Opposing expression gradients of calcitonin‐related polypeptide alpha (Calca/Cgrpα) and tyrosine hydroxylase (Th) in type II afferent neurons of the mouse cochlea

Type II spiral ganglion neurons (SGNs) are small caliber, unmyelinated afferents that extend dendritic arbors hundreds of microns along the cochlear spiral, contacting many outer hair cells (OHCs). Despite these many contacts, type II afferents are insensitive to sound and only weakly depolarized by glutamate release from OHCs. Recent studies suggest that type II afferents may be cochlear nociceptors, and can be excited by ATP released during tissue damage, by analogy to somatic pain‐sensing C‐fibers. The present work compares the expression patterns among cochlear type II afferents of two genes found in C‐fibers: calcitonin‐related polypeptide alpha (Calca/Cgrpα), specific to pain‐sensing C‐fibers, and tyrosine hydroxylase (Th), specific to low‐threshold mechanoreceptive C‐fibers, which was shown previously to be a selective biomarker of type II versus type I cochlear afferents (Vyas et al., 2016 ). Whole‐mount cochlear preparations from 3‐week‐ to 2‐month‐old CGRPα‐EGFP (GENSAT) mice showed expression of Cgrpα in a subset of SGNs with type II‐like peripheral dendrites extending beneath OHCs. Double labeling with other molecular markers confirmed that the labeled SGNs were neither type I SGNs nor olivocochlear efferents. Cgrpα starts to express in type II SGNs before hearing onset, but the expression level declines in the adult. The expression patterns of Cgrpα and Th formed opposing gradients, with Th being preferentially expressed in apical and Cgrpα in basal type II afferent neurons, indicating heterogeneity among type II afferent neurons. The expression of Th and Cgrpα was not mutually exclusive and co‐expression could be observed, most abundantly in the middle cochlear turn.     

Genetic access to neurons in the accessory optic system reveals a role for Sema6A in midbrain circuitry mediating motion perception

The accessory optic system (AOS) detects retinal image slip and reports it to the oculomotor system for reflexive image stabilization. Here, we characterize two Cre lines that permit genetic access to AOS circuits responding to vertical motion. The first (Pcdh9-Cre) labels only one of the four subtypes of ON direction-selective retinal ganglion cells (ON-DS RGCs), those preferring ventral retinal motion. Their axons diverge from the optic tract just behind the chiasm and selectively innervate the medial terminal nucleus (MTN) of the AOS. Unlike most RGC subtypes examined, they survive after optic nerve crush. The second Cre-driver line (Pdzk1ip1-Cre) labels postsynaptic neurons in the MTN. These project predominantly to the other major terminal nucleus of the AOS, the nucleus of the optic tract (NOT). We find that the transmembrane protein semaphorin 6A (Sema6A) is required for the formation of axonal projections from the MTN to the NOT, just as it is for the retinal innervation of the MTN. These new tools permit manipulation of specific circuits in the AOS and show that Sema6A is required for establishing AOS connections in multiple locations.     

Selective neuronal expression of the SoxE factor,Sox8, in direct pathway striatal projection neurons of the developing mouse brain

The striatum is the major component of the basal ganglia and is well known to play a key role in the control of motor function via balanced output from the indirect (iSPNs) and direct pathway striatal projection neurons (dSPNs). Little is known, however, about the molecular genetic mechanisms that control the formation of the iSPNs versus dSPNs. We show here that the SoxE family member, Sox8, is co‐expressed with the dSPN markers, Isl1 and Ebf1, in the developing striatum. Moreover, dSPNs, as marked by Isl1‐cre fate map, express Sox8 in the embryonic striatum and Sox8‐EGFP BAC transgenic mice specifically reveal the direct pathway axons during development. These EGFP⁺ axons are first observed to reach their midbrain target, the substantia nigra pars reticulata (SNr), at E14 in the mouse with a robust connection observed already at birth. The selective expression of EGFP in dSPNs of Sox8‐EGFP BAC mice is maintained at postnatal timepoints. Sox8 is known to be expressed in oligodendrocyte precursor cells (OPCs) together with other SoxE factors and we show here that the EGFP signal co‐localizes with the OPC markers throughout the brain. Finally, we show that Sox8‐EGFP BAC mice can be used to interrogate the altered dSPN development in Isl1 conditional mutants including aberrant axonal projections detected already at embryonic timepoints. Thus, Sox8 represents an early and specific marker of embryonic dSPNs and the Sox8‐EGFP BAC transgenic mice are an excellent tool to study the development of basal ganglia circuitry.     

The morphological characterization of orientation‐biased displaced large‐field ganglion cells in the central part of goldfish retina

The vertebrate retina has about 30 subtypes of ganglion cells. Each ganglion cell receives synaptic inputs from specific types of bipolar and amacrine cells ramifying at the same depth of the inner plexiform layer (IPL), each of which is thought to process a specific aspect of visual information. Here, we identified one type of displaced ganglion cell in the goldfish retina which had a large and elongated dendritic field. As a population, all of these ganglion cells were oriented in the horizontal axis and perpendicular to the dorsal–ventral axis of the goldfish eye in the central part of retina. This ganglion cell has previously been classified as Type 1.2. However, the circuit elements which synapse with this ganglion cell are not yet characterized. We found that this displaced ganglion cell was directly tracer‐coupled only with homologous ganglion cells at sites containing Cx35/36 puncta. We further illustrated that the processes of dopaminergic neurons often terminated next to intersections between processes of ganglion cells, close to where dopamine D1 receptors were localized. Finally, we showed that Mb1 ON bipolar cells had ribbon synapses in the axonal processes passing through the IPL and made ectopic synapses with this displaced ganglion cell that stratified into stratum 1 of the IPL. These results suggest that the displaced ganglion cell may synapse with both Mb1 cells using ectopic ribbon synapses and OFF cone bipolar cells with regular ribbon synapses in the IPL to function in both scotopic and photopic light conditions.     

Light‐evoked S‐nitrosylation in the retina

Nitric oxide (NO) synthesis in the retina is triggered by light stimulation. NO has been shown to modulate visual signal processing at multiple sites in the vertebrate retina, via activation of the most sensitive target of NO signaling, soluble guanylate cyclase. NO can also alter protein structure and function and exert biological effects directly by binding to free thiol groups of cysteine residues in a chemical reaction called S‐nitrosylation. However, in the central nervous system, including the retina, this reaction has not been considered to be significant under physiological conditions. Here we provide immunohistochemical evidence for extensive S‐nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity‐dependent manner, with a strikingly similar pattern in both species. Pretreatment with N‐ethylmaleimide (NEM), which occludes S‐nitrosylation, or with 1‐(2‐trifluromethylphenyl)imidazole (TRIM), an inhibitor of neuronal NO synthase, eliminated the light‐evoked increase in S‐nitrosylated protein immunofluorescence (SNI) in the retinas of both species. Similarly, light did not increase SNI, above basal levels, in retinas of transgenic mice lacking neuronal NO synthase. Qualitative analysis of the light‐adapted mouse retina with mass spectrometry revealed more than 300 proteins that were S‐nitrosylated upon illumination, many of which are known to participate directly in retinal signal processing. Our data strongly suggest that in the retina light‐evoked NO production leads to extensive S‐nitrosylation and that this process is a significant posttranslational modification affecting a wide range of proteins under physiological conditions. J. Comp. Neurol. 523:2082–2110, 2015. © 2015 Wiley Periodicals, Inc.     

Cell‐specific and developmental expression of lectican‐cleaving proteases in mouse hippocampus and neocortex

Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain and that this glycoprotein‐rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assemblies of these extracellular matrix glycoproteins are perineuronal nets, specialized lattice‐like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans, a family of chondroitin sulfate proteoglycans that includes aggrecan, brevican, neurocan, and versican. These lattice‐like structures emerge late in postnatal brain development, coinciding with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of the extracellular proteases that are responsible for their cleavage and turnover. A subset of a large family of extracellular proteases (called a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]) is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of two aggrecan‐degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in the hippocampus and neocortex. Here, we show that both lectican‐degrading metalloproteases are present in these brain regions and that each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by parvalbumin‐expressing interneurons during synaptogenesis, whereas Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development. J. Comp. Neurol. 523:629–648, 2015. © 2014 Wiley Periodicals, Inc.     

Molecular heterogeneity of aggrecan‐based perineuronal nets around five subclasses of parvalbumin‐expressing neurons in the mouse hippocampus

Subsets of GABAergic neurons are surrounded by perineuronal nets (PNNs), which play a critical role in the regulation of neural plasticity and neuroprotection. Although the plant lectin Wisteria floribunda agglutinin (WFA) has been commonly used to label PNNs, WFA only detects N‐acetyl‐d ‐galactosamine on aggrecan, a member of the lectican family. In this study, we used WFA and the antibody against the core protein of aggrecan (ACAN) to investigate the molecular heterogeneity of aggrecan‐based PNNs around five subclasses of parvalbumin‐expressing (PV+) γ‐aminobutyric acid (GABA)ergic neurons in the CA1 and CA3 regions of the mouse hippocampus. The vast majority of ACAN+ PNNs were colocalized with WFA in the stratum pyramidale, whereas a substantial population of ACAN+ PNNs lacked WFA labeling in the stratum oriens. We then defined the subclasses of PV+ neurons based on their cellular locations, molecular expression, and septal projection. Like the WFA+ PNNs, ACAN+ PNNs surrounded PV+ basket cells and bistratified cells but not axo‐axonic cells. Unlike the WFA+ PNNs, ACAN+ PNNs frequently surrounded PV+ oriens‐lacunosum moleculare cells and hippocampo‐septal cells. Interestingly, the relative densities of GABAergic synapses were higher around PV+ neurons with ACAN+ PNNs than around those without ACAN+ PNNs. Degradation of WFA+ PNNs by chondroitinase ABC did not affect the GABAergic synaptic densities around PV+ neurons. Our findings suggest that the molecular composition of aggrecan‐based PNNs around PV+ neurons may differ in a subclass‐specific manner, and also might help determine the functional involvement of PNNs in the regulation of GABAergic synapses around PV+ neurons in the hippocampus. J. Comp. Neurol. 525:1234–1249, 2017. © 2016 Wiley Periodicals, Inc.     

A critical period for the trophic actions of leptin on AgRP neurons in the arcuate nucleus of the hypothalamus

In the developing hypothalamus, the fat‐derived hormone leptin stimulates the growth of axons from the arcuate nucleus of the hypothalamus (ARH) to other regions that control energy balance. These projections are significantly reduced in leptin deficient (Lep^ob/ob) mice and this phenotype is largely rescued by neonatal leptin treatments. However, treatment of mature Lep^ob/ob mice is ineffective, suggesting that the trophic action of leptin is limited to a developmental critical period. To temporally delineate closure of this critical period for leptin‐stimulated growth, we treated Lep^ob/ob mice with exogenous leptin during a variety of discrete time periods, and measured the density of Agouti‐Related Peptide (AgRP) containing projections from the ARH to the ventral part of the dorsomedial nucleus of the hypothalamus (DMHv), and to the medial parvocellular part of the paraventricular nucleus (PVHmp). The results indicate that leptin loses its neurotrophic potential at or near postnatal day 28. The duration of leptin exposure appears to be important, with 9‐ or 11‐day treatments found to be more effective than shorter (5‐day) treatments. Furthermore, leptin treatment for 9 days or more was sufficient to restore AgRP innervation to both the PVHmp and DMHv in Lep^ob/ob females, but only to the DMHv in Lep^ob/ob males. Together, these findings reveal that the trophic actions of leptin are contingent upon timing and duration of leptin exposure, display both target and sex specificity, and that modulation of leptin‐dependent circuit formation by each of these factors may carry enduring consequences for feeding behavior, metabolism, and obesity risk.     

Morphological and functional changes in TRPM8‐expressing corneal cold thermoreceptor neurons during aging and their impact on tearing in mice

Morphological and functional alterations of peripheral somatosensory neurons during the aging process lead to a decline of somatosensory perception. Here, we analyze the changes occurring with aging in trigeminal ganglion (TG), TRPM8‐expressing cold thermoreceptor neurons innervating the mouse cornea, which participate in the regulation of basal tearing and blinking and have been implicated in the pathogenesis of dry eye disease (DED). TG cell bodies and axonal branches were examined in a mouse line (TRPM8^BAC‐EYFP) expressing a fluorescent reporter. In 3 months old animals, about 50% of TG cold thermoreceptor neurons were intensely fluorescent, likely providing strongly fluorescent axons and complex corneal nerve terminals with ongoing activity at 34°C and low‐threshold, robust responses to cooling. The remaining TRPM8⁺ corneal axons were weakly fluorescent with nonbeaded axons, sparsely ramified nerve terminals, and exhibited a low‐firing rate at 34°C, responding moderately to cooling pulses as do weakly fluorescent TG neurons. In aged (24 months) mice, the number of weakly fluorescent TG neurons was strikingly high while the morphology of TRPM8⁺ corneal axons changed drastically; 89% were weakly fluorescent, unbranched, and often ending in the basal epithelium. Functionally, 72.5% of aged cold terminals responded as those of young animals, but 27.5% exhibited very low‐background activity and abnormal responsiveness to cooling pulses. These morpho‐functional changes develop in parallel with an enhancement of tear's basal flow and osmolarity, suggesting that the aberrant sensory inflow to the brain from impaired peripheral cold thermoreceptors contributes to age‐induced abnormal tearing and to the high incidence of DED in elderly people.     

Mid‐life environmental enrichment increases synaptic density in CA1 in a mouse model of Aβ‐associated pathology and positively influences synaptic and cognitive health in healthy ageing

Early‐life cognitive enrichment may reduce the risk of experiencing cognitive deterioration and dementia in later‐life. However, an intervention to prevent or delay dementia is likely to be taken up in mid to later‐life. Hence, we investigated the effects of environmental enrichment in wildtype mice and in a mouse model of Aβ neuropathology (APP_SWE/PS1_dE9) from 6 months of age. After 6 months of housing in standard laboratory cages, APP_SWE/PS1_dE9 (n = 27) and healthy wildtype (n = 21) mice were randomly assigned to either enriched or standard housing. At 12 months of age, wildtype mice showed altered synaptic protein levels and relatively superior cognitive performance afforded by environmental enrichment. Environmental enrichment was not associated with alterations to Aβ plaque pathology in the neocortex or hippocampus of APP_SWE/PS1_dE9 mice. However, a significant increase in synaptophysin immunolabeled puncta in the hippocampal subregion, CA1, in APP_SWE/PS1_dE9 mice was detected, with no significant synaptic density changes observed in CA3, or the Fr2 region of the prefrontal cortex. Moreover, a significant increase in hippocampal BDNF was detected in APP_SWE/PS1_dE9 mice exposed to EE, however, no changes were detected in neocortex or between Wt animals. These results demonstrate that mid to later‐life cognitive enrichment has the potential to promote synaptic and cognitive health in ageing, and to enhance compensatory capacity for synaptic connectivity in pathological ageing associated with Aβ deposition.     

Pigment epithelium‐derived factor (PEDF) and PEDF‐receptor in the adult mouse brain: Differential spatial/temporal localization pattern

Pigment epithelium‐derived factor (PEDF) is a multifunctional protein which was initially described in the retina, although it is also present in other tissues. It functions as an antioxidant agent promoting neuronal survival. Recently, a PEDF receptor has shown an elevated binding affinity for PEDF. There are no relevant data regarding the distribution of both proteins in the brain, therefore the main goal of this work was to investigate the spatiotemporal presence of PEDF and PEDFR in the adult mouse brain, and to determine the PEDF blood level in mouse and human. The localization of both proteins was analyzed by different experimental methods such as immunohistochemistry, western‐blotting, and also by enzyme‐linked immunosorbent assay. Differential expression was found in some telencephalic structures and positive signals for both proteins were detected in the cerebellum. The magnitude of the PEDFR labeling pattern was higher than PEDF and included some cortical and subventricular areas. Age‐dependent changes in intensity of both protein immunoreactions were found in the cortical and hippocampal areas with greater reactivity between 4 and 8 months of age, whilst others, like the subventricular zones, these differences were more evident for PEDFR. Although ubiquitous presence was not found in the brain for these two proteins, their relevant functions must not be underestimated. It has been described that PEDF plays an important role in neuroprotection and data provided in the present work represents the first extensive study to understand the relevance of these two proteins in specific brain areas.     

Micro‐RNA‐30a regulates ischemia‐induced cell death by targeting heat shock protein HSPA5 in primary cultured cortical neurons and mouse brain after stroke

Micro‐RNAs (miRs) have emerged as key gene regulators in many diseases, including stroke. We recently reported that miR‐30a protects N2A cells against ischemic injury, in part through enhancing beclin 1‐mediated autophagy. The present study explores further the involvement of miR‐30a in ischemia‐induced apoptosis and its possible mechanisms in primary cortical neurons and stroked mouse brain. We demonstrate that miR‐30a level is significantly decreased in cortical neurons after 1‐hr oxygen–glucose deprivation (OGD)/24‐hr reoxygenation. Overexpression of miR‐30a aggravated the OGD‐induced neuronal cell death, whereas inhibition of miR‐30a attenuated necrosis and apoptosis as determined by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐di‐phenyl‐2H‐tetrazolium bromide, lactate dehydrogenase, TUNEL, and cleaved caspase‐3. The amount of HSPA5 protein, which is predicted to be a putative target of miR‐30a by TargetScan, could be reduced by pre‐miR‐30a, whereas it was increased by anti‐miR‐30a. Furthermore, the luciferase reporter assay confirmed that miR‐30a directly binds to the predicted 3′‐UTR target sites of the hspa5 gene. The cell injury regulated by miR‐30a in OGD‐treated cells could be aggravated by HSPA5 siRNA. We also observed an interaction of HSPA5 and caspase‐12 by coimmunoprecipitation and speculate that HSPA5 might be involved in endoplasmic reticulum stress‐induced apoptosis. In vivo, reduced miR‐30a increased the HSPA5 level and attenuated ischemic brain infarction in focal ischemia‐stroked mice. Downregulation of miR‐30a could prevent neural ischemic injury through upregulating HSPA5 protein expression, and decreased ER stress‐induced apoptosis might be one of the mechanisms underlying HSPA5‐mediated neuroprotection. © 2015 Wiley Periodicals, Inc.     

Target‐specific forebrain projections and appropriate synaptic inputs of hESC‐derived dopamine neurons grafted to the midbrain of parkinsonian rats

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non‐human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC‐derived progenitors were grafted into the midbrain of 6‐hydroxydopamine‐lesioned rats, and analyzed at 6, 18, and 24 weeks for a time‐course evaluation of specificity and extent of graft‐derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies‐based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft‐derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine‐induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC‐derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC‐derived DA neurons to the midbrain.     

Post‐crossing segment of dI1 commissural axons forms collateral branches to motor neurons in the developing spinal cord

The dI1 commissural axons in the developing spinal cord, upon crossing the midline through the floor plate, make a sharp turn to grow rostrally. These post‐crossing axons initially just extend adjacent to the floor plate without entering nearby motor columns. However, it remains poorly characterized how these post‐crossing dI1 axons behave subsequently to this process. In the present study, to address this issue, we examined in detail the behavior of post‐crossing dI1 axons in mice, using the Atoh1 enhancer‐based conditional expression system that enables selective and sparse labeling of individual dI1 axons, together with Hb9 and ChAT immunohistochemistry for precise identification of spinal motor neurons (MNs). We found unexpectedly that the post‐crossing segment of dI1 axons later gave off collateral branches that extended laterally to invade motor columns. Interestingly, these collateral branches emerged at around the time when their primary growth cones initiated invasion into motor columns. In addition, although the length of the laterally growing collateral branches increased with age, the majority of them remained within motor columns. Strikingly, these collateral branches further gave rise to multiple secondary branches in the region of MNs that innervate muscles close to the body axis. Moreover, these axonal branches formed presynaptic terminals on MNs. These observations demonstrate that dI1 commissural neurons develop axonal projection to spinal MNs via collateral branches arising later from the post‐crossing segment of these axons. Our findings thus reveal a previously unrecognized projection of dI1 commissural axons that may contribute directly to generating proper motor output.     

An optimized dosing regimen of cimaglermin (neuregulin 1β3, glial growth factor 2) enhances molecular markers of neuroplasticity and functional recovery after permanent ischemic stroke in rats

Cimaglermin (neuregulin 1β3, glial growth factor 2) is a neuregulin growth factor family member in clinical development for chronic heart failure. Previously, in a permanent middle cerebral artery occlusion (pMCAO) rat stroke model, systemic cimaglermin treatment initiated up to 7 days after ischemia onset promoted recovery without reduced lesion volume. Presented here to extend the evidence are two studies that use a rat stroke model to evaluate the effects of cimaglermin dose level and dose frequency initiated 24 hr after pMCAO. Forelimb‐ and hindlimb‐placing scores (proprioceptive behavioral tests), body‐swing symmetry, and infarct volume were compared between treatment groups (n = 12/group). Possible mechanisms underlying cimaglermin‐mediated neurologic recovery were examined through axonal growth and synapse formation histological markers. Cimaglermin was evaluated over a wider dose range (0.02, 0.1, or 1.0 mg/kg) than doses previously shown to be effective but used the same dosing regimen (2 weeks of daily intravenous administration, then 1 week without treatment). The dose‐frequency study used the dose‐ranging study's most effective dose (1.0 mg/kg) to compare daily, once per week, and twice per week dosing for 3 weeks (then 1 week without treatment). Dose‐ and frequency‐dependent functional improvements were observed with cimaglermin without reduced lesion volume. Cimaglermin treatment significantly increased growth‐associated protein 43 expression in both hemispheres (particularly somatosensory and motor cortices) and also increased synaptophysin expression. These data indicate that cimaglermin enhances recovery after stroke. Immunohistochemical changes were consistent with axonal sprouting and synapse formation but not acute neuroprotection. Cimaglermin represents a potential clinical development candidate for ischemic stroke treatment. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.