Long-range inhibitory neurons mediate cortical neurovascular coupling.

To meet the high energy demands of brain function, cerebral blood flow (CBF) parallels changes in neuronal activity by a mechanism known as neurovascular coupling (NVC). However, which neurons play a role in mediating NVC is not well understood. Here, we identify in mice and humans a specific population of cortical GABAergic neurons that co-express neuronal nitric oxide synthase and tachykinin receptor 1 (Tacr1). Through whole-tissue clearing, we demonstrate that Tacr1 neurons extend local and long-range projections across functionally connected cortical areas. We show that whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feedforward excitatory pathways. Additionally, through optogenetic experiments, we demonstrate that Tacr1 neurons are instrumental in mediating CBF through the relaxation of mural cells in a similar fashion to whisker stimulation. Finally, by electron microscopy, we observe that Tacr1 processes contact astrocytic endfeet. These findings suggest that Tacr1 neurons integrate cortical activity to mediate NVC.

Sex differences in glutamate AMPA receptor subunits mRNA with fast gating kinetics in the mouse cochlea.

Evidence shows that females have increased supra-threshold peripheral auditory processing compared to males. This is indicated by larger auditory brainstem responses (ABR) wave I amplitude, which measures afferent spiral ganglion neuron (SGN)-auditory nerve synchrony. However, the underlying molecular mechanisms of this sex difference are mostly unknown. We sought to elucidate sex differences in ABR wave I amplitude by examining molecular markers known to affect synaptic transmission kinetics. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate fast excitatory transmission in mature SGN afferent synapses. Each AMPAR channel is a tetramer composed of GluA2, 3, and 4 subunits (Gria2, 3, and 4 genes), and those lacking GluA2 subunits have larger currents, are calcium-permeable, and have faster gating kinetics. Moreover, alternatively spliced flip and flop isoforms of each AMPAR subunit affect channel kinetics, having faster kinetics those AMPARs containing Gria3 and Gria4 flop isoforms. We hypothesized that SGNs of females have more fast-gating AMPAR subunit mRNA than males, which could contribute to more temporally precise synaptic transmission and increased SGN synchrony. Our data show that the index of Gria3 relative to Gria2 transcripts on SGN was higher in females than males (females: 48%; males: 43%), suggesting that females have more SGNs with higher Gria3 mRNA relative to Gria2. Analysis of the relative abundance of the flip and flop alternatively spliced isoforms showed that females have a 2-fold increase in fast-gating Gria3 flop mRNA, while males have more slow-gating (2.5-fold) of the flip. We propose that Gria3 may in part mediate greater SGN synchrony in females. Significance Statement: Females of multiple vertebrate species, including fish and mammals, have been reported to have enhanced sound-evoked synchrony of afferents in the auditory nerve. However, the underlying molecular mediators of this physiologic sex difference are unknown. Elucidating potential molecular mechanisms related to sex differences in auditory processing is important for maintaining healthy ears and developing potential treatments for hearing loss in both sexes. This study found that females have a 2-fold increase in Gria3 flop mRNA, a fast-gating AMPA-type glutamate receptor subunit. This difference may contribute to greater neural synchrony in the auditory nerve of female mice compared to males, and this sex difference may be conserved in all vertebrates.

Embryonic medial ganglionic eminence cells survive and integrate into the inferior colliculus of adult mice.

Acoustic overexposure can lead to decreased inhibition in auditory centers, including the inferior colliculus (IC), and has been implicated in the development of central auditory pathologies. While systemic drugs that increase GABAergic transmission have been shown to provide symptomatic relief, their side effect profiles impose an upper-limit on the dose and duration of use. A treatment that locally increases inhibition in auditory nuclei could mitigate these side effects. One such approach could be transplantation of inhibitory precursor neurons derived from the medial ganglionic eminence (MGE). The present study investigated whether transplanted MGE cells can survive and integrate into the IC of non-noise exposed and noise exposed mice. MGE cells were harvested on embryonic days 12-14 and injected bilaterally into the IC of adult mice, with or without previous noise exposure. At one-week post transplantation, MGE cells possessed small, elongated soma and bipolar processes, characteristic of migrating cells. By 5 weeks, MGE cells exhibited a more mature morphology, with multiple branching processes and axons with boutons that stain positive for the vesicular GABA transporter (VGAT). The MGE survival rate after 14 weeks post transplantation was 1.7% in non-noise exposed subjects. MGE survival rate was not significantly affected by noise exposure (1.2%). In both groups the vast majority of transplanted MGE cells (>97%) expressed the vesicular GABA transporter. Furthermore, electronmicroscopic analysis indicated that transplanted MGE cells formed synapses with and received synaptic endings from host IC neurons. Acoustic stimulation lead to a significant increase in the percentage of endogenous inhibitory cells that express c-fos but had no effect on the percentage of c-fos expressing transplanted MGE cells. MGE cells were observed in the IC up to 22 weeks post transplantation, the longest time point investigated, suggesting long term survival and integration. These data provide the first evidence that transplantation of MGE cells is viable in the IC and provides a new strategy to explore treatment options for central hearing dysfunction following noise exposure.

Metabolism and global protein glycosylation are differentially expressed in healthy and osteoarthritic equine carpal synovial fluid.

BACKGROUND: Carpal osteochondral fragmentation and subsequent post-traumatic osteoarthritis (PTOA) are leading causes of wastage in the equine athlete. Identification of synovial fluid biomarkers could contribute to the diagnosis and understanding of osteoarthritis (OA) pathophysiology. OBJECTIVE: The aim of this study was to identify differentially expressed metabolic and glycosylation pathways in synovial fluid from healthy horses and horses with naturally occurring carpal OA. STUDY DESIGN: Cross-sectional, in vivo metabolomics and glycomics study. METHODS: In cohort 1, carpal synovial fluid (n = 12 horses; n = 6 healthy, n = 6 OA) was analysed using high-resolution liquid chromatography mass spectrometry (LC-MS). In cohort 2 (n = 40 horses; n = 20 healthy, n = 20 OA), carpal synovial fluid was analysed using lectin microarrays and a lubricin sandwich ELISA. RESULTS: Metabolomic analysis identified >4900 LC-MS features of which 84 identifiable metabolites were differentially expressed (P < .05) between healthy and OA joints, including key pathways related to inflammation (histidine and tryptophan metabolism), oxidative stress (arginine biosynthesis) and collagen metabolism (lysine metabolism). Principle Component Analysis and Partial Least Squares Discriminant Analysis demonstrated separation between healthy and OA synovial fluid. Lectin microarrays identified distinct glycosylation patterns between healthy and OA synovial fluid, including increased Core 1/Core 3 O-glycosylation, increased α-2,3 sialylation and decreased α-1,2 fucosylation in OA. O-glycans predominated over N-glycans in all synovial fluid samples, and synovial fluid lubricin was increased in OA joints as compared to controls. MAIN LIMITATIONS: The sample size in cohort 1 was limited, and there is inherent variation in severity and duration of joint injury in naturally occurring OA. However, LC-MS identified up to 5000 unique features. CONCLUSIONS: These data suggest new potential diagnostic and therapeutic targets for equine OA. Future targeted metabolomic and glycomic studies should be performed to verify these results. Lectin microarrays could be investigated as a potential screening tool for the diagnosis and therapeutic monitoring of equine OA.

Role of GluA4 in the acoustic and tactile startle responses.

The primary startle response (SR) is an innate reaction evoked by sudden and intense acoustic, tactile or visual stimuli. In rodents and humans the SR involves reflexive contractions of the face, neck and limb muscles. The acoustic startle response (ASR) pathway consists of auditory nerve fibers (AN), cochlear root neurons (CRNs) and giant neurons of the caudal pontine reticular nucleus (PnC), which synapse on cranial and spinal motor neurons. The tactile startle response (TSR) is transmitted by primary sensory neurons to the principal sensory (Pr5) and spinal (Sp5) trigeminal nuclei. The ventral part of Pr5 projects directly to the PnC neurons. The SR requires rapid transmission of sensory information to initiate a fast motor response. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) are necessary to transmit auditory information to the PnC neurons and elicit the SR. AMPARs containing the glutamate AMPAR subunit 4 (GluA4) have fast kinetics, which makes them ideal candidates to transmit the SR signal. This study examined the role of GluA4 within the primary SR pathway by using GluA4 knockout (GluA4-KO) mice. Deletion of GluA4 considerably decreased the amplitude and probability of successful ASR and TSR, indicating that the presence of this subunit is critical at a common station within the startle pathway. We conclude that deletion of GluA4 affects the transmission of sensory signals from acoustic and tactile pathways to the motor component of the startle reflex. Therefore, GluA4 is required for the full response and for reliable elicitation of the startle response.

Obesity-Associated Metabolic Disturbances Reverse the Antioxidant and Anti-Inflammatory Properties of High-Density Lipoproteins in Microglial Cells.

High-density lipoproteins (HDLs) play an important role in reverse cholesterol transport and present antioxidant properties, among others. In the central nervous system (CNS), there are HDLs, where these lipoproteins could influence brain health. Owing to the new evidence of HDL functionality remodeling in obese patients, and the fact that obesity-associated metabolic disturbances is pro-inflammatory and pro-oxidant, the aim of this study was to investigate if HDL functions are depleted in obese patients and obesity-associated microenvironment. HDLs were isolated from normal-weight healthy (nwHDL) and obese men (obHDL). The oxHDL level was measured by malondialdehyde and 4-hydroxynoneal peroxided products. BV2 microglial cells were exposed to different concentrations of nwHDL and obHDL in different obesity-associated pro-inflammatory microenvironments. Our results showed that hyperleptinemia increased oxHDL levels. In addition, nwHDLs reduced pro-inflammatory cytokines' release and M1 marker gene expression in BV2 microglial cells. Nevertheless, both nwHDL co-administered with LPS+leptin and obHDL promoted BV2 microglial activation and a higher pro-inflammatory cytokine production, thus confirming that obesity-associated metabolic disturbances reverse the antioxidant and anti-inflammatory properties of HDLs in microglial cells.

Central Auditory Plasticity from Molecules to Behavior.

Understanding how, when, and for how long the adult central auditory system adapts to hearing loss and aging is an important topic that is currently studied across the globe [...].

Outer Hair Cell Glutamate Signaling through Type II Spiral Ganglion Afferents Activates Neurons in the Cochlear Nucleus in Response to Nondamaging Sounds.

Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells, are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons: in this case, onto the sparse Type II SG neurons. However, unlike glutamate signaling at the inner hair cell-Type I SG neuron synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-Type II SG neuron synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-Type II SG pathway to signal to the brain in response to moderate, nondamaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the VGluTs associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to Type II SG neurons in KO mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus in response to sound. From these analyses, we show, for the first time, that glutamatergic signaling at the OHC-Type II SG neuron synapse is capable of activating cochlear nucleus neurons, even at moderate sound levels.SIGNIFICANCE STATEMENT Evidence suggests that cochlear outer hair cells (OHCs) release glutamate onto Type II spiral ganglion neurons only when exposed to loud sound, and that Type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGluT3 for synaptic glutamate release. We then used a genetically modified mouse line in which OHCs, but not inner hair cells, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC-Type II spiral ganglion pathway. Together, these data indicate that glutamate signaling at the OHC-Type II afferent synapse participates in auditory function at moderate sound levels.

Ultrastructural and molecular features of excitatory and glutamatergic synapses. The auditory nerve synapses.

Glutamatergic synapses mediate fast synaptic transmission in the central nervous system. New developments highlight the importance of the synapse structural and molecular remodeling during development, aging and in neurological disorders. This chapter summarizes key structural and molecular aspects of the presynaptic and postsynaptic components of glutamatergic synapses in the brain. In addition, this chapter describes how the structure of the postsynaptic density and ionotropic glutamate content contribute to the function of auditory nerve synapses in the lower auditory brainstem.

Role of GluA3 AMPA Receptor Subunits in the Presynaptic and Postsynaptic Maturation of Synaptic Transmission and Plasticity of Endbulb-Bushy Cell Synapses in the Cochlear Nucleus.

The AMPA receptor (AMPAR) subunit GluA3 has been suggested to shape synaptic transmission and activity-dependent plasticity in endbulb-bushy cell synapses (endbulb synapses) in the anteroventral cochlear nucleus, yet the specific roles of GluA3 in the synaptic transmission at endbulb synapses remains unexplored. Here, we compared WT and GluA3 KO mice of both sexes and identified several important roles of GluA3 in the maturation of synaptic transmission and short-term plasticity in endbulb synapses. We show that GluA3 largely determines the ultrafast kinetics of endbulb synapses glutamatergic currents by promoting the insertion of postsynaptic AMPARs that contain fast desensitizing flop subunits. In addition, GluA3 is also required for the normal function, structure, and development of the presynaptic terminal which leads to altered short term-depression in GluA3 KO mice. The presence of GluA3 reduces and slows synaptic depression, which is achieved by lowering the probability of vesicle release, promoting efficient vesicle replenishment, and increasing the readily releasable pool of synaptic vesicles. Surprisingly, GluA3 also makes the speed of synaptic depression rate-invariant. We propose that the slower and rate-invariant speed of depression allows an initial response window that still contains presynaptic firing rate information before the synapse is depressed. Because this response window is rate-invariant, GluA3 extends the range of presynaptic firing rates over which rate information in bushy cells can be preserved. This novel role of GluA3 may be important to allowing the postsynaptic targets of spherical bushy cells in mice use rate information for encoding sound intensity and sound localization.SIGNIFICANCE STATEMENT We report novel roles of the glutamate receptor subunit GluA3 in synaptic transmission in synapses between auditory nerve fibers and spherical bushy cells (BCs) in the cochlear nucleus. We show that GluA3 contributes to the generation of ultrafast glutamatergic currents at these synapses, which is important to preserve temporal information about the sound. Furthermore, we demonstrate that GluA3 contributes to the normal function and development of the presynaptic terminal, whose properties shape short-term plasticity. GluA3 slows and attenuates synaptic depression, and makes it less dependent on the presynaptic firing rates. This may help BCs to transfer information about the high rates of activity that occur at the synapse in vivo to postsynaptic targets that use rate information for sound localization.

Ultrastructural and functional changes at the tripartite synapse during epileptogenesis in a model of temporal lobe epilepsy.

The persistent unresponsiveness of many of the acquired epilepsies to traditional antiseizure medications has motivated the search for prophylactic drug therapies that could reduce the incidence of epilepsy in this at risk population. These studies are based on the idea of a period of epileptogenesis that can follow a wide variety of brain injuries. Epileptogenesis is hypothesized to involve changes in the brain not initially associated with seizures, but which result finally in seizure prone networks. Understanding these changes will provide crucial clues for the development of prophylactic drugs. Using the repeated low-dose kainate rat model of epilepsy, we have studied the period of epileptogenesis following status epilepticus, verifying the latent period with continuous EEG monitoring. Focusing on ultrastructural properties of the tripartite synapse in the CA1 region of hippocampus we found increased astrocyte ensheathment around both the presynaptic and postsynaptic elements, reduced synaptic AMPA receptor subunit and perisynaptic astrocyte GLT-1 expression, and increased number of docked vesicles at the presynaptic terminal. These findings were associated with an increase in frequency of the mEPSCs observed in patch clamp recordings of CA1 pyramidal cells. The results suggest a complex set of changes, some of which have been associated with increasingly excitable networks such as increased vesicles and mEPSC frequency, and some associated with compensatory mechanisms, such as increased astrocyte ensheathment. The diversity of ultrastructural and electrophysiological changes observed during epileptogeneiss suggests that potential drug targets for this period should be broadened to include all components of the tripartite synapse.

The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells.

The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

Impaired auditory processing and altered structure of the endbulb of Held synapse in mice lacking the GluA3 subunit of AMPA receptors.

AMPA glutamate receptor complexes with fast kinetics conferred by subunits like GluA3 and GluA4 are essential for temporal precision of synaptic transmission. The specific role of GluA3 in auditory processing and experience related changes in the auditory brainstem remain unknown. We investigated the role of the GluA3 in auditory processing by using wild type (WT) and GluA3 knockout (GluA3-KO) mice. We recorded auditory brainstem responses (ABR) to assess auditory function and used electron microscopy to evaluate the ultrastructure of the auditory nerve synapse on bushy cells (AN-BC synapse). Since labeling for GluA3 subunit increases on auditory nerve synapses within the cochlear nucleus in response to transient sound reduction, we investigated the role of GluA3 in experience-dependent changes in auditory processing. We induced transient sound reduction by plugging one ear and evaluated ABR threshold and peak amplitude recovery for up to 60 days after ear plug removal in WT and GluA3-KO mice. We found that the deletion of GluA3 leads to impaired auditory signaling that is reflected in decreased ABR peak amplitudes, an increased latency of peak 2, early onset hearing loss and reduced numbers and sizes of postsynaptic densities (PSDs) of AN-BC synapses. Additionally, the lack of GluA3 hampers ABR threshold recovery after transient ear plugging. We conclude that GluA3 is required for normal auditory signaling, normal ultrastructure of AN-BC synapses in the cochlear nucleus and normal experience-dependent changes in auditory processing after transient sound reduction.

Conductive Hearing Loss Has Long-Lasting Structural and Molecular Effects on Presynaptic and Postsynaptic Structures of Auditory Nerve Synapses in the Cochlear Nucleus.

UNLABELLED: Sound deprivation by conductive hearing loss increases hearing thresholds, but little is known about the response of the auditory brainstem during and after conductive hearing loss. Here, we show in young adult rats that 10 d of monaural conductive hearing loss (i.e., earplugging) leads to hearing deficits that persist after sound levels are restored. Hearing thresholds in response to clicks and frequencies higher than 8 kHz remain increased after a 10 d recovery period. Neural output from the cochlear nucleus measured at 10 dB above threshold is reduced and followed by an overcompensation at the level of the lateral lemniscus. We assessed whether structural and molecular substrates at auditory nerve (endbulb of Held) synapses in the cochlear nucleus could explain these long-lasting changes in hearing processing. During earplugging, vGluT1 expression in the presynaptic terminal decreased and synaptic vesicles were smaller. Together, there was an increase in postsynaptic density (PSD) thickness and an upregulation of GluA3 AMPA receptor subunits on bushy cells. After earplug removal and a 10 d recovery period, the density of synaptic vesicles increased, vesicles were also larger, and the PSD of endbulb synapses was larger and thicker. The upregulation of the GluA3 AMPAR subunit observed during earplugging was maintained after the recovery period. This suggests that GluA3 plays a role in plasticity in the cochlear nucleus. Our study demonstrates that sound deprivation has long-lasting alterations on structural and molecular presynaptic and postsynaptic components at the level of the first auditory nerve synapse in the auditory brainstem. SIGNIFICANCE STATEMENT: Despite being the second most prevalent form of hearing loss, conductive hearing loss and its effects on central synapses have received relatively little attention. Here, we show that 10 d of monaural conductive hearing loss leads to an increase in hearing thresholds, to an increased central gain upstream of the cochlear nucleus at the level of the lateral lemniscus, and to long-lasting presynaptic and postsynaptic structural and molecular effects at the endbulb of the Held synapse. Knowledge of the structural and molecular changes associated with decreased sensory experience, along with their potential reversibility, is important for the treatment of hearing deficits, such as hyperacusis and chronic otitis media with effusion, which is prevalent in young children with language acquisition or educational disabilities.

ERK1/2 Activation in Preexisting Oligodendrocytes of Adult Mice Drives New Myelin Synthesis and Enhanced CNS Function.

UNLABELLED: Growing evidence shows that mechanisms controlling CNS plasticity extend beyond the synapse and that alterations in myelin can modify conduction velocity, leading to changes in neural circuitry. Although it is widely accepted that newly generated oligodendrocytes (OLs) produce myelin in the adult CNS, the contribution of preexisting OLs to functional myelin remodeling is not known. Here, we show that sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in preexisting OLs of adult mice is sufficient to drive increased myelin thickness, faster conduction speeds, and enhanced hippocampal-dependent emotional learning. Although preexisting OLs do not normally contribute to remyelination, we show that sustained activation of ERK1/2 renders them able to do so. These data suggest that strategies designed to push mature OLs to reinitiate myelination may be beneficial both for enhancing remyelination in demyelinating diseases and for increasing neural plasticity in the adult CNS. SIGNIFICANCE STATEMENT: Myelin is a crucial regulator of CNS plasticity, function, and repair. Although it is generally accepted that new myelin production in the adult CNS is initiated by newly generated oligodendrocytes (OLs), great interest remains in additionally driving mature preexisting OLs to make myelin. The ability to induce myelination by the larger population of preexisting OLs carries the potential for enhanced remyelination in demyelinating diseases and increased neural plasticity in the adult CNS. Here, we show that sustained activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway is sufficient to drive mature OLs in the adult mouse CNS to reinitiate myelination, leading to new myelin wraps and functional changes.

Bhlhb5::flpo allele uncovers a requirement for Bhlhb5 for the development of the dorsal cochlear nucleus.

Auditory information is initially processed in the cochlear nuclei before being relayed to the brain. The cochlear nuclei are subdivided into dorsal, anterior ventral, and posterior ventral domains, each containing several subtypes of neurons that are thought to play discrete roles in the processing of sound. However, the ontogeny of these neurons is poorly understood, and this gap in knowledge hampers efforts to understand the basic neural circuitry of this nucleus. Here, we reveal that Bhlhb5 is expressed in both excitatory (unipolar brush cells) and inhibitory neurons (cartwheel cells) of the DCN during development. To gain genetic access to Bhlhb5-expressing neurons in the DCN, we generated a Bhlhb5::flpo knockin allele. Using an intersectional genetic strategy, we labeled cartwheel cells, thereby providing proof of concept that subpopulations of Bhlhb5-expressing neurons can be genetically targeted. Moreover, fate-mapping experiments using this allele revealed that Bhlhb5 is required for the proper development of the DCN, since mice lacking Bhlhb5 showed a dramatically diminished number of neurons, including unipolar brush and cartwheel cells. Intriguingly, the Bhlhb5::flpo allele also genetically labels numerous other regions of the nervous system that process sensory input, including the dorsal horn, the retina, and the nucleus of the lateral olfactory tract, hinting at a more general role for Bhlhb5 in the development of neurons that mediate sensory integration.

Excitation by Axon Terminal GABA Spillover in a Sound Localization Circuit.

Synapses from neurons of the medial nucleus of the trapezoid body (MNTB) onto neurons of the lateral superior olive (LSO) in the auditory brainstem are glycinergic in maturity, but also GABAergic and glutamatergic in development. The role for this neurotransmitter cotransmission is poorly understood. Here we use electrophysiological recordings in brainstem slices from P3-P21 mice to demonstrate that GABA release evoked from MNTB axons can spill over to neighboring MNTB axons and cause excitation by activating GABAAR. This spillover excitation generates patterns of staggered neurotransmitter release from different MNTB axons resulting in characteristic "doublet" postsynaptic currents in LSO neurons. Postembedding immunogold labeling and electron microscopy provide evidence that GABAARs are localized at MNTB axon terminals. Photolytic uncaging of p-hydroxyphenacyl (pHP) GABA demonstrates backpropagation of GABAAR-mediated depolarizations from MNTB axon terminals to the soma, some hundreds of microns away. These somatic depolarizations enhanced somatic excitability by increasing the probability of action potential generation. GABA spillover excitation between MNTB axon terminals may entrain neighboring MNTB neurons, which may play a role in the developmental refinement of the MNTB-LSO pathway. Axonal spillover excitation persisted beyond the second postnatal week, suggesting that this mechanism may play a role in sound localization, by providing new avenues of communication between MNTB neurons via their distal axonal projections. Significance statement: In this study, a new mechanism of neuronal communication between auditory synapses in the mammalian sound localization pathway is described. Evidence is provided that the inhibitory neurotransmitter GABA can spill over between axon terminals to cause excitation of nearby synapses to further stimulate neurotransmitter release. Excitatory GABA spillover between inhibitory axon terminals may have important implications for the development and refinement of this auditory circuit and may play a role in the ability to precisely localize sound sources.

Loss of VGLUT3 Produces Circadian-Dependent Hyperdopaminergia and Ameliorates Motor Dysfunction and l-Dopa-Mediated Dyskinesias in a Model of Parkinson's Disease.

The striatum is essential for many aspects of mammalian behavior, including motivation and movement, and is dysfunctional in motor disorders such as Parkinson's disease. The vesicular glutamate transporter 3 (VGLUT3) is expressed by striatal cholinergic interneurons (CINs) and is thus well positioned to regulate dopamine (DA) signaling and locomotor activity, a canonical measure of basal ganglia output. We now report that VGLUT3 knock-out (KO) mice show circadian-dependent hyperlocomotor activity that is restricted to the waking cycle and is due to an increase in striatal DA synthesis, packaging, and release. Using a conditional VGLUT3 KO mouse, we show that deletion of the transporter from CINs, surprisingly, does not alter evoked DA release in the dorsal striatum or baseline locomotor activity. The mice do, however, display changes in rearing behavior and sensorimotor gating. Elevation of DA release in the global KO raised the possibility that motor deficits in a Parkinson's disease model would be reduced. Remarkably, after a partial 6-hydroxydopamine (6-OHDA)-mediated DA depletion (∼70% in dorsal striatum), KO mice, in contrast to WT mice, showed normal motor behavior across the entire circadian cycle. l-3,4-dihydroxyphenylalanine-mediated dyskinesias were also significantly attenuated. These findings thus point to new mechanisms to regulate basal ganglia function and potentially treat Parkinson's disease and related disorders. SIGNIFICANCE STATEMENT: Dopaminergic signaling is critical for both motor and cognitive functions in the mammalian nervous system. Impairments, such as those found in Parkinson's disease patients, can lead to severe motor deficits. Vesicular glutamate transporter 3 (VGLUT3) loads glutamate into secretory vesicles for neurotransmission and is expressed by discrete neuron populations throughout the nervous system. Here, we report that the absence of VGLUT3 in mice leads to an upregulation of the midbrain dopamine system. Remarkably, in a Parkinson's disease model, the mice show normal motor behavior. They also show fewer abnormal motor behaviors (dyskinesias) in response to l-3,4-dihydroxyphenylalanine, the principal treatment for Parkinson's disease. The work thus suggests new avenues for the development of novel treatment strategies for Parkinson's disease and potentially other basal-ganglia-related disorders.

KV 10.1 opposes activity-dependent increase in Ca²âº influx into the presynaptic terminal of the parallel fibre-Purkinje cell synapse.

KEY POINTS: Voltage-gated KV 10.1 potassium channels are widely expressed in the mammalian brain but their function remains poorly understood. We report that KV 10.1 is enriched in the presynaptic terminals and does not take part in somatic action potentials. In parallel fibre synapses in the cerebellar cortex, we find that KV 10.1 regulates Ca(2+) influx and neurotransmitter release during repetitive high-frequency activity. Our results describe the physiological role of mammalian KV 10.1 for the first time and help understand the fine-tuning of synaptic transmission. The voltage-gated potassium channel KV 10.1 (Eag1) is widely expressed in the mammalian brain, but its physiological function is not yet understood. Previous studies revealed highest expression levels in hippocampus and cerebellum and suggested a synaptic localization of the channel. The distinct activation kinetics of KV 10.1 indicate a role during repetitive activity of the cell. Here, we confirm the synaptic localization of KV 10.1 both biochemically and functionally and that the channel is sufficiently fast at physiological temperature to take part in repolarization of the action potential (AP). We studied the role of the channel in cerebellar physiology using patch clamp and two-photon Ca(2+) imaging in KV 10.1-deficient and wild-type mice. The excitability and action potential waveform recorded at granule cell somata was unchanged, while Ca(2+) influx into axonal boutons was enhanced in mutants in response to stimulation with three APs, but not after a single AP. Furthermore, mutants exhibited a frequency-dependent increase in facilitation at the parallel fibre-Purkinje cell synapse at high firing rates. We propose that KV 10.1 acts as a modulator of local AP shape specifically during high-frequency burst firing when other potassium channels suffer cumulative inactivation.

Origin and function of short-latency inputs to the neural substrates underlying the acoustic startle reflex.

The acoustic startle reflex (ASR) is a survival mechanism of alarm, which rapidly alerts the organism to a sudden loud auditory stimulus. In rats, the primary ASR circuit encompasses three serially connected structures: cochlear root neurons (CRNs), neurons in the caudal pontine reticular nucleus (PnC), and motoneurons in the medulla and spinal cord. It is well-established that both CRNs and PnC neurons receive short-latency auditory inputs to mediate the ASR. Here, we investigated the anatomical origin and functional role of these inputs using a multidisciplinary approach that combines morphological, electrophysiological and behavioral techniques. Anterograde tracer injections into the cochlea suggest that CRNs somata and dendrites receive inputs depending, respectively, on their basal or apical cochlear origin. Confocal colocalization experiments demonstrated that these cochlear inputs are immunopositive for the vesicular glutamate transporter 1 (VGLUT1). Using extracellular recordings in vivo followed by subsequent tracer injections, we investigated the response of PnC neurons after contra-, ipsi-, and bilateral acoustic stimulation and identified the source of their auditory afferents. Our results showed that the binaural firing rate of PnC neurons was higher than the monaural, exhibiting higher spike discharges with contralateral than ipsilateral acoustic stimulations. Our histological analysis confirmed the CRNs as the principal source of short-latency acoustic inputs, and indicated that other areas of the cochlear nucleus complex are not likely to innervate PnC. Behaviorally, we observed a strong reduction of ASR amplitude in monaural earplugged rats that corresponds with the binaural summation process shown in our electrophysiological findings. Our study contributes to understand better the role of neuronal mechanisms in auditory alerting behaviors and provides strong evidence that the CRNs-PnC pathway mediates fast neurotransmission and binaural summation of the ASR.