Altered neurofilament protein expression in the lateral vestibular nucleus in Parkinson's disease.

A major cause of morbidity in Parkinson's disease (PD) is postural instability. The neuropathology underlying postural instability is unknown. Postural control is mediated by Deiters' neurons of the lateral vestibular nucleus (LVN), which are the brainstem origin of descending vestibulospinal reflexes. Deiters' neurons express the cytostructural protein, non-phosphorylated neurofilament protein (NPNFP). In PD, reduced expression of NPNFP in substantia nigra (SN) neurons is believed to contribute to dysfunction. It was the aim of this study to determine if there is altered expression of NPNFP in the LVN in PD. We immunolabeled NPNFP in brainstem sections of six aged controls (mean age 92 yo) and six PD donors (mean age 83 yo). Our results show there was a ~ 50% reduction in NPNFP-positive Deiters' neurons compared to controls (13 ± 2.0/section vs 25.7 ± 3.0/section; p < 0.01, repeated measures ANOVA). In contrast, there was no difference in NPNFP-positive counts in the facial nucleus between control and PD. The normalized intensity of NPNFP labeling in LVN was also reduced in PD (0.87 ± 0.05 vs 1.09 ± 0.03; p < 0.01). There was a 35% concurrent reduction in NPNFP-positive neuropil in PD relative to controls (p < 0.01). We also show there was an 84% increase (p < 0.05) in somatic lipofuscin in PD patients compared to control. Lipofuscin aggregation has been shown to increase not only with age but also with neurodegeneration. Furthermore, decreased NPNFP intensity was strongly correlated with increasing lipofuscin autofluorescence across all cases (R 2 = 0.81, p < 0.01). These results show two alterations in cellular content with PD, reduced expression and intensity of NPNFP and increased lipofuscin aggregation in Deiter's neurons. These changes may contribute to degeneration of postural reflexes observed in PD.

Age-dependent in vivo conversion of mouse cochlear pillar and Deiters' cells to immature hair cells by Atoh1 ectopic expression.

Unlike nonmammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters' cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in ∼10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1 followed by expression of 11 HC and synaptic markers, a process that took approximately 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for >2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression but caused cell loss of mature HCs. Together, our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age dependent and resembles normal HC development. Therefore, combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin-CASK interaction at the basal junction with the Deiter's cell.

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter's cell may contribute to efficient generation of somatic electromechanical force.

[Effects of fast-twisting long-retaining acupuncture therapy on apoptosis and expression of related proteins in vestibular nucleus in rats with vertigo induced by posterior circulation ischemia].

OBJECTIVE: To observe the effects of fast-twisting long-retaining (FTLR) acupuncture therapy on apoptosis of vestibular nucleus and expression of Caspase-3, Bcl-2 and Bax in rats with vertigo induced by posterior circulation ischemia. METHODS: A total of 70 healthy SD rats were randomly divided into a sham operation group, a model group, a medication group, a regular acupuncture group and a FTLR acupuncture group, 14 rats in each group. The rats in the model group, medication group, regular acupuncture group and FTLR acupuncture group were intervented with surgical ligation of the right common carotid artery (CCA) and the right subclavian artery (SCA) to establish the model of vertigo induced by posterior circulation ischemia; in the sham operation group, the right CCA and the right SCA were separated without ligation. The rats in the medication group were treated with gavage of flunarizine hydrochloride suspension (10 mL/kg). "Baihui" (GV 20), "Shuaigu" (GB 8) and "Fengchi" (GB 20) were selected in the two acupuncture groups. The rats in the regular acupuncture group were treated with routine acupuncture and the needles were retained for 30 min, while the rats in the FTLR acupuncture group were treated with quick twist (200-300 times/min) for 1 min and the needles were retained for 60 min. The rats in the sham operation group and the model group received no intervention. All the intervention was provided once a day for 10 days. The decline rate of local blood flow in vestibular nucleus was observed; the apoptosis of vestibular nucleus was observed by TUNEL method; the expression of Caspase-3, Bcl-2 and Bax proteins were detected by immunohistochemistry. RESULTS: Compared with the sham operation group, the decline rate of local blood flow in the right vestibular nucleus was significantly increased in the model group (P<0.01), and the apoptosis index (AI) of vestibular nucleus was significantly increased (P<0.01). Compared with the model group, the decline rates of local blood flow in the right vestibular nucleus in the two acupuncture groups and medication group were significantly reduced (P<0.01), and the AIs of vestibular nucleus cells were significantly reduced (P<0.01). The decline rate of local blood flow in the right vestibular nucleus in the FTLR acupuncture group was lower than those in the medication group and the regular acupuncture group (P<0.01, P<0.05), and the AI of vestibular nucleus was lower than those in the regular acupuncture group and the medication group (P<0.05). Compared with the sham operation group, the expression of Bcl-2 in the vestibular nucleus was significantly decreased in the model group (P<0.01), and the expressions of Bax and Caspase-3 were significantly increased (P<0.01). Compared with the model group, the expressions of Bcl-2 in the vestibular nucleus were significantly increased in the two acupuncture groups and medication group (P<0.01), and the expressions of Bax and Caspase-3 were significantly reduced (P<0.01). The expression of Bcl-2 in the vestibular nucleus in the FTLR acupuncture group was higher than those in the regular acupuncture group and the medication group (P<0.05), and the expressions of Bax and Caspase-3 were lower than those in the regular acupuncture group and the medication group (P<0.05). CONCLUSION: The FTLR acupuncture therapy could effectively inhibit the apoptosis of vestibular nucleus in rats with vertigo induced by posterior circulation ischemia, and its mechanism may be related to improving the blood supply of vestibular nucleus and regulating the expressions of Caspase-3, Bcl-2 and Bax proteins.

[Purkinje's cells in the vestibular and proprioceptive segments of rat's cerebellum following 14-day space flight].

Quantitative cytochemical and morphometric methods were used to investigate cytochrome oxidase activity and sizes of bodies and nuclei of Purkinje's cells in the medical nodulus and upper central lobule of the vermis obtained from rats sacrificed in 5-6 hours of landing after the 14-day SLS-2 mission of NASA space "shuttle" Columbia (STS-58). The reduced cytochrome oxidase activity was explained by suppression of the functional activity of Purkinje's cells in microgravity. Results of the investigations suggest weakening of the regulatory effect of the vermis Purkinje's cells on giant neurons of the dorsocaudal segment of Deiters nucleus. They also strengthen the earlier hypothesis that space flight decays the inhibitory effect of nodulus Purkinje's cells on the medial vestibular nucleus for the reason of change in the "velocity storage" in mammals during and after flight.

Morphometric analysis of the AMPA-type neurons in the Deiter's vestibular complex of the chick brain.

Chicken (Gallus gallus) brains were used to investigate the typology and the immunolabel pattern for the subunits composing the AMPA-type glutamate receptors (GluR) of hindbrain neurons of the dorsal (dND) and ventral nuclei (vND) of the Deiter's vestibular complex (CD), which is the avian correspondent of the lateral vestibular nucleus (LVN) of mammals. Our results revealed that neurons of both divisions were poor in GluR1. The vND, the GluR2/3+ and GluR4+ label presented no area or neuronal size preference, although most neurons were around 75%. The dND neurons expressing GluR2/3 are primarily around 85%, medium to large-sized 85%, and predominantly 60% located in the medial portion of the rostral pole and in the lateral portion of the caudal pole. The majority of dND neurons containing GluR4 are also around 75%, larger (70% are large and giant), exhibiting a distribution that seems to be complementary to that of GluR2/3+ neurons. This distinct arrangement indicates functional differences into and between the DC nuclei, also signaling that such variation could be attributed to the diverse nature of the subunit composition of the GluRs. Discussion addresses the morphological and functional correlation of the avian DC with the LVN of mammals in addition to the high morphological correspondence, To include these data into the modern comparative approach we propose to adopt a similar nomenclature for the avian divisions dND and vND that could be referred as dLVN and vLVN.

The spatial distribution pattern of Connexin26 expression in supporting cells and its role in outer hair cell survival.

Mutations in the GJB2 gene (which encodes Connexin26 (Cx26)) account for about a quarter of all cases of non-syndromic deafness. Previous studies have indicated that knockout (KO) of Gjb2 gene during early postnatal days can cause outer hair cell (OHC) loss in mouse models. However, the postnatal spatial distribution pattern of Cx26 in different types of supporting cells (SCs) and the role of such distributions for the survival of OHCs is still obscure. In this study, the spatial distribution patterns of Cx26 in SCs were observed, and based on these observations different spatial Cx26-null mouse models were established in order to determine the effect of changes in the spatial distribution of Cx26 in SCs on the survival of OHCs. At postnatal day (P)3, unlike the synchronous expression of Cx26 along both longitudinal and radial boundaries of most types of SCs, Cx26 expression was primarily observed along the longitudinal boundaries of rows of Deiter's cells (DCs). From P5 to P7, radial expression of Cx26 was gradually observed between adjacent rows of DCs. When Gjb2 gene was knocked out at random in different types of SCs, about 40% of the total DCs lost Cx26 expression and these Cx26-null DCs were distributed randomly in all three rows of DCs. The mice in this randomly Cx26-null group showed normal hearing and no significant OHC loss. When using a longitudinal KO pattern to induce knockout of Gjb2 gene specifically in the third row of DCs, about 33% of the total DCs lost Cx26 expression in this specific longitudinally Cx26-null group. The mice in this group showed late-onset hearing loss and significant OHC loss, however, the morphology of corresponding DCs was slightly altered. In both experimental groups, no substantial DC loss was observed. These results indicate that longitudinal Cx26-based channels are predominant in DCs during P3-P5. The Cx26 expression along rows of DCs might play a key role in the survival of OHCs, but this longitudinal KO pattern in DCs has a limited effect on DC survival or on its postnatal development.

Oestradiol effects on neuroendocrine responses induced by water deprivation in rats.

Water deprivation (WD) induces changes in plasma volume and osmolality, which in turn activate several responses, including thirst, the activation of the renin-angiotensin system (RAS) and vasopressin (AVP) and oxytocin (OT) secretion. These systems seem to be influenced by oestradiol, as evidenced by the expression of its receptor in brain areas that control fluid balance. Thus, we investigated the effects of oestradiol treatment on behavioural and neuroendocrine changes of ovariectomized rats in response to WD. We observed that in response to WD, oestradiol treatment attenuated water intake, plasma osmolality and haematocrit but did not change urinary volume or osmolality. Moreover, oestradiol potentiated WD-induced AVP secretion, but did not alter the plasma OT or angiotensin II (Ang II) concentrations. Immunohistochemical data showed that oestradiol potentiated vasopressinergic neuronal activation in the lateral magnocellular PVN (PaLM) and supraoptic (SON) nuclei but did not induce further changes in Fos expression in the median preoptic nucleus (MnPO) or subfornical organ (SFO) or in oxytocinergic neuronal activation in the SON and PVN of WD rats. Regarding mRNA expression, oestradiol increased OT mRNA expression in the SON and PVN under basal conditions and after WD, but did not induce additional changes in the mRNA expression for AVP in the SON or PVN. It also did not affect the mRNA expression of RAS components in the PVN. In conclusion, our results show that oestradiol acts mainly on the vasopressinergic system in response to WD, potentiating vasopressinergic neuronal activation and AVP secretion without altering AVP mRNA expression.

Brainstem projection of the vestibular nerve in the guinea pig: an HRP (horseradish peroxidase) and WGA-HRP (wheat germ agglutinin-HRP) study.

We explored the course and termination of primary vestibular afferent fibers within the brainstem of the guinea pig by means of anterograde axonal transport of horseradish peroxidase (HRP) and wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Primary vestibular afferent fibers distribute within the entire vestibular nuclear complex, with the exception of the dorsal part of the lateral vestibular nucleus. The superior vestibular nucleus is characterized by the concentration of terminals within its central part. Although terminal labeling is weaker within the periphery, no area completely lacks primary input. The lateral vestibular nucleus can be divided into a ventral and a dorsal part; within the ventral part small and giant cells receive primary afferent fibers, whereas no significant terminal labeling occurs in the dorsal part. The medial vestibular nucleus shows the most uniform labeling, although the lateral part of its rostral third has a few more terminals than the medial half. Primary projection to the descending vestibular nucleus is widespread, although in its rostrodorsal part it is less impressive. Of the small cell groups commonly associated with the vestibular nuclear complex, only group y receives abundant primary input. Whereas group z completely lacks labeled fibers as well as terminals, single primary axons can be observed passing groups x and f. However, no terminals can be found within the borders of these two cell groups. Scanty projections can be detected within the prepositus hypoglossi nucleus, as well as within the external cuneate nucleus, the cochlear nucleus, the abducent nucleus, and parts of the reticular formation.

Inhibition by sodium valproate of the transport of GABA through the Deiters' neurone plasma membrane.

The transport of GABA through the microdissected plasma membrane Deiters' neurone reflects the physiological event of postsynaptic uptake of GABA by its uptake carrier. Sodium valproate at concentrations greater than or equal to 2.4 mM was able to decrease markedly (57%) such a transport. This effect, which reduces the efficiency of the GABA postsynaptic inactivation process, might be a mechanism for the potentiation by valproate of the synaptic action of GABA.

P2X receptors in cochlear Deiters' cells.

1. The ionotropic purinoceptors in isolated Deiters' cells of guinea-pig cochlea were characterized by use of the whole-cell variant of the patch-clamp technique. 2. Extracellular application of adenosine 5'-triphosphate (ATP) induced a dose-dependent inward current when the cells were voltage-clamped at -80 mV. The ATP-induced current showed desensitization and had a reversal potential around -4 mV. 3. Increasing intracellular free Ca2+ by decreasing the concentration of EGTA in the pipette solution reduced the amplitude of the ATP-gated current. 4. The order of agonist potency was: 2-methylthioATP (2-meSATP)>ATP>benzoylbenzoyl-ATP (BzATP)>alpha,beta-methyleneATP (alpha,beta,meATP>adenosine 5'-diphosphate (ADP)>uridine 5'-triphosphate (UTP)>adenosine 5'-monophosphate (AMP)=adenosine (Ad). 5. Pretreatment with forskolin (10 microM), 8-bromoadenosine-3',5'-cyclophosphate (8-Br-cyclic AMP, 1 mM), 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or phorbol-12-myristate-13-acetate (PMA, 1 microM) reversibly reduced the ATP-induced peak current. 6. The results are consistent with molecular biological data which indicate that P2X2 purinoceptors are present in Deiters' cells. In addition, the reduction of the ATP-gated current by activators of protein kinase A and protein kinase C indicates that these P2X2 purinoceptors can be functionally modulated by receptor phosphorylation.

Increased binding of GABA to its post-synaptic carrier sites on the plasma membrane of Deiters' neurons after a learning experiment in rats.

A new micromethod for studying the interaction of gamma-aminobutyric acid (GABA) with its post-synaptic uptake in a defined type of nerve cell is described. The method involves the isolation by free hand microdissection of Deiters' nerve cells from the rabbit lateral vestibular nucleus and their incubation with tritiated GABA in the presence of 100 mM Na+ at 4 degrees C. From the binding data a Kd was calculated for this interaction of 104 nM and a BMax of 8.4 X 10(5) sites/neuron. The method was applied to the study of the modifications of the binding of GABA to Deiters' neurons from control rats and rats trained to balance on a steel wire in order to reach food. This performance is a powerful stimulation to the vestibular system. The results show that, in the binding experiment with 100 nM [3H]GABA, the amount of GABA which specifically binds to the Deiters' neurons is increased by 38% in the trained rats. Analogously, when the incubation with GABA was performed at 37 degrees C, involving an intake of GABA into the neurons, the amount of GABA taken up increases by 50% in the trained group. No GABA-binding or uptake increase was found when animals were subjected to intense vestibular stimulation for a short period without learning. These results indicate that when rats learn a behavioral test which involves an improved vestibular control, there is a specific neurochemical modification in the neurons of the lateral vestibular nucleus. This modification seems to be of importance for the physiology of the neuronal circuits controlling the vestibular function in the rat.

Increase in chloride ion permeability across the nerve cell membrane after the endogenous antigen S-100 incorporation.

36Cl fluxes through microdissected Deiters' neuronal membranes have been studied in a microchamber device simulating the extra- and intracellular compartments. GABA stimulates Cl- permeability through the membranes by 24%. Also, S-100/Ca2+ incorporation into the Deiters' membrane increases 36Cl- permeation to a similar extent. The two effects do not appear to be additive. This circumstance is interpreted as indicating that S-100/Ca2+ exerts its effects via postsynaptic GABAA receptor complexes.

The effects of centrifugation on the morphology of the lateral vestibular nucleus in the rat: a light and electron microscopic study.

Very little is known about structural changes in the central nervous system following exposure to increased g forces. Sprague-Dawley rats were centrifuged at Ames Research Center at 2.76-4.15 x g for periods ranging from 4 days to 21 months. The lateral vestibular nuclei were processed for electron microscopy and examined for evidence of structural alteration as a result of centrifugation. The number of filamentous nuclear inclusions, varicosities, and relative number of axosomatic synaptic terminals containing flattened vesicles (presumed inhibitory in function) increased in centrifuged rats. Altered mitochondria also were noted in that cell bodies of Deiters' neurons. Neuroaxonal dystrophy (NAD) was found in long-term centrifuged rats and was characterized by axons filled with reticulated mitochondria. The NAD found in the lateral vestibular nuclei of centrifuged rats is different from that seen in the dorsal column nuclei of aged mice.

Co-localization of NMDA receptors and AMPA receptors in neurons of the vestibular nuclei of rats.

We are interested in studying the co-localization of NMDA glutamate receptor subunits (NR1, NR2A/B) and AMPA glutamate receptor subunits (GluR1, GluR2, GluR2/3 and GluR4) in individual neurons of the rat vestibular nuclei. Immunoreactivity for NR1, NR2A/B, GluR1, GluR2, GluR2/3 and GluR4 was found in the somata and dendrites of neurons in the four major subdivisions (superior, medial, lateral, and spinal vestibular nuclei) and in two minor groups (groups x and y) of the vestibular nuclei. Double immunofluorescence showed that all the NR1-containing neurons exhibited NR2A/B immunoreactivity, indicating that native NMDA receptors are composed of NR1 and NR2A/B in a hetero-oligomeric configuration. Co-expression of NMDA receptor subunits and AMPA receptor subunits was demonstrated by double labeling of NR1/GluR1, NR1/GluR2/3, NR1/GluR4 and NR2A/B/GluR2 in individual vestibular nuclear neurons. All NR1-containing neurons expressed GluR2/3 immunoreactivity, and all NR2A/B-containing neurons expressed GluR2 immunoreactivity. However, only about 52% of NR1-immunoreactive neurons exhibited GluR1 immunoreactivity and 46% of NR1-containing neurons showed GluR4 immunoreactivity. The present data reveal that NMDA receptors are co-localized with variants of AMPA receptors in a large proportion of vestibular nuclear neurons. These results suggest that cross-modulation between NMDA receptors and AMPA receptors may occur in individual neurons of the vestibular nuclei during glutamate-mediated excitatory neurotransmission and may in turn contribute to synaptic plasticity within the vestibular nuclei.

Modulation by acute stress of chloride permeation across microdissected vestibular neurons membranes: different results in two rabbit strains and CRF involvement.

Free hand isolation of adult rabbit vestibular Deiters' neurons and dissection of their single membranes allows the study of their ionic permeability characteristics in a microchambers device. In the case of hare-like rabbits, the dissection of such membranes presents evidence of a high basal permeation of labelled chloride, possibly related to mechanical disturbance of the plasma membrane-related cytoskeleton and activation of chloride channels. This did not apply to the laboratory strain of white New Zealand rabbits. However, membranes from hare-like rabbits which were stressed by being rotated on a platform before the experiment, behaved like those from the New Zealand strain. Vice versa, habituation to handling day after day of New Zealand rabbits resulted in a chloride permeation equal to that of unstressed hare-like rabbits. We propose that the stressful conditions result in the release of neurochemical messages to the vestibular Deiters' cells which influence their electrophysiological behavior. The corticotropin releasing factor (CRF), a stress-related peptide present in the climbing fibers, actually blocks the basal chloride permeation across the Deiters' membranes and this effect is partially reversed by its receptor antagonist, alpha-helical CRF [9-41].

Spontaneous activity in rat vestibular nuclei in brain slices and effects of acetylcholine agonists and antagonists.

Extracellular recording was used to investigate spontaneously active neurons in all four major nuclei of the rat vestibular nuclear complex (VNC) in brainstem slices. The density of spontaneously active neurons was highest in the medial vestibular nucleus (MVN), slightly lower in the superior (SuVN) and spinal (SpVN) nuclei, and lowest in the lateral vestibular nucleus (LVN). We compared the effects of acetylcholine agonists and antagonists on spontaneously discharging neurons in MVN, SuVN, and SpVN with those in the nearby dorsal cochlear nucleus (DCN). The proportion of neurons responding to carbachol was greatest in DCN and smallest in SpVN. Unlike in DCN, some neurons in MVN, SuVN, and SpVN showed decreased firing during carbachol or muscarine. Magnitudes of responses to carbachol and muscarine were closely correlated (P<0.01). MVN neurons possessed nicotinic as well as muscarinic receptors. Activation of either type was unaffected by blocking synaptic transmission. The IC(50) values for the muscarinic subtype-preferential antagonists were compared, and tropicamide, preferential for M(4), was the most potent. Our results suggest that: (1) the relative numbers of spontaneously active neurons in rat VNC differ among nuclei; (2) acetylcholine agonists elicit changes in mean firing rates of neurons in MVN, SuVN and SpVN, but fewer neurons respond, and responses are smaller than in DCN; (3) both muscarinic and nicotinic acetylcholine receptors are present on MVN neurons, but muscarinic receptors may be more prominent.

Failure to demonstrate retrograde labelling of cerebellar Purkinje cells after injection of [3H]GABA in Deiters' nucleus.

After microinjections of [3H]GABA into the dorsal part of the Deiters' nucleus no retrograde labelling could be observed in the cerebellar Purkinje cells projecting to this nucleus. Tentatively, this failure to demonstrate transmitter specific retrograde labelling is attributed to lack of high affinity uptake mechanisms in the GABAergic Purkinje axon terminals. These observations indicate a very important role of terminal uptake mechanisms in initiation of transmitter specific retrograde labelling. Anterograde axonal labelling was observed along several tracts originating in the injected area, but was not limited to connections with GABA as likely transmitter candidate.

S100-glia regulation of GABA transport across the nerve cell membrane.

A technique has been devised to isolate and prepare fresh nerve cell plasma membranes in order to study the transport of biologically active substances across the membrane and in the two opposite directions. The membrane is placed tightly over a 30-micrometer diameter hole in a thin glass plate forming a partition between two compartments of a micro-chamber made from silicon rubber. The plasma membrane is usually placed with the outer surface facing the upper compartment. We have studied the transport of labeled GABA across the plasma membrane of Deiters' nerve cells and the effect of the brain-specific protein S-100 in its calcium form on this process. 100 nl samples were separated by thin layer chromatography and each sample analyzed by an instrument especially made for low level 3H- and 14C-measurements. The S-100, Ca2+ protein significantly increased the GABA transport across the nerve cell membrane by maximally 25% and against a gradient. The kinetics of the transport process, and inhibition by 2-4 diaminobutyric acid, furthermore supported the conclusion that the S-100, Ca2+-stimulated GABA transport was an active process. When a thin layer of the nerve cell's S-100-synthesizing glia was placed in contact with the plasma membrane - as in the vivo situation - the stimulation of GABA transport was abrogated. The S-100, Ca2+ protein, if absorbed on the nerve cell membrane, stimulates GABA transport across the membrane. This phenomenon seems to be regulated by the glia which cover all parts of the plasma membrane except the post-synaptic areas.

Outward rectifying potassium currents are the dominant voltage activated currents present in Deiters' cells.

Supporting cells in the cochlea are thought to maintain the homeostasis of the organ of Corti and contribute to the electrical and micromechanical environment of the hair cells. Of the different types of supporting cells, Deiters' cells form a structure that holds the outer hair cells (OHCs) at their base and apex. This structure may play an important role in modifying cochlear mechanics by influencing the force produced by sound induced motion of the OHCs which in turn may be modulated by ATP acting on ligand gated cation channels on the Deiters' cells. Also, a glia-like role of buffering external K+ concentration for the Deiters' cells has been suggested. We studied Deiters' cells' electrical properties and ion conductances using the whole cell variant of the patch clamp technique since they must play an important role in the function of these cells. It was found that isolated Deiters' cells possess a large voltage activated, outwardly rectifying K+ selective conductance. Voltage activated Ca2+ currents and non-selective currents were not detected and voltage activated inward currents were very small. The outward K+ currents were found to be dependent on voltage but not on Ca2+ for their activation. Nimodipine and 4-aminopyridine (4-AP) were shown to interact directly with the K+ channels in a voltage dependent manner. It is suggested that the K+ selective channels in Deiters' cells may be similar to the Kv1.5 type channel. However, based on the voltage dependence of the channels that was described by double Boltzmann equation and on the alteration of that dependence by 4-AP, it is possible that more than one type of K+ selective channel exists.