Antibody formation against beta-tubulin class III in response to brain trauma.

Brain trauma typically leads to neuronal damage and loss. Assuming a transient autoimmune response to debris of the damaged neurones, we have monitored serum titres of IgG and IgM antibodies to beta-tubulin class III (betaTcIII), which is almost exclusively found in neuronal cytoskeletons. In 15 out of 18 patients, the peak of the IgG or IgM antibody titre appeared in the serum within 3 weeks of a brain trauma.

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.

Corticofugal connections between the cerebral cortex and the vestibular nuclei in the rat.

The distribution of cortical efferent connections to the vestibular nuclei was quantitatively analyzed by means of retrograde axonal transport of horseradish peroxidase, wheat germ agglutinin-horseradish peroxidase, and Fast Blue in rats. The tracer substances were injected into the spinal vestibular nucleus (SpVe), the caudal part of the medial vestibular nucleus (MVe), and nucleus X of Brown Norwegian rats. Projections to the vestibular nuclei were revealed bilaterally, but predominantly contralaterally from five cortical areas: (1) the parietotemporal region (PT) which occupied the caudal two-thirds of the secondary somatosensory area and spread over the caudal part of the primary somatosensory area and the visceral cortex; (2) the anterior forelimb (AF) overlapping the anterior part of the forelimb area and the transitional zone; (3) the anterior hindlimb (AH) overlapping the anterior part of the hindlimb area and the transitional zone; (4) the lateral forelimb (LF) centered in the intercalated zone lateral to the forelimb area; and (5) the ventrotemporal region (VT) located at the ventral part of the temporal cortex. In addition to these cortical fields, the frontal cortex was found to project directly to the vestibular nuclei. These corticofugal projections were verified in experiments in which biocytin was injected into the rat PT. Anterogradely labelled fibers were traced predominantly contralaterally to the SpVe, caudal part of the MVe, and nucleus X. It is suggested that the rat corticofugal projections to the caudal vestibular nuclei modify vestibular reflexes to assist in coordinating eye, head and body movements during locomotion.

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.

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.

Impaired inhibition of epileptiform activity by baclofen, but not by adenosine in the weaver hippocampus.

The weaver defect results in a loss of baclofen- and adenosine-gated K+ conductance in the hippocampus of adult homozygous (wv/wv) mice. In addition, suppression of hippocampal epileptiform activity by baclofen is impaired (Jarolimek, W., Bäurle, J., Misgeld, U., 1998. Pore mutation in a G protein-gated inwardly rectifying K+ channel subunit causes loss of K+ dependent inhibition in weaver hippocampus. Journal of Neuroscience 18, 4001-4007). We used wv/wv and wild-type (+/+) mice to determine whether K+ conductance increases are essential for the suppression of epileptiform activity by R-baclofen and adenosine in disinhibited hippocampal slices. In wv/wv mice R-baclofen was less potent by two orders of magnitude in reducing the frequency of spontaneous synchronous burst discharges than in +/+ mice. Endogenous adenosine and adenosine A1 receptor agonists differed only slightly in their efficacy to inhibit spontaneous synchronous burst discharges in wv/wv and +/+ mice. The findings on adenosine A1 receptors suggest that the varied efficacy of R-baclofen in wv/wv and +/+ mice may not be explained solely on the basis of a loss of ligand-gated K+ conductance. Therefore, we investigated the affinity of GABA(B) receptors for the antagonist CGP55845A in wv/wv and +/+ hippocampi. Schild plot analysis revealed a K(D) for the GABA(B) antagonist CGP55845A 10 fold higher in wv/wv than in +/+ mice. The data suggest that an alteration of GABA(B) receptors could contribute to the reduced efficacy of R-baclofen to suppress hippocampal epileptiform activity in weaver mice, while the suppression by adenosine remains largely unaffected.

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.

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.

Vestibular ganglion neurons survive the loss of their cerebellar targets.

Neuronal survival during mammalian development crucially depends on target-derived neurotrophic factors. Target loss removes this trophic support and leads in most cases to the transsynaptic retrograde degeneration of the respective afferents. Primary vestibular afferents (PVA) originating from bipolar neurons in the vestibular ganglion (VG) are the first mossy fibers that enter the cerebellum, but little is known about the survival requirements of VG neurons. In the present study the influence of the differential granule cell (GC) target loss on the survival of VG neurons was studied quantitatively using unbiased stereological methods in the cerebellar mutants Purkinje cell degeneration (pcd/pcd), Lurcher (Lc/+), and Weaver (wv/wv). Neither the secondary GC loss in the Purkinje cell deficient mutants pcd/pcd and Lc/+, nor the primary loss of GCs in wv/wv produced any significant reduction in the total number of bipolar neurons in the VG compared to controls. So, PVA neurons are highly resistant to cerebellar target deprivation and survive in the absence of cerebellar granule and Purkinje cells, regardless of whether the target loss occurs before (in wv/wv), during (in Lc/+) or after (in pcd/pcd) the mossy fiber-granule cell synaptogenesis.

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.

Co-localization of glycine and calbindin D-28k in the vestibular ganglion of the rat.

Bipolar neurons of the vestibular ganglion (VG) are biochemically heterogeneous. The calcium-binding protein calbindin D-28k (Calb) is present only in a subset of particularly large neurons, and the amino acid glycine (Gly) has been immunocytochemically detected in a group of similarly sized cells. The close correspondence in size and number of cells in these two subgroups suggests that the Calb- and Gly-positive populations may be identical. In order to test this hypothesis, we performed direct and indirect double-labeling for Calb and Gly in the VG of the rat. The results confirm the existence of a distinct subpopulation of Calb-immunoreactive neurons, consisting of the largest cells in the VG. In contrast, the vast majority of neurons in the VG display some degree of Gly immunoreactivity, which gradually decreases from intense to almost unlabeled. Direct evidence is provided that the fraction of cells most heavily labeled by Gly antibodies is not identical with the Calb-positive subpopulation. Although some correlation between soma diameter and labeling intensity exists, Gly immunoreactivity is clearly not restricted to large neurons. The findings imply that the functional mechanisms in which Gly is potentially involved may be shared by a large spectrum of primary vestibular afferents with a broad range of physiological properties.

Co-localization of glutamate, choline acetyltransferase and glycine in the mammalian vestibular ganglion and periphery.

Glutamate (Glu) is considered to be the main transmitter at the central synapses of primary vestibular afferents (PVA) and glycine (Gly) is assumed to play a modulatory role. In the vestibular periphery a transmitter role for acetylcholine (ACh) has been attributed chiefly to vestibular efferents (VE), however only a subset of VE neurons displays immunoreactivity (ir) for choline acetyltransferase (ChAT) and acetylcholine esterase (AChE). Controversial results exist on the presence of these two enzymes in PVA. In this study the presence of Glu, ChAT, Gly and their co-localization in the vestibular ganglia (VG) and end organs of mouse, rat, guinea pig and squirrel monkey were investigated. In the VG all bipolar neurons display strong Glu-ir and the majority of cells show a graded ChAT-ir and Gly-ir in all species examined. ChAT and Gly are present in highly overlapping neuronal populations and with a similar gradation. In the end organs ChAT and Gly are again co-localized in the same sets of fibers and endings. In conclusion, in the vestibular ganglion and end organs ChAT appears also to be present in primary afferents rather than being restricted to efferent processes. ChAT in primary afferents might indicate a modulatory or co-transmitter function of acetylcholine.

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.

Unbiased number of vestibular ganglion neurons in the mouse.

Vestibular sensory information from the labyrinth and otolith organs is conducted to the central nervous system exclusively via primary vestibular afferents (PVA) originating from neurons located in the vestibular ganglion (VG). In the present study, the total number of VG neurons was determined in two different wild-type mouse strains using the principles of unbiased stereological counting methods by means of the physical disector. 3316 (+/-225 SD) neurons were present in the VG of the B6CBA-strain and 3551 (+/-239 SD) in C57BL/6J-mice. Since no statistical difference was detected between the two strains, the pooled mean number was 3433 (+/-232 SD) neurons. This is the first unbiased estimate of VG neurons aimed at providing a numerical basis for comparative studies and for the impact of experimental, pharmacological and pathological conditions as well as ageing on the survival and maintenance of VG neurons.

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.

Gamma-aminobutyric acid(B) autoreceptors in substantia nigra and neostriatum of the weaver mutant mouse.

The weaver mutation causes cell loss in the center of the substantia nigra, pars compacta. We compared the depression of gamma-aminobutyric acid (GABA)(A) synaptic currents by the GABA(B) agonist R-baclofen in pars compacta neurons of weaver mice which were largely spared from cell degeneration and of wild-type mice. In weaver neurons the suppression of GABA(A) synaptic currents by R-baclofen was reduced compared to wild-type neurons. The EC(50) of R-baclofen was 6.3 times higher in weaver than in wild-type mice. In the neostriatum, which is not a target of the mutation, such a difference did not exist. We conclude that in the pars compacta the weaver mutation leads to a reduced presynaptic autoinhibition through GABA(B) receptors which may promote survival of a subset of weaver neurons in the pars compacta.

Axonal torpedoes in cerebellar Purkinje cells of two normal mouse strains during aging.

The present study systematically investigated the proportional evolution of Purkinje cell (PC) axonal swellings, also termed torpedoes, during aging of the two unrelated mouse strains B6CBA and C57BL/6J. Torpedoes were identified using monoclonal antibodies against the calcium-binding protein calbindin D-28k in mice ranging in age from 8 days postnatally up to 32 months. The relative density of PCs bearing torpedoes in animals up to 6 months of age was less than 0.1%. The number increases between 6-8 months and rises further in older mice almost linearly up to 13.7% affected PCs in the oldest animal (32 months) studied. In contrast, PC loss, as indicated by parvalbumin-immunoreactive empty baskets, is only at a very moderate level (less than 0.5%) in these strains. While the proximal axonic segments often show two and occasionally up to five swellings and frequently appear to be hypertrophied as a whole, the dendritic trees and neuronal somata of the affected PCs exhibit normal morphology. On rare occasions adaptive reactions indicated by "arciform axons" and enlarged varicosities of recurrent collaterals were observed. The results demonstrate that in addition to age-related PC loss of whatever degree, axonal disturbances of PCs, indicated by torpedoes, are present, leading most probably to a graded loss of cerebellar cortico-fungal projections.