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Altered somatosensory reactivity is frequently observed among individuals with autism spectrum disorders (ASDs). Here, we report that while multiple mouse models of ASD exhibit aberrant somatosensory behaviors in adulthood, some models exhibit altered tactile reactivity as early as embryonic development, while in others, altered reactivity emerges later in life. Additionally, tactile over-reactivity during neonatal development is associated with anxiety-like behaviors and social interaction deficits in adulthood, whereas tactile over-reactivity that emerges later in life is not. The locus of circuit disruption dictates the timing of aberrant tactile behaviors: altered feedback or presynaptic inhibition of peripheral mechanosensory neurons leads to abnormal tactile reactivity during neonatal development, while disruptions in feedforward inhibition in the spinal cord lead to touch reactivity alterations that manifest later in life. Thus, the developmental timing of aberrant touch processing can predict the manifestation of ASD-associated behaviors in mouse models, and differential timing of sensory disturbance onset may contribute to phenotypic diversity across individuals with ASD.

Specialized mechanosensory end organs within mammalian skin—hair follicle-associated lanceolate complexes, Meissner corpuscles, and Pacinian corpuscles—enable our perception of light, dynamic touch1. In each of these end organs, fast-conducting mechanically sensitive neurons, called Aβ low-threshold mechanoreceptors (Aβ LTMRs), associate with resident glial cells, known as terminal Schwann cells (TSCs) or lamellar cells, to form complex axon ending structures. Lanceolate-forming and corpuscle-innervating Aβ LTMRs share a low threshold for mechanical activation, a rapidly adapting (RA) response to force indentation, and high sensitivity to dynamic stimuli1–6. How mechanical stimuli lead to activation of the requisite mechanotransduction channel Piezo27–15and Aβ RA-LTMR excitation across the morphologically dissimilar mechanosensory end organ structures is not understood. Here, we report the precise subcellular distribution of Piezo2 and high-resolution, isotropic 3D reconstructions of all three end organs formed by Aβ RA-LTMRs determined by large volume enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging. We found that within each end organ, Piezo2 is enriched along the sensory axon membrane and is minimally or not expressed in TSCs and lamellar cells. We also observed a large number of small cytoplasmic protrusions enriched along the Aβ RA-LTMR axon terminals associated with hair follicles, Meissner corpuscles, and Pacinian corpuscles. These axon protrusions reside within close proximity to axonal Piezo2, occasionally contain the channel, and often form adherens junctions with nearby non-neuronal cells. Our findings support a unified model for Aβ RA-LTMR activation in which axon protrusions anchor Aβ RA-LTMR axon terminals to specialized end organ cells, enabling mechanical stimuli to stretch the axon in hundreds to thousands of sites across an individual end organ and leading to activation of proximal Piezo2 channels and excitation of the neuron.

The somatosensory system decodes a range of tactile stimuli to generate a coherent sense of touch. Discriminative touch of the body depends on signals conveyed from peripheral mechanoreceptors to the brain through the spinal cord dorsal column and its brainstem target, the dorsal column nuclei (DCN)1,2. Models of somatosensation emphasize that fast-conducting low-threshold mechanoreceptors (LTMRs) innervating the skin drive the DCN3,4. However, postsynaptic dorsal column (PSDC) neurons within the spinal cord dorsal horn also collect mechanoreceptor signals and form a second major input to the DCN5–7. The significance of PSDC neurons and their contributions to the coding of touch have remained unclear since their discovery. Here we show that direct LTMR input to the DCN conveys vibrotactile stimuli with high temporal precision. Conversely, PSDC neurons primarily encode touch onset and the intensity of sustained contact into the high-force range. LTMR and PSDC signals topographically realign in the DCN to preserve precise spatial detail. Different DCN neuron subtypes have specialized responses that are generated by distinct combinations of LTMR and PSDC inputs. Thus, LTMR and PSDC subdivisions of the dorsal column encode different tactile features and differentially converge in the DCN to generate specific ascending sensory processing streams.

The somatosensory system decodes a range of tactile stimuli to generate a coherent sense of touch. Discriminative touch of the body depends on signals conveyed from peripheral mechanoreceptors to the brain through the spinal cord dorsal column and its brainstem target, the dorsal column nuclei (DCN)

Summary: Synaptotagmin 7 (Syt7) is a high-affinity calcium sensor that is implicated in multiple aspects of synaptic transmission. Here, we study the influence of Syt7 on the climbing fiber (CF) to Purkinje cell (PC) synapse. We find that small facilitation and prominent calcium-dependent recovery from depression at this synapse do not rely on Syt7 and that Syt7 is not normally present in CFs. We expressed Syt7 in CFs to assess the consequences of introducing Syt7 to a synapse that normally lacks Syt7. Syt7 expression does not promote asynchronous release or accelerate recovery from depression. Syt7 decreases the excitatory postsynaptic current (EPSC) magnitude, consistent with a decrease in the initial probability of release (PR). Syt7 also increases synaptic facilitation to such a large extent that it could not arise solely as an indirect consequence of decreased PR. Thus, the primary consequence of Syt7 expression in CFs, which normally lack Syt7, is to promote synaptic facilitation.

The inferior olive (IO) is comprised of electrically-coupled neurons that make climbing fiber synapses onto Purkinje cells. Neurons in different IO subnuclei are inhibited by synapses with wide ranging release kinetics. Inhibition can be exclusively synchronous, asynchronous, or a mixture of both. Whether the same boutons, neurons or sources provide these kinetically distinct types of inhibition was not known. We find that the deep cerebellar nuclei (DCN) and vestibular nuclei (VN) are two major sources of inhibition to the IO that are specialized to provide inhibitory input with distinct kinetics. DCN to IO synapses lack fast synaptotagmin isoforms, release neurotransmitter asynchronously, and are exclusively GABAergic. VN to IO synapses contain fast synaptotagmin isoforms, release neurotransmitter synchronously, and are mediated by combined GABAergic and glycinergic transmission. These findings indicate that VN and DCN inhibitory inputs to the IO are suited to control different aspects of IO activity.

The calcium-sensing protein synaptotagmin 7 mediates facilitation that is masked by depression, but supports frequency-invariant transmission in mouse cerebellar and vestibular synapses. In most parts of the brain, synaptic strength varies as a function of neuronal firing frequency, a phenomenon known as 'short-term synaptic plasticity'. This fundamental nonlinearity in synaptic transmission has to be compensated for to achieve frequency-independent transmission in some auditory, vestibular and cerebellar circuits. Now Wade Regehr and colleagues show that synaptotagmin 7, a calcium-sensing protein, supports a short-term facilitation that increases with firing frequency and thus compensates for use-dependent depression at synapses in the cerebellum and in the vestibular system. The resulting constant synaptic strength over a wide range of firing frequencies allows for efficient encoding of neuronal firing rates. At most synapses in the brain, short-term plasticity dynamically modulates synaptic strength. Rapid frequency-dependent changes in synaptic strength have key roles in sensory adaptation, gain control and many other neural computations1,2. However, some auditory, vestibular and cerebellar synapses maintain constant strength over a wide range of firing frequencies3,4,5, and as a result efficiently encode firing rates. Despite its apparent simplicity, frequency-invariant transmission is difficult to achieve because of inherent synaptic nonlinearities6. Here we study frequency-invariant transmission at synapses from Purkinje cells to deep cerebellar nuclei and at vestibular synapses in mice. Prolonged activation of these synapses leads to initial depression, which is followed by steady-state responses that are frequency invariant for their physiological activity range. We find that synaptotagmin 7 (Syt7), a calcium sensor for short-term facilitation7, is present at both synapses. It was unclear why a sensor for facilitation would be present at these and other depressing synapses. We find that at Purkinje cell and vestibular synapses, Syt7 supports facilitation that is normally masked by depression, which can be revealed in wild-type mice but is absent in Syt7 knockout mice. In wild-type mice, facilitation increases with firing frequency and counteracts depression to produce frequency-invariant transmission. In Syt7-knockout mice, Purkinje cell and vestibular synapses exhibit conventional use-dependent depression, weakening to a greater extent as the firing frequency is increased. Presynaptic rescue of Syt7 expression restores both facilitation and frequency-invariant transmission. Our results identify a function for Syt7 at synapses that exhibit overall depression, and demonstrate that facilitation has an unexpected and important function in producing frequency-invariant transmission.

The output of the cerebellar cortex is conveyed to the deep cerebellar nuclei (DCN) by Purkinje cells (PCs). Here we characterize the properties of the PC-DCN synapse in juvenile and adult mice and find that prolonged high-frequency stimulation leads to steady-state responses that become increasingly frequency-independent within the physiological firing range of PCs in older animals, resulting in a linear relationship between charge transfer and activation frequency. We use a low affinity antagonist to show that GABAA receptor saturation occurs at this synapse, but it does not underlie frequency invariant transmission. We propose that PC-DCN synapses have two components of release: one prominent early in trains, and another specialized to maintain transmission during prolonged activation. Short-term facilitation offsets partial vesicle depletion to produce frequency-independent transmission.

In albino rats, it has been reported that lateral striate cortex (V1) is highly binocular, and that input from the ipsilateral eye to this region comes through the callosum. In contrast, in Long Evans rats, this region is nearly exclusively dominated by the contralateral eye even though it is richly innervated by the callosum (Laing, Turecek, Takahata, & Olavarria, 2015). We hypothesized that the inability of callosal connections to relay ipsilateral eye input to lateral V1 in Long Evans rats is a consequence of the existence of ocular dominance columns (ODCs), and of callosal patches in register with ipsilateral ODCs in the binocular region of V1 (Laing et al., 2015). We therefore predicted that in albino rats input from both eyes intermix in the binocular region, without segregating into ODCs, and that callosal connections are not patchy. Confirming our predictions, we found that inputs from both eyes, studied with the transneuronal tracer WGA-HRP, are intermixed in the binocular zone of albinos, without segregating into ODCs. Similarly, we found that callosal connections in albino rats are not patchy but instead are distributed homogeneously throughout the callosal region in V1. We propose that these changes allow the transcallosal passage of ipsilateral eye input to lateral striate cortex, increasing its binocularity. Thus, the binocular region in V1 of albino rats includes lateral striate cortex, being therefore about 25% larger in area than the binocular region in Long Evans rats. Our findings provide insight on the role of callosal connections in generating binocular cells.

Neurotransmitter release can be synchronous and occur within milliseconds of action potential invasion, or asynchronous and persist for tens of milliseconds. The molecular determinants of release kinetics remain poorly understood. It has been hypothesized that asynchronous release dominates when fast Synaptotagmin isoforms are far from calcium channels or when specialized sensors, such as Synaptotagmin 7, are abundant. Here we test these hypotheses for GABAergic projections onto neurons of the inferior olive, where release in different subnuclei ranges from synchronous to asynchronous. Surprisingly, neither of the leading hypotheses accounts for release kinetics. Instead, we find that rapid Synaptotagmin isoforms are abundant in subnuclei with synchronous release, but absent where release is asynchronous. Viral expression of Synaptotagmin 1 transforms asynchronous synapses into synchronous ones. Thus, the nervous system controls levels of fast Synaptotagmin isoforms to regulate release kinetics, and thereby controls the ability of synapses to encode spike rates or precise timing.

When an action potential invades a presynaptic terminal it evokes large, brief Ca2+signals that trigger vesicle fusion within milliseconds that is followed by a small residual Ca2+(Cares) signal. At many synapses Caresproduces synaptic facilitation that lasts up to hundreds of milliseconds and, although less common, Carescan also evoke asynchronous release (AR) that persists for tens of milliseconds. The properties of facilitation and AR are very different, which suggests that they are mediated by distinct mechanisms. However, recently it has been shown that the slow calcium sensor synaptotagmin 7 (Syt7) mediates facilitation at many synapses where AR does not occur, and conversely Syt7 can mediate AR without mediating facilitation. Here we study cerebellar granule cell synapses onto stellate cells and Purkinje cells in mice of both sexes to assess the role of Syt7 in these phenomena at the same synapse. This is of particular interest at granule cell synapses where AR is much more calcium dependent and shorter-lived than facilitation. We find that Syt7 can mediate these two processes despite their divergent properties. In Syt7 knock-out animals, facilitation and AR are smaller and shorter lived than in wild-type animals, even though the initial probability of release and Caressignals are unchanged. Although there are short-lived Syt7-independent mechanisms that mediate facilitation and AR in Syt7 KO animals, we find that at granule cell synapses AR and facilitation are both mediated primarily by Syt7.SIGNIFICANCE STATEMENTAt synapses made by cerebellar granule cells, presynaptic activity elevates calcium for tens of milliseconds, which in turn evokes both asynchronous release (AR) and synaptic facilitation. AR is more calcium sensitive and shorter-lived than facilitation at these synapses, suggesting that they are mediated by different mechanisms. However, we find that the slow calcium sensor synaptotagmin 7 mediates both of these phenomena. Small, rapidly decaying components of AR and facilitation are present in Syt7 KO animals, indicating that additional mechanisms can contribute to both AR and facilitation at these synapses.

Neurons of the inferior olive (IO) are electrically coupled and show subthreshold oscillations (STOs) in membrane potential that enhance spike synchrony. Spike synchrony among IO neurons is relevant for the fine temporal control of movement and the activation of muscles as collectives, or as “muscle synergies,” during complex movements. Multiple lines of evidence indicate that STOs are an emergent property of the IO network enabled by weak, but prevalent, electrical coupling. We have demonstrated N-methyl- d -aspartate (NMDA) receptor-mediated strengthening of electrical coupling between IO neurons that synchronizes STOs and increases their amplitude. We argue that: (1) weak electrical coupling in the IO makes available a wide dynamic range upon which a strengthening process can modulate STO amplitude and synchrony to have a strong functional impact; and (2) the upward modulation of STO amplitude and synchrony by NMDA receptor-mediated strengthening of electrical synapses may underlie the ability to acquire, through motor learning, the highest degree of temporal precision of synergistic muscle activity during skilled movement.

Ocular dominance columns (ODCs) exist in many primates and carnivores, but it is believed that they do not exist in rodents. Using a combination of transneuronal tracing, in situ hybridization for Zif268 and electrophysiological recordings, we show that inputs from both eyes are largely segregated in the binocular region of V1 in Long Evans rats. We also show that, interposed between this binocular region and the lateral border of V1, there lies a strip of cortex that is strongly dominated by the contralateral eye. Finally, we show that callosal connections colocalize primarily with ipsilateral eye domains in the binocular region and with contralateral eye input in the lateral cortical strip, mirroring the relationship between patchy callosal connections and specific sets of ODCs described previously in the cat. Our results suggest that development of cortical modular architecture is more conserved among rodents, carnivores, and primates than previously thought.

RNA interference (RNAi) to knockdown N-methyl-D-aspartate receptor (NMDAR) function is being investigated to address disorders associated with pathological brain rhythms. A motivating finding has been that pharmacological block of NMDARs inhibited oscillations in neuronal membrane potential that entrain rhythmic bursts of action potentials. To determine whether transient effects of NMDAR antagonist drugs to inhibit neuronal rhythmicity can be stably induced with genetic specificity, we examined the effects of RNAi of GluN1 protein on the subthreshold oscillations (STOs) of neurons in the inferior olive (IO), a pacemaking nucleus necessary for motor and cognitive timing. Western blot of dissociated neurons demonstrated 90% knockdown of GluN1 after a strong in vivo transduction by a dual-microRNA lentiviral vector. GluN1 RNAi in whole-cell-patched IO neurons blocked both membrane depolarization and STOs typically induced by NMDAR activation for up to 54 days without affecting input resistance, membrane capacitance, action potential firing, high-threshold Ca(2+) spikes, the hyperpolarization-activated current Ih, or the activation of the low-threshold Ca(2+) current I(T). Although an off-target effect on Cav3 expression was ruled out also by BlastN query, we found that GluN1 RNAi chronically eliminated I(T)-dependent STOs at resting membrane potential, well below the activation threshold of the NMDAR channel. In the context of a recent report showing that NMDAR activation induces STOs as it strengthens electrical coupling, the long-term block of STOs by GluN1 RNAi may relate to the loss of an essential support mechanism. Lentivector-mediated RNAi of GluN1 provides a novel technique for future investigations of NMDAR involvement in electrical oscillations and behavior.

SUMMARY Activation of CaV2.1 voltage-gated calcium channels is facilitated by preceding calcium entry. Such self-modulatory facilitation is thought to contribute to synaptic facilitation. Using knockin mice with mutated CaV2.1 channels that do not facilitate (Ca IM-AA mice), we surprisingly found that, under conditions of physiological calcium and near-physiological temperatures, synaptic facilitation at hippocampal CA3 to CA1 synapses was not attenuated in Ca IM-AA mice and facilitation was paradoxically more prominent at two cerebellar synapses. Enhanced facilitation at these synapses is consistent with a decrease in initial calcium entry, suggested by an action-potential-evoked CaV2.1 current reduction in Purkinje cells from Ca IM-AA mice. In wild-type mice, CaV2.1 facilitation during high-frequency action potential trains was very small. Thus, for the synapses studied, facilitation of calcium entry through CaV2.1 channels makes surprisingly little contribution to synaptic facilitation under physiological conditions. Instead, CaV2.1 facilitation offsets CaV2.1 inactivation to produce remarkably stable calcium influx during high-frequency activation., Graphical Abstract, In Brief Weyrer et al. use Ca IM-AA mice in which CaV2.1 calcium channel facilitation is eliminated to study synaptic facilitation at hippocampal and cerebellar synapses. Under conditions of physiological temperature, external calcium, and presynaptic waveforms, facilitation of CaV2.1 channels is small and does not contribute to synaptic facilitation at these synapses.

Inferior olive (IO) neurons are critical for motor coordination and exhibit oscillations in membrane potential that are subthreshold for spiking. The prevalence, coherence, and continuity of those subthreshold oscillations (STOs) depend upon resonant interactions between neighboring neurons supported by electrical coupling. Many studies of the olivocerebellar system in rodents, in which STOs were related to tremor, whisking, and licking, fueled a debate over whether IO STOs were relevant for primates whose repertoire of movement is generally less periodic. The debate was never well informed due to the lack of a direct examination of the physiological properties of primate IO neurons. Here, we obtained dual patch-clamp recordings of neighboring IO neurons from young adult macaques in brainstem slices and compared them to identical recordings from rats. Macaque IO neurons exhibited an equivalent prevalence of continuous STOs as rats (45 vs 54%, respectively). However, macaque STOs were slower (1–4 Hz) and did not overlap with the dominant 4–9 Hz frequency of rats. The slower STO frequency of macaques was at least partially due to a prolonged membrane time constant and increased membrane capacitance that could be attributed to stronger electrical coupling and greater total dendritic length. The presence of synchronized STOs in the IO of adult macaques, coincident with strong and prevalent electrical coupling, answers a fundamental outstanding question in cerebellar neuroscience and is consistent with a prominent role for synchronized oscillation in primate sensory-motor control. SIGNIFICANCE STATEMENT It was debated whether inferior olive (IO) neurons of primates behave as synchronized oscillators as was found for rodents using intracellular, optical, and multielectrode recordings. An inability to resolve this issue using single-Purkinje cell extracellular recordings in monkeys limited our understanding of timing mechanisms in the primate brain. Using dual whole-cell recordings from the IO of young adult rhesus macaques in acutely prepared brainstem slices, our work demonstrates that pairs of primate IO neurons show synchronized oscillations in membrane potential. The findings have strong mechanistic and translational relevance, as IO activation has been implicated in humans9 perceptual timing of sensory events and motricity.

It has been known for more than 70 years that synaptic strength is dynamically regulated in a use-dependent manner. At synapses with a low initial release probability, closely spaced presynaptic action potentials can result in facilitation, a short-term form of enhancement in which each subsequent action potential evokes greater neurotransmitter release. Facilitation can enhance neurotransmitter release considerably and can profoundly influence information transfer across synapses, but the underlying mechanism remains a mystery. One proposed mechanism is that a specialized calcium sensor for facilitation transiently increases the probability of release, and this sensor is distinct from the fast sensors that mediate rapid neurotransmitter release. Yet such a sensor has never been identified, and its very existence has been disputed. Here we show that synaptotagmin 7 (Syt7) is a calcium sensor that is required for facilitation at several central synapses. In Syt7-knockout mice, facilitation is eliminated even though the initial probability of release and the presynaptic residual calcium signals are unaltered. Expression of wild-type Syt7 in presynaptic neurons restored facilitation, whereas expression of a mutated Syt7 with a calcium-insensitive C2A domain did not. By revealing the role of Syt7 in synaptic facilitation, these results resolve a longstanding debate about a widespread form of short-term plasticity, and will enable future studies that may lead to a deeper understanding of the functional importance of facilitation.

We employed a transgenic mouse having conditional expression of ChR2(H134R) in neurons of the inferior olive to facilitate understanding of the role of electrical coupling and oscillation in central nervous system function. Two-photon excitation of ChR2-expressing neurons using 64 laser beams restricted to single inferior olive cell bodies depolarized neurons and evoked voltage deflections in neighboring neurons demonstrating electrical coupling. Broader illumination of neuronal ensembles using blue light induced an optical clamp of endogenous electrical rhythms in the inferior olive of acutely-prepared brain slices, which when applied in vivo directly modulated the local field potential activity and induced tremor. The experiments demonstrate novel methods to optically manipulate electrically coupled potentials and rhythmogenesis within a neuronal ensemble. From a functional perspective, the experiments shed light on the cellular and circuitry mechanisms of essential tremor, a prevalent neurological condition, by indicating time- and frequencydependence of tremor upon varying rhythms of inferior olive stimulation. The experiments indicate analog control of a brain rhythm that may be used to enhance our understanding of the functional consequences of central rhythmogenesis.

Summary Electrical synapses are formed by gap junctions and permit electrical coupling, which shapes the synchrony of neuronal ensembles. Here, we provide a direct demonstration of receptor-mediated strengthening of electrical coupling in mammalian brain. Electrical coupling in the inferior olive of rats was strengthened by activation of NMDA-type glutamate receptors (NMDARs), which were found at synaptic loci and at extrasynaptic loci 20–100 nm proximal to gap junctions. Electrical coupling was strengthened by pharmacological and synaptic activation of NMDARs, whereas costimulation of ionotropic non-NMDAR glutamate receptors transiently antagonized the effect of NMDAR activation. NMDAR-dependent strengthening (1) occurred despite increased input conductance, (2) induced Ca 2+ -influx microdomains near dendritic spines, (3) required activation of the Ca 2+ /calmodulin-dependent protein-kinase II, (4) was restricted to neurons that were weakly coupled, and (5) thus strengthened coupling, mainly between nonadjacent neurons. This provided a mechanism to expand the synchronization of rhythmic membrane potential oscillations by chemical neurotransmitter input.