffective inhibition on target neurons by surviving PCs; and (2) enhancement of intranuclear inhibition in the deep cerebellar and vestibular nuclei. Which of the two strategies evolves is dependent upon the composition of the residual cell types in the cerebellum and on the degree of PC input loss in a given area of the target nuclei. Motor behaviour seems to deteriorate under the first of these mechanisms whereas it may benefit from the second. This is substantiated by stereotaxic removal of the remaining PC input, which eliminates the influence of the first mechanism and is able to induce the second strategy. As a consequence, motor performance improves considerably. In this review, results leading to the above conclusions are presented and links forged to human cerebellar diseases.

Differential number of glycine- and GABA-immunopositive neurons and terminals in the deep cerebellar nuclei of normal and Purkinje cell degeneration mutant mice.

The total number of glycine-immunopositive (Gly+) neurons in the deep cerebellar nuclei (DCN) was quantified under normal conditions in wild-types (B6C3Fe) and compared with the Purkinje cell-deprived situation in Purkinje cell degeneration (PCD)-mutants by using an unbiased stereological method, the disector. In addition, the size and density of Gly+ terminals, the number of gamma-aminobutyric acid immunopositive (GABA+) somata and the somatal colocalization of Gly and GABA were determined. In both wild-types and PCD mutants, Gly+ somata are distributed relatively homogeneously among the different subdivisions of the DCN. However, in the complete volume of the DCN, which is reduced in PCD mutants by 52%, 8,582 Gly+ neuronal somata are present in wild-types and 14,637 in PCD mutants, which corresponds to an increase of 70.5% in the mutant. In contrast, the total number of GABA+ somata is almost the same in wild-types (16,713) and in PCD mutants (15,339). The number of neurons that colocalize both Gly and GABA is again almost identical in wild-types (3,976) and PCD mutants (3,861). Moreover, the size and number of Gly+ terminals contacting DCN neurons of PCD mutants are increased significantly compared to the wild-types. These data define for the first time the normal distribution of glycine and its somatal colocalization with GABA in the DCN of the mouse. In addition, it is shown that the Purkinje cell loss in PCD mutants leads to a significant increase in Gly+ somata and to a larger size and number of Gly+ boutons in the DCN. This suggests that the respective neurons are capable of exerting an enhanced inhibitory synaptic activity on their target neurons, substituting, at least in part, for the lost Purkinje cell (PC) inhibition. Probable correlations of these findings with the mildness of the motor disturbances found in PCD mutants are discussed.

Course and targets of the calbindin D-28k subpopulation of primary vestibular afferents.

The subpopulation of primary vestibular afferents (PVA) displaying immunoreactivity for the calcium binding protein Calbindin D-28k (Calb) is constituted of particularly large bipolar neurons in the vestibular ganglion (VG) that innervate the central regions of the vestibular end organs exclusively via calyx endings on type I vestibular hair cells. These large-diameter PVA are characterized by irregular spontaneous discharge patterns and predominantly phasic firing properties with respect to natural vestibular stimulation. The present study describes the complete course and terminations of Calb+ PVA in the cerebellar cortex, the cerebellar (CN) and vestibular nuclei (VN) of the mouse. To eliminate the two sources of Calb+ fibers in the cerebellum, i.e., the Calb+ primary vestibular input and the axons of cerebellar Purkinje cells (PC), in their totality, a unilateral eighth nerve transection was performed in the PC-deficient mutant mice, Purkinje cell degeneration (pcd/pcd) and Lurcher (Lc/+). Neurectomy in these mutants results in a complete ipsilateral loss of Calb+ fibers in the cerebellar cortex, the CN and VN. The Calb+ primary vestibular input on the contralateral side terminates solely in the rostral half of the ventral uvula and in the nodulus of the cerebellar cortex. Calb+ fibers traverse all three subdivisions of the CN, but terminations were found only in the lateral and medial cerebellar nuclei. In the VN, Calb+ PVA terminations were restricted to the superior, the ventral part of the lateral, the lateral portion of the medial, and the inferior vestibular nuclei. Calb + terminals were also present in the small cell group Y and Cajal's interstitial nucleus of the vestibular nerve as well as in defined areas of the reticular formation. All Calb + PVA are strictly unilateral. The results show that the Calb+ subpopulation of VG neurons is the sole source of Calb+ fibers and terminals in the PC-deficient cerebellum and the VN. The central input of this distinct subgroup of PVA is distributed in narrow posterior vermal areas and parts of the CN and VN. The cerebellar mutants, Purkinje cell degeneration and Lurcher, provide excellent tools to selectively investigate the subgroup of Calb+ PVA in the mouse in its entirety.

Diverse effects of Purkinje cell loss on deep cerebellar and vestibular nuclei neurons in Purkinje cell degeneration mutant mice: a possible compensatory mechanism.

The genetic defect in the Purkinje cell degeneration (PCD) mutant mouse completely disrupts the cerebellar corticonuclear connection through intrinsic action on the final integrating unit of the cerebellar cortex, the Purkinje cell (PC). The postsynaptic target neurons of the PC in the deep cerebellar nuclei (DCN) and the vestibular nuclei (VN) are denervated by this PC loss by more than two-thirds of their total y-aminobutyric acid (GABA)-ergic innervation. This massive disinhibition should be reflected in an increased and thus electrophysiologically detectable activity of the respective neurons. To address this question, we performed extracellular recordings of PCD mutant and corresponding wild-type VN neurons under sinusoidal vestibular stimulation. The response amplitudes (neuronal response to sinusoidal rotation) of VN neurons in PCD mutant mice showed a decrease rather than the expected increase. The same was true for the mean resting rate, whereas the phase relationships were unaffected for the most part. This finding is a clear indication of compensatory reactions in the VN that substitute quantitatively for the lost PC inhibition. The expression of the calcium-binding protein parvalbumin (Parv) is assumed to correlate with the physiological activity of neurons, and Parv is localized predominantly in inhibitory neurons. Because inhibitory inter- or projecting neurons are also largely denervated by the PC loss, Parv immunocytochemistry also was performed. In wild-type mice, only very few Parv-immunopositive (Parv+) somata were present in the VN, and none were present in the DCN. In PCD mutant mice, a substantial number of Parv+ neuronal somata were visible in the VN, and even more were visible in the DCN. This increase in Parv+ somata in PCD mutant mice is closely related temporally and spatially to the extent of denervation caused by the PCD. Parv+ neuronal somata are first visible in the dentate nucleus at postnatal day (P) 24 and appear in the other cerebellar and VN up to P29. Direct double labeling of Parv and GABA and of Parv and glycine reveals that the large majority of Parv + neurons colocalize GABA, glycine, or both inhibitory transmitters. These results show that neurons that are postsynaptic to cerebellar PC develop diverse physiological and biochemical reactions in the course of genetically determined PCD. These mechanisms are likely to contribute to the phenotypically mild motor disturbances observed in PCD mutant mice.

Dependence of parvalbumin expression on Purkinje cell input in the deep cerebellar nuclei.

A complete loss of Purkinje cell (PC) input leads to an increase in expression of the calcium-binding protein parvalbumin (Parv) in neurons of the deep cerebellar nuclei (DCN) of PC degeneration (pcd) mutants. To verify this apparent dependence of Parv expression on PC input in the DCN, the patterns of expression in five other cerebellar mutants (weaver, staggerer, leaner, nervous, and lurcher) with differing grades and chronologies of PC loss were compared. Degree and time course of PC loss and the subsequent denervation of DCN neurons were monitored by using Calbindin D-28k (Calb) immunocytochemistry. Similar to pcd mice, somatal Parv in lurcher mutants increased massively throughout the cerebellar nuclei. In nervous and leaner mutants, somatal Parv was restricted to almost completely denervated nuclear areas, whereas areas with appreciable remnants of PC input were spared. The first appearance of Parv+ somata was closely correlated with the time course of PC degeneration--postnatal day 19 in lurcher mutants and postnatal day 23 in nervous mutants. In staggerer mice, neurons immunopositive for Parv as well as for Calb were present in outer DCN areas, likely representing ectopic PCs rather than DCN neurons. No Parv+ DCN somata were found in weaver mutants at any time. In conclusion, increased expression of somatal Parv in DCN neurons is not restricted to the specific histopathology in pcd mutants but is a common mechanism that is dependent on the topography and severeness of PC-input loss. The functional significance of the Parv increase and its possible contribution to the degree of motor disability among the different mutants are discussed.

Vermectomy enhances parvalbumin expression and improves motor performance in weaver mutant mice: an animal model for cerebellar ataxia.

In the Weaver mutant mouse (wv/wv), an animal model for hereditary cerebellar ataxia, electrophysiological experiments have revealed a disorganized output of cerebellar Purkinje cells (the latter using GABA as an inhibitory transmitter) which, by a cascade of mechanisms, was thought to be the cause of the poor motor abilities. In Purkinje cell degeneration mice (pcd/pcd) lacking nearly all Purkinje cells and displaying milder motor deficiencies than wv, in comparison to wild-type mice, a strong increase in parvalbumin- and (co-localized with parvalbumin) glycine-immunopositive somata in the deep cerebellar and vestibular nuclei has recently been found. It was therefore intriguing to investigate whether motor performance in weaver mutants could be ameliorated by applying cerebellar lesions to eliminate the faulty output and to look for a change in transmitter weighting, indicated by a strong increase in parvalbumin-positive somata in areas (the respective target areas) which were formerly devoid of it. Ten Weaver mutants were subjected to cerebellar lesions. After removal of the vermis a total abolition of tremor, a definite improvement in the balance of affected body parts, an increase in locomotor activity when tested in an open-field matrix, and a strong increase in parvalbumin expression in Weaver mutant deep cerebellar and vestibular nuclei in comparison to wild-types have indeed been found. Increase in motor activity (or explorative behaviour) has been placed in relation to learning mechanisms. The increase in parvalbumin expression and the observed improvement in motor abilities and mechanisms probably related to learning underline the hypothesis that any change in the physiological equilibrium of the brain function by removal of input or output related to an assembly of nerve cells leads to a cascade of changes at the transmitter and neuronal level in near or distant connected brain structures.

Autoradiographic analysis of benzodiazepine receptors in mutant mice with cerebellar defects.

[3H]Flunitrazepam binding sites were analysed in the cerebellar cortex, deep cerebellar and vestibular nuclei of Purkinje cell deficit (pcd) mutant mice which have an almost complete postnatal loss of Purkinje cells, and weaver mutant mice, in which there is a postnatal degeneration of granule cells. Increases in [3H]flunitrazepam binding site densities were found in the molecular and granule cell layer of weaver mutant mice, whereas decreases were observed in pcd mutant mice when compared to wildtype 'control' mice. Apparently unaltered benzodiazepine receptor densities were found in the flocculus of pcd mutant mice, whereas increases were seen in the flocculus of weaver mutant mice. The densities of benzodiazepine binding sites in the medial and lateral deep cerebellar nuclei of both mutants significantly exceeded those in control mice. Significant increases in flunitrazepam binding sites were also found in the superior and spinal nucleus of the vestibular complex of pcd mice as compared to wildtype. In the weaver mutants the benzodiazepine receptor density is enhanced in the superior and medial vestibular nucleus. Apparently unaltered numbers of such receptors compared to the wildtype control group were found in the remaining vestibular nuclei of both mutants.

Enlargement of GAD-immunopositive terminals in the lateral vestibular nucleus (LVN) of weaver mutant mice.

Reorganization of cerebellar circuitry due to specific cell loss in Weaver mutants causes physiological and morphological alterations in the terminal domains of the GABAergic cerebellar corticovestibular projections. In this study sizes of anti-GAD immunopositive terminals in the dorsal part of the lateral vestibular nucleus (dLVN) of normal mice and Weaver mutants were quantified morphometrically. In anti-GAD-immunoreacted material terminal sizes in the dLVN of Weaver exceed significantly those of coprocessed wildtypes. This suggests that the cerebellar disturbances in Weaver predispose the normal synaptic remodelling observed in wildtypes towards the formation of enlarged terminals.

DNA fragmentation and activation of c-Jun in the cerebellum of mutant mice (weaver, Purkinje cell degeneration).

Weaver and Purkinje cell degeneration (pcd) are autosomal recessive mutations in the mouse characterized by an almost complete loss of cerebellar Purkinje neurones and granule cells, respectively. Developmental neuronal death occurs by activation of an apoptotic pathway and chromatin condensation has been observed in degenerating granule cells of weaver mutants. In the present study we demonstrate nuclear DNA fragmentation in Purkinje cells of pcd mice and in granule cells of weaver mutants during the period of neuronal degeneration using in situ end labelling by terminal transferase and fluorescein-dUTP. Furthermore, activation of candidate cell death effector gene c-jun has been detected exclusively within the affected cell populations by immunohistochemistry. Both labelled DNA fragments and nuclear c-Jun immunoreactivity were virtually absent in wild-type animals. Thus, genetically determined cell death in pcd and weaver mutant mice has features of apoptosis and may require activation of cell death effector genes.

Improvement in motor performance of Weaver mutant mice following lesions of the cerebellum.

In Weaver mutants (B6CBA wv/wv) cerebellar granule cells degenerate almost completely postnatally. A partial loss of Purkinje cells (PC) and a degeneration of dopaminergic cells in the substantia nigra have also been found. Weaver mice suffer from striking motor symptoms, including difficulty in walking without toppling over. In an attempt to influence the poor motor performance, the cerebellum in young animals was removed, thus eliminating the faulty output of surviving PCs, demonstrated electrophysiologically. Unoperated Weaver, lesioned wildtypes and one sham mouse were used as controls. Before and after operation, a battery of behavioural tests was performed. In Weaver mice, tumbling to the side (t) and the relation of t to the motor activity (k) while traversing an open-field matrix, (t/k), improved considerably, as did manoeuvring on a slanted wire mesh, but keeping balance on a wooden bench did not to the same degree. Locomotor activity alone improved in some animals. In wildtypes no significant changes occurred after operation, with the exception of a strong reduction in locomotor activity. The experiments demonstrate that the motor performance in Weaver mutant mice benefits from removal of their cerebellum.

Plasticity of GABAergic terminals in Deiters' nucleus of weaver mutant and normal mice: a quantitative light microscopic study.

This study reports on the developmental changes in size and the average density of GABAergic axonal boutons bordering on the somata of large neurons in the dorsal part of the lateral vestibular nucleus (Deiters' nucleus) in normal and mutant mice. Weaver mutants, PCD mutants and the corresponding wild types were used to test for size alterations and differences in the number of GABA-immunopositive terminals. Hemicerebellectomized animals were examined in addition. Quantification of bouton profile size was performed from 30-microns-thick vibratome and 0.5-micron Araldite-embedded semi-thin sections immunoreacted for GABA from 7 days postnatally up to an age of 9 months. Terminal density was determined at the 5-6 month stage from semi-thin sections only. Morphometric analysis over the lifetime of normal animals (B6CBA) revealed a progressive increase in the size of bouton profiles, which peaked at 5-6 months and reached sizes of 2-3 microns2. In weaver mutants a parallel development in terminal size was found to be present, but the size of the largest terminals exceeded those of the controls by 75-100%, reaching 3-6 microns2 with the same time course. PCD mutants, with an almost total absence of Purkinje cells had, on the contrary, small bouton profiles that reached a maximum of only 2 microns2. The hemicerebellectomized animals responded with decreased bouton profile size ipsilaterally. The terminal numbers per unit membrane length were surprisingly similar in wild types and weaver mutants, despite a reduction in Purkinje cells of almost 50% in the weaver anterior lobe.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiology and GABA-immunocytochemistry in the vestibular nuclei of normal (C57BL/6J) and Leaner mutant mice.

Neurological disorders of genetic origin that lead to distinct disarrangements of the cerebellar wiring and cause a specific motor behaviour are likely to differentially influence the response properties and activity of postsynaptic cerebellar target neurons in the deep cerebellar (DCN) and vestibular nuclei (VN). Comparative electrophysiological and morphological analyses of these neurons in different mutants may increase our understanding of the physiological consequences of cell damage to the cerebellum and help to elucidate the relationships between histopathology and severeness of motor impairment. The Leaner mutation removes GABAergic inhibitory input to the VN predominantly originating from Purkinje cells (PC) located in the anterior lobe of the cerebellum and causes extremely severe motor disability when compared to other cerebellar mutants. In the present study the electrophysiological properties of naturally stimulated (sinusoidal head and body rotation) neurons in the VN of Leaner mutants and their corresponding wild-types (C57BL/6J) were investigated. Neuronal activity of VN single units in Leaner mutants is significantly increased over that of wild-types (frequency range 0.2-0.6 Hz) and more pronounced for type I neurons than for type II. Phase relationships and spontaneous activity are similar at these frequencies in both groups. To elucidate the degree of GABAergic input loss of VN-neurons, quantitative morphometric and numerical analyses of GABA-immunopositive synaptic boutons in the lateral VN of Leaner mutants were performed in addition and revealed significantly smaller terminals and a massive decrease (80%) in Leaner mutant terminal numbers compared to controls. In the context of the findings recently obtained in Weaver and Purkinje cell degeneration (PCD) mutants, the results in Leaner suggest that the loss of inhibition due to the PC degeneration in the anterior vermis leads to a differential enhancement of type I and type II target neuron activity in the VN. The overall activity in Leaner is intermediate between PCD (no increase in activity) and Weaver (strong increase of type I but no increase of type II). GABA-immunocytochemistry supports the idea that in Leaner the lost GABAergic PC-innervation of the lateral VN has not been replaced by surviving PCs, which is in contrast to Weaver where sprouting of GABAergic terminals in this nucleus was observed. Substitution of lost cerebellar inhibition by non-cerebellar sources, as suggested in the case of PCD-mutants, is uncertain in Leaner due to the increased activity of type I target neurons. These conditions may contribute, among others, to the severe motor disturbances in Leaner.

Neurons in the spinal cord of the frog responding to moving visual stimuli.

Impulse discharges were recorded extracellularly with microelectrodes from the spinal cord during visual stimulation. With a sinusoidally moved surround three classes of neurons could be discerned: S-1 neurons activated by movement of the visual stimulus to the right, S-2 neurons by movement to the left and S-3 neurons activated by stimulus movement in both directions. Maximum activation was observed at movements between 0.08 and 0.1 Hz and a marked decrease in activation above 0.2 Hz. All neurons responding to visual stimulation were also activated by vestibular stimulation.

Calbindin D-28k in the lateral vestibular nucleus of mutant mice as a tool to reveal Purkinje cell plasticity.

Antibodies against the calcium-binding protein Calbindin D-28k (CaBP) are specific markers of cerebellar Purkinje cells (PC). To identify the origin of CaBP-immunopositive (CaBP+) terminals and fibres in the dorsal part of the lateral vestibular nucleus (dLVN), brains of Purkinje cell degeneration mutants (PCD) were immunoreacted for CaBP using the avidin-biotin method (ABC). In PCD an almost complete loss of CaBP+ fibres and terminals in the dLVN compared to the wildtype and the Weaver mutant was present. Morphometric analysis of CaBP+ synaptic terminals in the dLVN of adult Weaver mutants showed that the maximum and mean terminal size exceeded those in wildtypes by almost twice, which is a far larger difference than in GABA-immunoreacted material. The results show that CaBP-immunoreactivity and terminal size expansion in Weaver are both mainly attributable to PCs. Moreover, it can be concluded that the colocalization of CaBP and GABA in fibres and terminals of the dLVN in normal animals is almost entirely restricted to the PC-innervation of this nucleus. Therefore CaBP-immunocytochemistry is an excellent tool to selectively investigate the direct PC-projections in the dLVN, as it sets off the GABAergic PC-innervation from the total GABAergic innervation of this area.

Responses of retinal ganglion cells to eyeball deformation: a neurophysiological basis for "pressure phosphenes".

By means of microelectrodes, the activity of single neurons (on-center, off-center ganglion cells, latency class I and class II neurons) was recorded from the optic tract of anesthetized cats. Eyeball deformation in total darkness led fairly consistently to an activation of the on-center ganglion cells, while off-center ganglion cells were inhibited. The latency and strength of this activation or inhibition seemed to be mainly dependent on the strength of eyeball indentation and the location of the neurons relative to the point of eyeball indentation. Some on-center neurons (mostly latency class I) also exhibited a short activation at "deformation off". For comparison, the responses of retinal ganglion cells to eyeball deformation in a hydrostatically open system and to a sudden increase in the intraocular pressure (closed system) are described. The neurophysiological data are explained by the assumption that eyeball indentation leads to a nonuniform tangential stretch of the retina, which exerts a locally variable depolarization of horizontal cells. This horizontal cell depolarization leads either directly or via a feedback loop through cone pedicles to a depolarization of on-bipolars and a hyperpolarization of off-bipolars. These effects determine in turn the responses seen at the ganglion cell level. It is emphasized that eyeball deformation can be used as an independent tool in transmitter studies of the retina.

Neurophysiological properties of the retinal ganglion cell classes of the Cuban treefrog, Hyla septentrionalis.

The properties of the retinal ganglion cell classes in the cuban treefrog Hyla septentrionalis were studied qualitatively and quantitatively. In the superficial layers of the optic tectum three main classes of afferent optic nerve fibers could be distinguished, class-1*, class-3 and class-4 neurons. Hyla displays a more "classical" organization of the receptive fields in class-1* neurons and a weaker inhibitory surround and lower thresholds with respect to velocity, size and contrast than in Bufo or ranid frogs. The functions for velocity, contrast, size of stimulus, neuronal adaptation and adaptation to background luminance level were evaluated. Experiments with monochromatic light spots are mentioned. The results are compared to those of other amphibia and the diversity of the retinal ganglion cell properties in the different species is stressed as an important factor in the processing of the various ganglion cell types at the tectal level.