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Skin biopsies from patients with neuropathic pain often show changes in epidermal innervation, although it remains to be elucidated to what extent such changes can be linked to a particular subgroup of nerve fibers and how these changes are correlated with pain intensity. Here, we investigated to what extent behavioral signs of hyperalgesia are correlated with immunohistochemical changes of peptidergic and non-peptidergic epidermal nerve fibers in a rat model of nerve injury-induced pain. Rats subjected to unilateral partial ligation of the sciatic nerve developed significant mechanical and thermal hyperalgesia as tested by the withdrawal responses of the ipsilateral footpad to von Frey hairs and hotplate stimulation. At day 14, epidermal nerve fiber density (IENFD) and total epidermal nerve fiber length/mm(2) (ENFL) were significantly and consistently reduced compared to the contralateral side, following testing and re-testing by two blinded observers. The expression of calcitonin gene-related peptide (CGRP), a marker for peptidergic nerve fibers, was not significantly changed on the ipsilateral side. In contrast, the expression of the P2X3 receptor, a marker for non-peptidergic nerve fibers, was not only significantly reduced but could also be correlated with behavioral hyperalgesia. When labeling both peptidergic and non-peptidergic nerve fibers with the pan-neuronal marker PGP9.5, the expression was significantly reduced, albeit without a significant correlation with behavioral hyperalgesia. In conjunction, our data suggest that the pathology of the P2X3 epidermal nerve fibers can be selectively linked to neuropathy, highlighting the possibility that it is the degeneration of these fibers that drives hyperalgesia. This article is protected by copyright. All rights reserved.

Compensatory eye movements elicited by head rotation, also known as vestibulo-ocular reflex (VOR), can be adapted with the use of visual feedback. The cerebellum is essential for this type of movement adaptation, but its neuronal correlates remain to be elucidated. Here we show that the direction of vestibular input determines the magnitude of eye movement adaptation induced by mismatched visual input in mice, with larger changes during contraversive head rotation. Moreover, the location of the neural correlate of this changed behaviour depends on the type of paradigm. Gain-increase paradigms induce increased simple spike (SS) activity in ipsilateral cerebellar Purkinje cells (PC), which is in line with eye movements triggered by optogenetic PC activation. In contrast, gain-decrease paradigms do not induce changes in SS activity, highlighting that the murine vestibulo-cerebellar cortical circuitry is optimally designed to enhance ipsiversive eye movements. This article is protected by copyright. All rights reserved

Classical delay eyeblink conditioning is likely the most commonly used paradigm to study cerebellar learning. As yet, few studies have focused on extinction and savings of conditioned eyeblink responses (CRs). Saving effects, which are reflected in a reacquisition after extinction that is faster than the initial acquisition, suggest that learned associations are at least partly preserved during extinction. In this study, we tested the hypothesis that acquisition-related plasticity is nihilated during extinction in the cerebellar cortex, but retained in the cerebellar nuclei, allowing for faster reacquisition. Changes of 7 T functional magnetic resonance imaging (fMRI) signals were investigated in the cerebellar cortex and nuclei of young and healthy human subjects. Main effects of acquisition, extinction, and reacquisition against rest were calculated in conditioned stimulus-only trials. First-level β values were determined for a spherical region of interest (ROI) around the acquisition peak voxel in lobule VI, and dentate and interposed nuclei ipsilateral to the unconditioned stimulus. In the cerebellar cortex and nuclei, fMRI signals were significantly lower in extinction compared to acquisition and reacquisition, but not significantly different between acquisition and reacquisition. These findings are consistent with the theory of bidirectional learning in both the cerebellar cortex and nuclei. It cannot explain, however, why conditioned responses reappear almost immediately in reacquisition following extinction. Although the present data do not exclude that part of the initial memory remains in the cerebellum in extinction, future studies should also explore changes in extracerebellar regions as a potential substrate of saving effects. Hum Brain Mapp, 2017. © 2017 Wiley Periodicals, Inc.

Cerebellar cortical throughput involved in motor control comprises granule cells (GCs) and Purkinje cells (PCs), both of which receive inhibitory GABAergic input from interneurons. The GABAergic input to PCs is essential for learning and consolidation of the vestibulo-ocular reflex, but the role of GC excitability remains unclear. We now disrupted the Kcc2 K-Cl cotransporter specifically in either cell type to manipulate their excitability and inhibition by GABAA-receptor Cl− channels. Although Kcc2 may have a morphogenic role in synapse development, Kcc2 disruption neither changed synapse density nor spine morphology. In both GCs and PCs, disruption of Kcc2, but not Kcc3, increased [Cl−]i roughly two-fold. The reduced Cl− gradient nearly abolished GABA-induced hyperpolarization in PCs, but in GCs it merely affected excitability by membrane depolarization. Ablation of Kcc2 from GCs impaired consolidation of long-term phase learning of the vestibulo-ocular reflex, whereas baseline performance, short-term gain-decrease learning and gain consolidation remained intact. These functions, however, were affected by disruption of Kcc2 in PCs. GC excitability plays a previously unknown, but specific role in consolidation of phase learning.

Objective. To determine the functional recovery in patients with severe transient peripheral facial motor paralysis (Bell palsy). Study Design. Prospective controlled trial. Setting. Academic medical center. Subjects and Methods. Blink recovery was studied in 2 groups of severely affected Bell palsy patients during a follow-up period of 84 weeks. The patients in one group received prednisolone within the first week after the onset of symptoms. No medication was given to the other group. A control group of healthy subjects was also included. Simultaneous orbicularis oculi muscle activity and eyelid kinematics were recorded by surface electromyographic (EMG) recording and eyelid search coils, respectively. Results. At the beginning of the paralysis, very little integrated orbicularis oculi muscle activity and eyelid movement was measured at the palsied side of the face. Thirteen weeks later, the integrated orbicularis oculi EMG and functional blink recovery gradually improved until 39 weeks. Beyond, only the integrated orbicularis oculi EMG slightly increased. At 84 weeks, the integrated orbicularis oculi EMG was significantly larger in the prednisolone group compared with the control group. The integrated EMG of the nonmedicated group recovered to normal values. Curiously enough, the functional blink recovery at the palsied side remained reduced to 64% compared with the healthy controls in the prednisolone-treated group and to 36% in the nonmedicated group. Conclusion. The authors demonstrate that prednisolone significantly increased the orbicularis oculi muscle activity and significantly improved functional blink recovery in severely affected Bell palsy patients. However, the increase of muscle activity was insufficient to restore functional blinking to normal values.

The cerebellar cortex is crucial for sensorimotor integration. Sensorimotor inputs converge on cerebellar Purkinje cells via two afferent pathways: the climbing fibre pathway triggering complex spikes, and the mossy fibre–parallel fibre pathway, modulating the simple spike activities of Purkinje cells. We used, for the first time, the mouse whisker system as a model system to study the encoding of somatosensory input by Purkinje cells. We show that most Purkinje cells in ipsilateral crus 1 and crus 2 of awake mice respond to whisker stimulation with complex spike and/or simple spike responses. Single-whisker stimulation in anaesthetised mice revealed that the receptive fields of complex spike and simple spike responses were strikingly different. Complex spike responses, which proved to be sensitive to the amplitude, speed and direction of whisker movement, were evoked by only one or a few whiskers. Simple spike responses, which were not affected by the direction of movement, could be evoked by many individual whiskers. The receptive fields of Purkinje cells were largely intermingled, and we suggest that this facilitates the rapid integration of sensory inputs from different sources. Furthermore, we describe that individual Purkinje cells, at least under anaesthesia, may be bound in two functional ensembles based on the receptive fields and the synchrony of the complex spike and simple spike responses. The ‘complex spike ensembles’ were oriented in the sagittal plane, following the anatomical organization of the climbing fibres, while the ‘simple spike ensembles’ were oriented in the transversal plane, as are the beams of parallel fibres.

Human Bartter syndrome IV is an autosomal recessive disorder characterized by congenital deafness and severe renal salt and fluid loss. It is caused by mutations in BSND, which encodes barttin, a beta-subunit of ClC-Ka and ClC-Kb chloride channels. Inner-ear-specific disruption of Bsnd in mice now reveals that the positive potential, but not the high potassium concentration, of the scala media depends on the presence of these channels in the epithelium of the stria vascularis. The reduced driving force for K(+)-entry through mechanosensitive channels into sensory hair cells entails a profound congenital hearing loss and subtle vestibular symptoms. Although retaining all cell types and intact tight junctions, the thickness of the stria is reduced early on. Cochlear outer hair cells degenerate over several months. A collapse of endolymphatic space was seen when mice had additionally renal salt and fluid loss due to partial barttin deletion in the kidney. Bsnd(-/-) mice thus demonstrate a novel function of Cl(-) channels in generating the endocochlear potential and reveal the mechanism leading to deafness in human Bartter syndrome IV.

Identification of genes involved in hereditary vestibular disease is growing at a remarkable pace. Mutant mouse technology can be an important tool for understanding the biological mechanism of human vestibular diseases.

The homeostasis of intracellular Cl(-) concentration ([Cl(-)](i)) is critical for neuronal function, including gamma-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl(-)](i) using a transgenetically expressed Cl(-)-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 microm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl(-)-free solution, demonstrating that the EYFP signal represented an increase in [Cl(-)](i). Similar to glutamate, a rise in [Cl(-)](i) was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K(+)], indicating that an increase in driving force for Cl(-) suffices to increase [Cl(-)](i). To elucidate the membrane mechanisms mediating the Cl(-) influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl(-)](i) was resistant to furosemide, bumetanide and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl(-)](i) via several pathways involving NFA- and NPPB-sensitive anion channels and GABA(A) receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl(-) transporters. The present study highlights the vulnerability of [Cl(-)](i) homeostasis after membrane depolarization in neurons.

Targeted deletion of the connexin36 (Cx36) gene in the mouse genome leads to visual transmission defects, weakened synchrony of rhythmic inhibitory potentials in the neocortex, and disruption of -frequency network oscillations. We have generated transgenic mice in which a reporter protein consisting of the exon1 coded N-terminal part of Cx36 fused to -galactosidase (N36--gal) is expressed instead of Cx36. Here, we have used these mice for a detailed analysis of the reporter gene expression. By -gal staining of adult retina, we found expression of the lacZ reporter gene in the ganglion cell layer, in two rows of the inner nuclear layer, and in the photoreceptor layer. In the brain, -gal staining was present in -aminobutyric acid (GABA)ergic neurons of the cerebellar nuclei, in non-GABAergic neurons of the inferior olive, in mitral cells of the olfactory bulb, and in parvalbumin-positive cells of the cerebral cortex. Outside the central nervous system, N36--gal signals were detected in insulin producing -cells of the pancreas and in the medulla of the adrenal gland of adult Cx36 /del[LacZ] mice. This expression pattern suggests that Cx36 fulfills functional roles not only in several types of neurons in the retina and central nervous system but also in excitable cells of the pancreas and adrenal gland. J. Comp. Neurol. 473:511–525, 2004. © 2004 Wiley-Liss, Inc. Indexing terms: retina; inferior olive; cerebellum; cortex; pancreas; adrenal gland

Bicaudal D is an evolutionarily conserved protein, which is involved in dynein-mediated motility both in Drosophila and in mammals. Here we report that the N–terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. Here, we use the BICD2 N–terminal domain as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N–terminal domain is reduced within the full-length molecule, indicating that the C–terminal part of the protein might regulate the interaction between BICD2 and the motor complex. Our findings provide a novel model system for dissection of the molecular mechanism of dynein motility.

Patients with Hodgkin's disease can develop paraneoplastic cerebellar ataxia because of the generation of autoantibodies against mGluR1 (mGluR1-Abs). Yet, the pathophysiological mechanisms underlying their motor coordination deficits remain to be elucidated. Here, we show that application of IgG purified from the patients' serum to cerebellar slices of mice acutely reduces the basal activity of Purkinje cells, whereas application to the flocculus of mice in vivo evokes acute disturbances in the performance of their compensatory eye movements. In addition, the mGluR1-Abs block induction of long-term depression in cultured mouse Purkinje cells, whereas the cerebellar motor learning behavior of the patients is affected in that they show impaired adaptation of their saccadic eye movements. Finally, postmortem analysis of the cerebellum of a paraneoplastic cerebellar ataxia patient showed that the number of Purkinje cells was significantly reduced by approximately two thirds compared with three controls. We conclude that autoantibodies against mGluR1 can cause cerebellar motor coordination deficits caused by a combination of rapid effects on both acute and plastic responses of Purkinje cells and chronic degenerative effects.

The epistatic circler mouse (Ecl mouse) is a preexisting mutant, which displays a circling phenotype and hyperactivity. It has been shown that the circling phenotype in this mutant results from a complex inheritance pattern, but the vestibular pathology has not been analyzed. The present study deals with the morphological and functional basis responsible for the circling behavior in the Ecl mouse. Morphological examination of the inner ears revealed a bilateral malformation of the horizontal (lateral) semicircular canal and duct. No cochlear abnormalities were detected, and auditory brainstem response (ABR) measurements indicated that the auditory system is not affected. Investigation of the vestibuloocular reflex (VOR) in Ecl mice showed that their horizontal VOR on stimulation is virtually absent, which correlates with the morphological findings.

Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes.

Patterning the vertebrate ear requires the coordinated expression of genes that are involved in morphogenesis, neurogenesis, and hair cell formation. The zinc finger gene GATA-3 is expressed both in the inner ear and in afferent and efferent auditory neurons. Specifically, GATA-3 is expressed in a population of neurons in rhombomere 4 that extend their axons across the floor plate of rhombomere 4 (r4) at embryonic day 10 (E10) and reach the sensory epithelia of the ear by E13.5. The distribution of their cell bodies corresponds to that of the cell bodies of the cochlear and vestibular efferent neurons as revealed by labeling with tracers. Both GATA-3 heterozygous and GATA-3 null mutant mice show unusual axonal projections, such as misrouted crossing fibers and fibers in the facial nerve, that are absent in wild-type littermates. This suggests that GATA-3 is involved in the pathfinding of efferent neuron axons that navigate to the ear. In the ear, GATA-3 is expressed inside the otocyst and the surrounding periotic mesenchyme. The latter expression is in areas of branching of the developing ear leading to the formation of semicircular canals. Ears of GATA-3 null mutants remain cystic, with a single extension of the endolymphatic duct and no formation of semicircular canals or saccular and utricular recesses. Thus, both the distribution of GATA-3 and the effects of null mutations on the ear suggest involvement of GATA-3 in morphogenesis of the ear. This study shows for the first time that a zinc finger factor is involved in axonal navigation of the inner ear efferent neurons and, simultaneously, in the morphogenesis of the inner ear.

Earlier behavioural studies have shown that the expression of the immediate-early gene c-fos, as visualized by the immunohistochemical detection of Fos, in the inferior olive (IO) correlated closely with expression in related areas of the cerebellar nuclei. It has been speculated that the expression of c-fos within the cerebellar nuclei may be induced by enhanced spiking activity of the immunopositive neurons in the inferior olive. Two potential mechanisms may play a role in this process: a direct induction by way of the collaterals of the olivary climbing fibres to the cerebellar nuclei, or indirectly, by climbing fibre activity-induced depression of mossy fibre-parallel fibre-induced simple spike frequency of the Purkinje cells resulting in a subsequent disinhibition of the related parts of the cerebellar nuclei. In an attempt to distinguish between these possible mechanisms, we analysed Fos immunoreactivity in the olivocerebellar system of wild-type mice and in the mutant mouse Lurcher which lacks Purkinje cells. The tremorgenic agent harmaline, which is known to induce enhanced and rhythmic firing of olivary neurons was given intraperitoneally to anaesthetized mice of both genotypes. Harmaline application coincides with the induction of Fos-immunoreactive neurons in most areas of the IO in both wild-type and Lurcher mice. Both types of mice also showed enhanced expression in the larger neurons of the cerebellar nuclei. However, in the smaller, GABAergic nucleo-olivary neurons, increased c-fos expression was only observed in the wild-type mice. We conclude that: (i) increased olivary activity indeed may result in increased c-Fos expression in related areas of the cerebellar nuclei; (ii) because the indirect mode of induction is not operative in Lurcher mice, the olivary collateral innervation of the cerebellar nuclei is sufficient for c-fos induction in the larger nucleobulbar neurons in Lurcher and potentially also in wild-type mice; however (iii) for the nucleo-olivary cells an intact cerebellar cortical input is necessary to evoke increased expression of c-fos following harmaline application.

Apoptosis is of paramount importance during embryonic development. This insight stems from early studies which correlated cell death to normal developmental processes and now has been confirmed by linking aberrant cell death patterns to aberrant development. Linking apoptosis to the phenotype of a developing organism requires spatial information on the localization of the dying cells, making in situ detection essential This prerequisite limits the tools available for such studies (1) to vital dyes, which can be detected at the whole mount level only; (2) to detection based upon apoptotic morphology by routine light microscopy and electron microscopy; and (3) to staining for apoptosis associated DNA fragmentation via, e.g., the TUNEL procedure, which marks cells in a relative late phase of apoptosis. New apoptosis markers need to be specific and should preferably detect cells early during this process. In the present study we show that the recently discovered in vitro marker of apoptosis, Annexin V meets these requirements for in vivo detection. Through intracardiac injections of biotin labeled Annexin V, a Ca2+ dependent phosphatidylserine binding protein, we were able to visualize apoptotic cells derived from each germ layer in the developing mouse embryo from the whole mount level up to the ultrastructural level. Double-labeling on paraffin sections for both this method and TUNEL revealed that cells become Annexin V-biotin labeled early during the process of apoptosis.

An exciting decade of two-photon fluorescence imaging has revealed that the shape and turnover of dendritic spines are dynamic and modulated by synaptic plasticity (Matsuzaki et al., 2004). Such structural changes can be induced via focal glutamate uncaging at individual spines, but they can also be driven by experience (Holtmaat et al., 2006). Indeed, the size of both the head and neck of dendritic spines can be modulated by neuronal activity (Matsuzaki et al., 2004; Bloodgood & Sabatini, 2005). In turn, the geometry of spines will affect the compartmentalization of signals generated at the spine head and the extent to which ions and second messengers can spread via the neck to the parent dendrite and nearby spines. Interestingly, Santamaria et al. (2006) demonstrated in an earlier study that diffusion in spiny dendrites cannot be described by the standard diffusion equation. Instead, spines temporarily ‘trap’ molecules causing anomalous diffusion in the parent dendrite (Fig. 1). Uncaging dextran-conjugated fluorescent probes and imaging the fluorescence changes over time along a stretch of dendrite enabled calculation of the spatial variance of the diffusing molecules and estimation of the anomalous exponent dw. In smooth dendrites diffusion had an anomalous exponent of dw = 2, whereas in spiny dendrites dw turned out to be high (dw > 2) resulting in slowed diffusion. The importance of this finding is that the diffusion of second messengers within dendrites could be controlled by changes in spine shape or density. It is known that spine density in CA1 pyramidal neurons varies considerably across their dendritic arbors, with a progressive increase in spine density and spine size in the distal segments of apical dendrites (Konur et al., 2003). Thus, different dendritic compartments may exhibit different rates of diffusion of postsynaptic signaling molecules. In their study published in this issue of EJN, Santamaria et al. (2011) show that, in both Purkinje cells and CA1 pyramidal neurons, the anomalous exponent dw scales linearly with spine density. Anomalous diffusion in dendrites appears to be a hallmark of all neurons with spines. In addition, these authors provide empirical evidence that neither branching geometry nor dendrite diameter account for anomalous diffusion, confirming their previous theoretical models (Santamaria et al., 2006). The mechanistic studies by Santamaria et al. (2011) might be relevant to explain, for example, the functional impact of estradiol on memory formation (Andreescu et al., 2007) or of the pathological impact of a lack of fragile X mental retardation protein on motor behavior (Koekkoek et al., 2005). In both cases, altered geometry of spines and ⁄ or their inputs are likely to affect the diffusion of second messengers in the dendritic network. Therefore, it will be of general interest to identify the molecules for which anomalous diffusion plays a relevant role under physiological and pathophysiological conditions. For instance, active extrusion of calcium is likely to supersede the effects of spine geometry on diffusion of this ion in dendrites, whereas diffusion of inositol triphosphate (IP3) is slowed down (Santamaria et al., 2006) and that of GTPase signaling proteins varies among their family (Yasuda & Murakoshi, 2011). Anomalous diffusion imposed by spine geometry may thus represent a mechanism modulating the function of a selected set of signaling molecules.