Phenotypical, genotypical and pathological characterization of the moonwalker mouse, a model of ataxia.

We performed a comprehensive study of the morphological, functional, and genetic features of moonwalker (MWK) mice, a mouse model of spinocerebellar ataxia caused by a gain of function of the TRPC3 channel. These mice show numerous behavioral symptoms including tremor, altered gait, circling behavior, impaired motor coordination, impaired motor learning and decreased limb strength. Cerebellar pathology is characterized by early and almost complete loss of unipolar brush cells as well as slowly progressive, moderate loss of Purkinje cell (PCs). Structural damage also includes loss of synaptic contacts from parallel fibers, swollen ER structures, and degenerating axons. Interestingly, no obvious correlation was observed between PC loss and severity of the symptoms, as the phenotype stabilizes around 2 months of age, while the cerebellar pathology is progressive. This is probably due to the fact that PC function is severely impaired much earlier than the appearance of PC loss. Indeed, PC firing is already impaired in 3 weeks old mice. An interesting feature of the MWK pathology that still remains to be explained consists in a strong lobule selectivity of the PC loss, which is puzzling considering that TRPC is expressed in every PC. Intriguingly, genetic analysis of MWK cerebella shows, among other alterations, changes in the expression of both apoptosis inducing and resistance factors possibly suggesting that damaged PCs initiate specific cellular pathways that protect them from overt cell loss.

Hypernegative GABAA Reversal Potential in Pyramidal Cells Contributes to Medial Prefrontal Cortex Deactivation in a Mouse Model of Neuropathic Pain.

Deactivation of the medial prefrontal cortex (mPFC) has been broadly reported in both neuropathic pain models and human chronic pain patients. Several cellular mechanisms may contribute to the inhibition of mPFC activity, including enhanced GABAergic inhibition. The functional effect of GABAA(γ-aminobutyric acid type A)-receptor activation depends on the concentration of intracellular chloride in the postsynaptic neuron, which is mainly regulated by the activity of Na-K-2Cl cotransporter isoform 1 (NKCC1) and K-Cl cotransporter isoform 2 (KCC2), 2 potassium-chloride cotransporters that import and extrude chloride, respectively. Recent work has shown that the NKCC1-KCC2 ratio is affected in numerous pathological conditions, and we hypothesized that it may contribute to the alteration of mPFC function in neuropathic pain. We used quantitative in situ hybridization to assess the level of expression of NKCC1 and KCC2 in the mPFC of a mouse model of neuropathic pain (spared nerve injury), and we found that KCC2 transcript is increased in the mPFC of spared nerve injury mice while NKCC1 is not affected. Perforated patch recordings further showed that this results in the hypernegative reversal potential of the GABAA current in pyramidal neurons of the mPFC. Computational simulations suggested that this change in GABAA reversal potential is sufficient to significantly reduce the overall activity of the cortical network. Thus, our results identify a novel pathological modulation of GABAA function and a new mechanism by which mPFC function is inhibited in neuropathic pain. Our data also help explain previous findings showing that activation of mPFC interneurons has proalgesic effect in neuropathic, but not in control conditions. PERSPECTIVE: Chronic pain is associated with the presence of depolarizing GABAA current in the spinal cord, suggesting that pharmacological NKCC1 antagonism has analgesic effects. However, our results show that in neuropathic pain, GABAA current is actually hyperinhibitory in the mPFC, where it contributes to the mPFC functional deactivation. This suggests caution in the use of NKCC1 antagonism to treat pain.

A Leptin-Mediated Neural Mechanism Linking Breathing to Metabolism.

Breathing is coupled to metabolism. Leptin, a peptide mainly secreted in proportion to adipose tissue mass, increases energy expenditure with a parallel increase in breathing. We demonstrate that optogenetic activation of LepRb neurons in the nucleus of the solitary tract (NTS) mimics the respiratory stimulation after systemic leptin administration. We show that leptin activates the sodium leak channel (NALCN), thereby depolarizing a subset of glutamatergic (VGluT2) LepRb NTS neurons expressing galanin. Mice with selective deletion of NALCN in LepRb neurons have increased breathing irregularity and central apneas. On a high-fat diet, these mice gain weight with an associated depression of minute ventilation and tidal volume, which are not detected in control littermates. Anatomical mapping reveals LepRb NTS-originating glutamatergic axon terminals in a brainstem inspiratory premotor region (rVRG) and dorsomedial hypothalamus. These findings directly link a defined subset of NTS LepRb cells to the matching of ventilation to energy balance.

Absence of UCHL 1 function leads to selective motor neuropathy.

OBJECTIVE: The aim of this study was to investigate the role of ubiquitin C-terminal hydrolase-L1 (UCHL1) for motor neuron circuitry and especially in spinal motor neuron (SMN) health, function, and connectivity. METHODS: Since mutations in UCHL1 gene leads to motor dysfunction in patients, we investigated the role of UCHL1 on SMN survival, axon health, and connectivity with the muscle, by employing molecular and cellular marker expression analysis and electrophysiological recordings, in healthy wild-type and Uchl1 (nm3419) (UCHL1-/-) mice, which lack all UCHL1 function. RESULTS: There is pure motor neuropathy with selective degeneration of the motor, but not sensory axons in the absence of UCHL1 function. Neuromuscular junctions (NMJ) are impaired in muscle groups that are innervated by slow-twitch or fast-twitch SMN. However, unlike corticospinal motor neurons, SMN cell bodies remain intact with no signs of elevated endoplasmic reticulum (ER) stress. INTERPRETATION: Presence of NMJ defects and progressive retrograde axonal degeneration in the absence of major SMN soma loss suggest that defining pathology as a function of neuron number is misleading and that upper and lower motor neurons utilize UCHL1 function in different cellular events. In line with findings in patients with mutations in UCHL1 gene, our results suggest a unique role of UCHL1, especially for motor neuron circuitry. SMN require UCHL1 to maintain NMJ and motor axon health, and that observed motor dysfunction in the absence of UCHL1 is not due to SMN loss, but mostly due to disintegrated circuitry.

Absence of alsin function leads to corticospinal motor neuron vulnerability via novel disease mechanisms.

Mutations in the ALS2 gene result in early-onset amyotrophic lateral sclerosis, infantile-onset ascending hereditary spastic paraplegia and juvenile primary lateral sclerosis, suggesting prominent upper motor neuron involvement. However, the importance of alsin function for corticospinal motor neuron (CSMN) health and stability remains unknown. To date, four separate alsin knockout (Alsin(KO)) mouse models have been generated, and despite hopes of mimicking human pathology, none displayed profound motor function defects. This, however, does not rule out the possibility of neuronal defects within CSMN, which is not easy to detect in these mice. Detailed cellular analysis of CSMN has been hampered due to their limited numbers and the complex and heterogeneous structure of the cerebral cortex. In an effort to visualize CSMN in vivo and to investigate precise aspects of neuronal abnormalities in the absence of alsin function, we generated Alsin(KO)-UeGFP mice, by crossing Alsin(KO) and UCHL1-eGFP mice, a CSMN reporter line. We find that CSMN display vacuolated apical dendrites with increased autophagy, shrinkage of soma size and axonal pathology even in the pons region. Immunocytochemistry coupled with electron microscopy reveal that alsin is important for maintaining cellular cytoarchitecture and integrity of cellular organelles. In its absence, CSMN displays selective defects both in mitochondria and Golgi apparatus. UCHL1-eGFP mice help understand the underlying cellular factors that lead to CSMN vulnerability in diseases, and our findings reveal unique importance of alsin function for CSMN health and stability.

Consensus Paper: Cerebellar Development.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

α-Synuclein expression in the mouse cerebellum is restricted to VGluT1 excitatory terminals and is enriched in unipolar brush cells.

α-Synuclein has a crucial role in synaptic vesicle release and synaptic membrane recycling. Although its general expression pattern has been described in the cerebellum, the precise cerebellar structures where α-synuclein is localized are poorly understood. To address this question, we used α-synuclein immunohistochemistry in adult mice cerebellar sections. We found that α-synuclein labels glutamatergic but not glycinergic and GABAergic synaptic terminals in the molecular and granule cell layers. α-Synuclein was preferentially expressed in parallel and mossy fiber synaptic terminals that also express vesicular glutamate transporter 1 (VGluT1), while it was not detected in VGluT2-positive climbing fibers. α-Synuclein was particularly enriched in lobules IX and X, a region known to contain a high density of unipolar brush cells (UBCs). To elucidate whether the α-synuclein-positive mossy fibers belong to UBCs, we double-labeled cerebellar sections with antibodies to α-synuclein and UBC-type-specific markers (calretinin for type I and metabotropic glutamate receptor 1α (mGluR1α) for type II UBCs) and took advantage of organotypic cerebellar cultures (in which all mossy fibers are UBC axons) and moonwalker mice (in which almost all UBCs are ablated) and found that both type I and type II UBCs express α-synuclein. In moonwalker mutant cerebella, the α-synuclein/VGluT1 immunolabeling showed a dramatic decrease in the vestibulocerebellum that correlated with the absence of UBC. α-Synuclein appears to be an excellent marker for intrinsic mossy fibers of the VGluT1 subset in conjunction with UBCs of both subtypes.

The Spontaneous Ataxic Mouse Mutant Tippy is Characterized by a Novel Purkinje Cell Morphogenesis and Degeneration Phenotype.

This study represents the first detailed analysis of the spontaneous neurological mouse mutant, tippy, uncovering its unique cerebellar phenotype. Homozygous tippy mutant mice are small, ataxic, and die around weaning. Although the cerebellum shows grossly normal foliation, tippy mutants display a complex cerebellar Purkinje cell phenotype consisting of abnormal dendritic branching with immature spine features and patchy, non-apoptotic cell death that is associated with widespread dystrophy and degeneration of the Purkinje cell axons throughout the white matter, the cerebellar nuclei, and the vestibular nuclei. Moderate anatomical abnormalities of climbing fiber innervation of tippy mutant Purkinje cells were not associated with changes in climbing fiber-EPSC amplitudes. However, decreased ESPC amplitudes were observed in response to parallel fiber stimulation and correlated well with anatomical evidence for patchy dark cell degeneration of Purkinje cell dendrites in the molecular layer. The data suggest that the Purkinje neurons are a primary target of the tippy mutation. Furthermore, we hypothesize that the Purkinje cell axonal pathology together with disruptions in the balance of climbing fiber and parallel fiber-Purkinje cell input in the cerebellar cortex underlie the ataxic phenotype in these mice. The constellation of Purkinje cell dendritic malformation and degeneration phenotypes in tippy mutants is unique and has not been reported in any other neurologic mutant. Fine mapping of the tippy mutation to a 2.1 MB region of distal chromosome 9, which does not encompass any gene previously implicated in cerebellar development or neuronal degeneration, confirms that the tippy mutation identifies novel biology and gene function.

Differential distribution of phospholipase C beta isoforms and diaglycerol kinase-beta in rodents cerebella corroborates the division of unipolar brush cells into two major subtypes.

Sublineage diversification of specific neural cell classes occurs in complex as well as simply organized regions of the central and peripheral nervous systems; the significance of the phenomenon, however, remains insufficiently understood. The unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons that occur at high density in vestibulocerebellum. As they are classified into subsets that differ in chemical phenotypes, intrinsic properties, and lobular distribution, they represent a valuable neuronal model to study subclass diversification. In this study, we show that cerebellar UBCs of adult rats and mice form two subclasses-type I and type II UBCs-defined by somatodendritic expression of calretinin (CR), mGluR1α, phospholipases PLCß1 and PLCß4, and diacylglycerol kinase-beta (DGKß). We demonstrate that PLCß1 is associated only with the CR(+) type I UBCs, while PLCß4 and DGKß are exclusively present in mGluR1α(+) type II UBCs. Notably, all PLCß4(+) UBCs, representing about 2/3 of entire UBC population, also express mGluR1α. Furthermore, our data show that the sum of CR(+) type I UBCs and mGluR1α(+) type II UBCs accounts for the entire UBC class identified with Tbr2 immunolabeling. The two UBC subtypes also show a very different albeit somehow overlapping topographical distribution as illustrated by detailed cerebellar maps in this study. Our data not only complement and extend the previous knowledge on the diversity and subclass specificity of the chemical phenotypes within the UBC population, but also provide a new angle to the understanding of the signaling networks in type I and type II UBCs.

Early onset of ataxia in moonwalker mice is accompanied by complete ablation of type II unipolar brush cells and Purkinje cell dysfunction.

Transient receptor potential "canonical" cation channels (TRPC) are involved in many cellular activities, including neuronal synaptic transmission. These channels couple lipid metabolism, calcium homeostasis, and electrophysiological properties as they are calcium permeable and activated through the phospholipase C pathway and by diacylglycerol. The TRPC3 subunit is abundantly expressed in Purkinje cells (PCs), where it mediates slow metabotropic glutamate receptor-mediated synaptic responses. Recently, it has been shown that heterozygous moonwalker mice, which are a model of cerebellar ataxia, carry a dominant gain-of-function mutation (T635A) in the TRPC3 gene. This mutation leads to PC loss and dysmorphism, which have been suggested to cause the ataxia. However, the ataxic phenotype is present from a very early stage (before weaning), whereas PC loss does not appear until several months of age. Here we show that another class of cerebellar neurons, the type II unipolar brush cells (UBCs), express functional TRPC3 channels; intriguingly, these cells are ablated in moonwalker mice by 1 month of age. Additionally, we show that in moonwalker mice, intrinsic excitability of PCs is altered as early as 3 weeks after birth. We suggest that this altered excitability and the TRPC3-mediated loss of type II UBCs may both contribute to the ataxic phenotype of these mice and that different calcium handling in PCs and type II UBCs may account for the dramatic differences in sensitivity to the moonwalker mutation between these cell types.

Electrophysiological, morphological, and topological properties of two histochemically distinct subpopulations of cerebellar unipolar brush cells.

Unipolar brush cells (UBCs) are excitatory cerebellar granular layer interneurons whose brush-like dendrites receive one-to-one mossy fiber inputs. Subclasses of UBCs differ primarily by expressing metabotropic glutamate receptor (mGluR) 1α or calretinin. We used GENSAT Tg(Grp-EGFP) BAC transgenic mice, which selectively express enhanced green fluorescent protein (EGFP) in mGluR1α-positive UBCs to compare the functional properties of the two subclasses. Compared to EGFP-negative UBCs, which include the calretinin-positive cells, EGFP-positive UBCs had smaller somata (area 48 vs 63 µm(2)), lower specific membrane resistance (6.4 vs. 13.7 KΩ cm(2)), were less prone to intrinsic firing, and showed more irregular firing (in cell-attached ~49 % were firing vs. ~88 %, and the CV was 0.53 vs. 0.32 for EGFP-negative cells). Some of these differences are attributable to higher density of background K(+) currents in EGFP-positive cells (at -120 mV, the barium-sensitive current was 94 vs. 37 pA in EGFP-negative cells); Ih, on the contrary, was more abundantly expressed in EGFP-negative cells (at -140 mV, it was -122 vs. -54 pA in EGFP-positive neurons); furthermore, while group II mGluR modulation of the background potassium current in EGFP-negative UBCs was maintained after intracellular dialysis, mGluR modulation in EGFP-positive UBCs was lost in whole-cell recordings. Finally, cell-attached firing was reversibly abolished by the GABA(B) activation in EGFP-positive, but not in EGFP-negative UBCs. Immunohistochemistry showed that EGFP-negative UBCs express GIRK2 at high density, while mGluR1α UBCs are GIRK2 negative, suggesting that GIRK2 mediates the mGluR-sensitive current in EGFP-negative UBCs. These data suggest that the two subclasses perform different functions in the cerebellar microcircuits.

Roles of the espin actin-bundling proteins in the morphogenesis and stabilization of hair cell stereocilia revealed in CBA/CaJ congenic jerker mice.

Hearing and vestibular function depend on mechanosensory staircase collections of hair cell stereocilia, which are produced from microvillus-like precursors as their parallel actin bundle scaffolds increase in diameter and elongate or shorten. Hair cell stereocilia contain multiple classes of actin-bundling protein, but little is known about what each class contributes. To investigate the roles of the espin class of actin-bundling protein, we used a genetic approach that benefited from a judicious selection of mouse background strain and an examination of the effects of heterozygosity. A congenic jerker mouse line was prepared by repeated backcrossing into the inbred CBA/CaJ strain, which is known for excellent hearing and minimal age-related hearing loss. We compared stereocilia in wild-type CBA/CaJ mice, jerker homozygotes that lack espin proteins owing to a frameshift mutation in the espin gene, and jerker heterozygotes that contain reduced espin levels. The lack of espins radically impaired stereociliary morphogenesis, resulting in stereocilia that were abnormally thin and short, with reduced differential elongation to form a staircase. Mean stereociliary diameter did not increase beyond ∼0.10-0.14 µm, making stereocilia ∼30%-60% thinner than wild type and suggesting that they contained ∼50%-85% fewer actin filaments. These characteristics indicate a requirement for espins in the appositional growth and differential elongation of the stereociliary parallel actin bundle and fit the known biological activities of espins in vitro and in transfected cells. The stereocilia of jerker heterozygotes showed a transient proximal-distal tapering suggestive of haploinsufficiency and a slowing of morphogenesis that revealed previously unrecognized assembly steps and intermediates. The lack of espins also led to a region-dependent degeneration of stereocilia involving shortening and collapse. We conclude that the espin actin-bundling proteins are required for the assembly and stabilization of the stereociliary parallel actin bundle.

The unipolar brush cell: a remarkable neuron finally receiving deserved attention.

Unipolar brush cells (UBC) are small, glutamatergic neurons residing in the granular layer of the cerebellar cortex and the granule cell domain of the cochlear nuclear complex. Recent studies indicate that this neuronal class consists of three or more subsets characterized by distinct chemical phenotypes, as well as by intrinsic properties that may shape their synaptic responses and firing patterns. Yet, all UBCs have a unique morphology, as both the dendritic brush and the large endings of the axonal branches participate in the formation of glomeruli. Although UBCs and granule cells may share the same excitatory and inhibitory inputs, the two cell types are distinctively differentiated. Typically, whereas the granule cell has 4-5 dendrites that are innervated by different mossy fibers, and an axon that divides only once to form parallel fibers after ascending to the molecular layer, the UBC has but one short dendrite whose brush engages in synaptic contact with a single mossy fiber terminal, and an axon that branches locally in the granular layer; branches of UBC axons form a non-canonical, cortex-intrinsic category of mossy fibers synapsing with granule cells and other UBCs. This is thought to generate a feed-forward amplification of single mossy fiber afferent signals that would reach the overlying Purkinje cells via ascending granule cell axons and their parallel fibers. In sharp contrast to other classes of cerebellar neurons, UBCs are not distributed homogeneously across cerebellar lobules, and subsets of UBCs also show different, albeit overlapping, distributions. UBCs are conspicuously rare in the expansive lateral cerebellar areas targeted by the cortico-ponto-cerebellar pathway, while they are a constant component of the vermis and the flocculonodular lobe. The presence of UBCs in cerebellar regions involved in the sensorimotor processes that regulate body, head and eye position, as well as in regions of the cochlear nucleus that process sensorimotor information suggests a key role in these critical functions; it also invites further efforts to clarify the cellular biology of the UBCs and their specific functions in the neuronal microcircuits in which they are embedded. High density of UBCs in specific regions of the cerebellar cortex is a feature largely conserved across mammals and suggests an involvement of these neurons in fundamental aspects of the input/output organization as well as in clinical manifestation of focal cerebellar disease.

Espin actin-cytoskeletal proteins are in rat type I spiral ganglion neurons and include splice-isoforms with a functional nuclear localization signal.

The espins are Ca(2+)-resistant actin-bundling proteins that are enriched in hair cell stereocilia and sensory cell microvilli. Here, we report a novel localization of espins to a large proportion of rat type I spiral ganglion neurons (SGNs) and their projections to the cochlear nucleus (CN). Moreover, we show that a fraction of these espins is in the nucleus of SGNs owing to the presence of splice-isoforms that contain a functional nuclear localization signal (NLS). Espin antibody labeled approximately 83% of type I SGNs, and the labeling intensity increased dramatically during early postnatal development. Type II SGNs and vestibular ganglion neurons were unlabeled. In the CN, espin-positive auditory nerve fibers showed a projection pattern typical of type I SGNs, with intense labeling in the nerve root region and posteroventral CN (PVCN). The anteroventral CN (AVCN) showed moderate labeling, whereas the dorsal CN showed weak labeling that was restricted to the deep layer. Espin-positive synaptic terminals were enriched around nerve root neurons and octopus cells in the PVCN and were also found on globular bushy cells and multipolar neurons in the PVCN and AVCN. SGNs expressed multiple espin transcripts and proteins, including splice-isoforms that contain a nonapeptide, which is rich in positively charged amino acids and creates a bipartite NLS. The nonapeptide was necessary to target espin isoforms to the nucleus and was sufficient to target an unrelated protein to the nucleus when joined with the upstream di-arginine-containing octapeptide. The presence of cytoplasmic and nuclear espins in SGNs suggests additional roles for espins in auditory neuroscience.

Spinal astrocyte glutamate receptor 1 overexpression after ischemic insult facilitates behavioral signs of spasticity and rigidity.

Using a rat model of ischemic paraplegia, we examined the expression of spinal AMPA receptors and their role in mediating spasticity and rigidity. Spinal ischemia was induced by transient occlusion of the descending aorta combined with systemic hypotension. Spasticity/rigidity were identified by simultaneous measurements of peripheral muscle resistance (PMR) and electromyography (EMG) before and during ankle flexion. In addition, Hoffman reflex (H-reflex) and motor evoked potentials (MEPs) were recorded from the gastrocnemius muscle. Animals were implanted with intrathecal catheters for drug delivery and injected with the AMPA receptor antagonist NGX424 (tezampanel), glutamate receptor 1 (GluR1) antisense, or vehicle. Where intrathecal vehicle had no effect, intrathecal NGX424 produced a dose-dependent suppression of PMR [ED50 of 0.44 microg (0.33-0.58)], as well as tonic and ankle flexion-evoked EMG activity. Similar suppression of MEP and H-reflex were also seen. Western blot analyses of lumbar spinal cord tissue from spastic animals showed a significant increase in GluR1 but decreased GluR2 and GluR4 proteins. Confocal and electron microscopic analyses of spinal cord sections from spastic animals revealed increased GluR1 immunoreactivity in reactive astrocytes. Selective GluR1 knockdown by intrathecal antisense treatment resulted in a potent reduction of spasticiy and rigidity and concurrent downregulation of neuronal/astrocytic GluR1 in the lumbar spinal cord. Treatment of rat astrocyte cultures with AMPA led to dose-dependent glutamate release, an effect blocked by NGX424. These data suggest that an AMPA/kainate receptor antagonist can represent a novel therapy in modulating spasticity/rigidity of spinal origin and that astrocytes may be a potential target for such treatment.

Targeted wild-type and jerker espins reveal a novel, WH2-domain-dependent way to make actin bundles in cells.

The espin actin-bundling proteins, which are the target of deafness mutations, are present in the parallel actin bundles of stereocilia and microvilli and appear to increase their steady-state length. Here, we report a new activity of the espins, one that depends on their enigmatic WH2 domain: the ability to assemble a large actin bundle when targeted to a specific subcellular location. This activity was observed for wild-type espins targeted to the centrosome in transfected neuronal cells and for jerker espins targeted to the nucleolus in a wide variety of transfected cells as a result of the frameshifted peptide introduced into the espin C-terminus by the jerker deafness mutation. This activity, which appears specific to espins, requires two espin F-actin-binding sites and the actin-monomer-binding activity of the espin WH2 domain, but can be mimicked by adding a WH2 domain to an unrelated actin-bundling protein, villin. Espins do not activate the Arp2/3 complex in vitro, and bundle assembly is not indicative of in-vitro nucleation activity. Our results suggest a novel way to build actin bundles at specific sites in cells.

Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear.

The espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation.

Otolith organ or semicircular canal stimulation induces c-fos expression in unipolar brush cells and granule cells of cat and squirrel monkey.

Immediate early gene expression in the cerebellar vermis of cats and squirrel monkeys was stimulated by prolonged whole body rotations. Continuous, earth-horizontal axis rotations that excited only otoliths or high velocity vertical axis rotations that excited only semicircular canals resulted in c-fos immunoreactive nuclei concentrated in the granular layer of lobules X and ventral IX (the nodulus and ventral uvula), which represent the medial parts of the vestibulo-cerebellum. Large clusters of labeled nuclei consisting mainly of granule cells and calretinin-positive unipolar brush cells were present in the granular layer, whereas Purkinje cell nuclei were unlabeled, and labeled basket and stellate cell nuclei were scattered in the molecular layer. In other vermal lobules there was a significant but less dense label than in the nodulus and ventral uvula. Generally, the extent of c-fos labeling of molecular layer interneurons was in relation to nuclear labeling of granular layer neurons: labeling of both basket and stellate cells accompanied nuclear labeling of neurons throughout the depth of the granular layer, whereas only stellate cells were labeled when nuclear labeling was restricted to the superficial granular layer. Yaw horizontal or roll vertical rotations each stimulated c-fos expression in the cat medial vestibulo-cerebellum to approximately the same extent. Low-velocity rotations resulted in much less c-fos expression. Similar, albeit less intense, patterns of c-fos activation were observed in monkeys. Concentrated c-fos expression in the medial vestibulo-cerebellum after exposure to a strong head velocity signal that could originate from either otolith or canal excitation suggests that granule and unipolar brush cells participate in a neuronal network for estimating head velocity, irrespective of the signal source.

Espin cytoskeletal proteins in the sensory cells of rodent taste buds.

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 +/- 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 +/- 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP(3)R(3))(,) alpha-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP(3)R(3), PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP(3)R(3), alpha-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of approximately 3 mum-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10-12 mum into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells.