Bidirectional homeostatic plasticity induced by interneuron cell death and transplantation in vivo

Chronic changes in excitability and activity can induce homeostatic plasticity. These perturbations may be associated with neurological disorders, particularly those involving loss or dysfunction of GABA interneurons. In distal-less homeobox 1 (Dlx1^−/−) mice with late-onset interneuron loss and reduced inhibition, we observed both excitatory synaptic silencing and decreased intrinsic neuronal excitability. These homeostatic changes do not fully restore normal circuit function, because synaptic silencing results in enhanced potential for long-term potentiation and abnormal gamma oscillations. Transplanting medial ganglionic eminence interneuron progenitors to introduce new GABAergic interneurons, we demonstrate restoration of hippocampal function. Specifically, miniature excitatory postsynaptic currents, input resistance, hippocampal long-term potentiation, and gamma oscillations are all normalized. Thus, in vivo homeostatic plasticity is a highly dynamic and bidirectional process that responds to changes in inhibition.Prolonged changes in activity levels induce bidirectional changes in neuronal excitability and synaptic activity known as homeostatic plasticity (1, 2). This phenomenon has been described well at excitatory synapses and functions to maintain activity within a preferred dynamic range. Maintaining excitatory/inhibitory synaptic balance is critical for neuronal information processing and a potential problem when confronted with aberrant states of excitability, such as those associated with autism, schizophrenia, Alzheimer’s disease, or epilepsy (3–12).Chronic manipulation of synaptic input and/or action potential (AP) output rates in cortical and hippocampal cell cultures induces homeostatic synaptic scaling, in which the amplitude and then the frequency of pyramidal neuron miniature excitatory postsynaptic currents (mEPSCs) increase when activity is lowered or decrease when activity is raised (13–16). Recent studies have begun to reveal the underlying molecular mechanisms of homeostatic synaptic changes, including the AMPA receptor subunits, synapse-associated calcium-binding proteins, and intracellular signaling cascades involved (14, 17, 18). Changes to activity also trigger homeostatic plasticity of inhibitory synaptic transmission (19–23). Homozygous deletion of glutamate decarboxylase 1 (Gad1), the rate-limiting enzyme in the synthesis of GABA, reduced miniature inhibitory postsynaptic current (mIPSC) amplitudes in cultured hippocampal neurons but also blocked further homeostatic changes to mIPSCs. This suggests a key role for regulation of Gad1 expression in inhibitory homeostatic plasticity (23). Intrinsic excitability is also homeostatically regulated by activity. Changes in input resistance (Rin) and voltage-activated K⁺ and Na⁺ channel number (24–27), and in Na⁺ channel compartmentalization (28, 29), have been described following manipulations that chronically alter neuronal activity. Finally, in vivo manipulation of neuronal activity with TTX results in larger mEPSC amplitudes and reduced Rin of CA1 pyramidal neurons (30), suggesting that multiple mechanisms of homeostatic plasticity can occur simultaneously in the intact nervous system.Loss of GABAergic interneurons is common across different neurological disorders. It is unknown whether homeostatic plasticity can be induced by changes in activity related to interneuronopathy or how the combination of interneuron cell death and compensation alters circuit function. To begin to address these issues, we studied synaptic and intrinsic excitability in a hippocampal circuit in which a subpopulation of interneurons is reduced [i.e., distal-less homeobox 1 (Dlx1^−/−) mice] (31–33). At around 30 d of age, these mice lose a subset of somatostatin (Sst)-, calretinin (CR)-, vasoactive intestinal peptide-, and neuropeptide Y (NPY)-positive interneurons; exhibit decreased inhibitory synaptic activity in some brain regions; and subsequently develop epilepsy (31). Our results show that secondary to the in vivo interneuron loss is a homeostatic reduction in mEPSC frequency, decreased AMPA/NMDA ratio, and decreased intrinsic excitability in CA1 pyramidal neurons (that do not express Dlx1). Transplantation of GABA progenitor cells from the medial ganglionic eminence (MGE) (34) causes a reversal of the homeostatic changes in excitatory synaptic activity and Rin. Additionally, we describe unique changes in Dlx1^−/− circuit function that homeostatic compensation does not correct: enhanced long-term potentiation (LTP) and altered gamma frequency oscillations (GFOs). The severity of these phenotypes is reduced by interneuron transplantation. These studies demonstrate the responsiveness of excitatory circuitry to changes in inhibition, using homeostatic plasticity as a mechanism for maintaining excitatory/inhibitory balance.     

Fine-tuning synaptic plasticity by modulation of CaV2.1 channels with Ca2+ sensor proteins

Modulation of P/Q-type Ca²⁺ currents through presynaptic voltage-gated calcium channels (Ca_V2.1) by binding of Ca²⁺/calmodulin contributes to short-term synaptic plasticity. Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca²⁺ sensor proteins that differentially modulate Ca_V2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic Ca_V2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca²⁺-dependent facilitation of P/Q-type Ca²⁺ current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca²⁺-dependent inactivation of P/Q-type Ca²⁺ current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic Ca_V2.1 channels by differentially expressed Ca²⁺ sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity.Neurons fire repetitively in different frequencies and patterns, and activity-dependent alterations in synaptic strength result in diverse forms of short-term synaptic plasticity that are crucial for information processing in the nervous system (1–3). Short-term synaptic plasticity on the time scale of milliseconds to seconds leads to facilitation or depression of synaptic transmission through changes in neurotransmitter release. This form of plasticity is thought to result from residual Ca²⁺ that builds up in synapses during repetitive action potentials and binds to a Ca²⁺ sensor distinct from the one that evokes neurotransmitter release (1, 2, 4, 5). However, it remains unclear how changes in residual Ca²⁺ cause short-term synaptic plasticity and how neurotransmitter release is regulated to generate distinct patterns of short-term plasticity.In central neurons, voltage-gated calcium (Ca_V2.1) channels are localized in high density in presynaptic active zones where their P/Q-type Ca²⁺ current triggers neurotransmitter release (6–11). Because synaptic transmission is proportional to the third or fourth power of Ca²⁺ entry through presynaptic Ca_V2.1 channels, small changes in Ca²⁺ current have profound effects on synaptic transmission (2, 12). Studies at the calyx of Held synapse have provided important insights into the contribution of presynaptic Ca²⁺ current to short-term synaptic plasticity (13–17). Ca_V2.1 channels are required for synaptic facilitation, and Ca²⁺-dependent facilitation and inactivation of the P/Q-type Ca²⁺ currents are correlated temporally with synaptic facilitation and rapid synaptic depression (13–17).Molecular interactions between Ca²⁺/calmodulin (CaM) and Ca_V2.1 channels induce sequential Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents in nonneuronal cells (18–21). Facilitation and inactivation of P/Q-type currents are dependent on Ca²⁺/CaM binding to the IQ-like motif (IM) and CaM-binding domain (CBD) of the Ca_V2.1 channel, respectively (20, 21). This bidirectional regulation serves to enhance channel activity in response to short bursts of depolarizations and then to decrease activity in response to long bursts. In synapses of superior cervical ganglion (SCG) neurons expressing exogenous Ca_V2.1 channels, synaptic facilitation is induced by repetitive action potentials, and mutation of the IM and CBD motifs prevents synaptic facilitation and inhibits the rapid phase of synaptic depression (22). Thus, in this model synapse, regulation of presynaptic Ca_V2.1 channels by binding of Ca²⁺/CaM can contribute substantially to the induction of short-term synaptic plasticity by residual Ca²⁺.CaM is expressed ubiquitously, but short-term plasticity has great diversity among synapses, and the potential sources of this diversity are unknown. How could activity-dependent regulation of presynaptic Ca_V2.1 channels contribute to the diversity of short-term synaptic plasticity? CaM is the founding member of a large family of Ca²⁺ sensor (CaS) proteins that are differentially expressed in central neurons (23–25). Two CaS proteins, Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2(VILIP-2), modulate facilitation and inactivation of Ca_V2.1 channels in opposite directions through interaction with the bipartite regulatory site in the C-terminal domain (26, 27), and they have varied expression in different types of central neurons (23, 25, 28). CaBP1 strongly enhances inactivation and prevents facilitation of Ca_V2.1 channel currents, whereas VILIP-2 slows inactivation and enhances facilitation of Ca_V2.1 currents during trains of stimuli (26, 27). Molecular analyses show that the N-terminal myristoylation site and the properties of individual EF-hand motifs in CaBP1 and VILIP-2 determine their differential regulation of Ca_V2.1 channels (27, 29–31). However, the role of CaBP1 and VILIP-2 in the diversity of short-term synaptic plasticity is unknown, and the high density of Ca²⁺ channels and unique Ca²⁺ dynamics at the presynaptic active zone make extrapolation of results from studies in nonneuronal cells uncertain. We addressed this important question directly by expressing CaBP1 and VILIP-2 in presynaptic SCG neurons and analyzing their effects on synaptic plasticity. Our results show that CaM-related CaS proteins can serve as sensitive bidirectional switches that fine-tune the input–output relationships of synapses depending on their profile of activity and thereby maintain the balance of facilitation versus depression by the regulation of presynaptic Ca_V2.1 channels.     

Rapid homeostasis by disinhibition during whisker map plasticity

How homeostatic processes contribute to map plasticity and stability in sensory cortex is not well-understood. Classically, sensory deprivation first drives rapid Hebbian weakening of spiking responses to deprived inputs, which is followed days later by a slow homeostatic increase in spiking responses mediated by excitatory synaptic scaling. Recently, more rapid homeostasis by inhibitory circuit plasticity has been discovered in visual cortex, but whether this process occurs in other brain areas is not known. We tested for rapid homeostasis in layer 2/3 (L2/3) of rodent somatosensory cortex, where D-row whisker deprivation drives Hebbian weakening of whisker-evoked spiking responses after an unexplained initial delay, but no homeostasis of deprived whisker responses is known. We hypothesized that the delay reflects rapid homeostasis through disinhibition, which masks the onset of Hebbian weakening of L2/3 excitatory input. We found that deprivation (3 d) transiently increased whisker-evoked spiking responses in L2/3 single units before classical Hebbian weakening (≥5 d), whereas whisker-evoked synaptic input was reduced during both periods. This finding suggests a transient homeostatic increase in L2/3 excitability. In whole-cell recordings from L2/3 neurons in vivo, brief deprivation decreased whisker-evoked inhibition more than excitation and increased the excitation–inhibition ratio. In contrast, synaptic scaling and increased intrinsic excitability were absent. Thus, disinhibition is a rapid homeostatic plasticity mechanism in rodent somatosensory cortex that transiently maintains whisker-evoked spiking in L2/3, despite the onset of Hebbian weakening of excitatory input.During deprivation-induced sensory map plasticity in cerebral cortex, changes in sensory input trigger both homeostatic plasticity mechanisms that maintain stable cortical firing rates and Hebbian mechanisms, in which inactive inputs lose (and active inputs gain) representation in sensory maps (1). Diverse mechanisms for homeostasis exist, including synaptic scaling (2–4), plasticity of intrinsic excitability (5, 6), and changes in sensory-evoked inhibition and excitation–inhibition (E-I) ratio (7–11). How homeostatic and Hebbian mechanisms interact to control map stability and plasticity remains unclear.One key unknown is the relative dynamics of homeostatic and Hebbian plasticity. Homeostasis mediated by synaptic scaling is slow, occurring over hours in vitro and days in vivo. This process is evident in visual cortex, where eyelid closure during the critical period classically drives Hebbian weakening of closed eye spiking responses (after 2 d of deprivation) followed several days later by a slower homeostatic increase in visual responses (12, 13) mediated by excitatory synaptic scaling (3, 14, 15). We investigated whether more rapid forms of homeostasis also exist that shape the earliest stages of cortical plasticity. Recent results in visual cortex show that eyelid closure rapidly weakens inhibitory circuits (within 1 d), and this process increases network excitability and, therefore, is an initial homeostatic response to deprivation (10, 16). This disinhibition correlates with rapid structural plasticity in inhibitory axons and dendrites (17) and is mediated by a reduction in excitatory drive to parvalbumin-positive interneurons (10). Whether rapid homeostasis by disinhibition or other mechanisms is a general feature of cortical plasticity outside the visual cortex is unknown. Theoretical work shows that rapid homeostasis by inhibitory and/or intrinsic plasticity can guide development of realistic sensory tuning and sparse sensory coding in cortical networks, suggesting broad relevance (18).We tested for rapid homeostasis during the onset of whisker map plasticity in the rodent primary somatosensory (S1) cortex, a major model of cortical plasticity. Each cortical column in the S1 whisker map corresponds to one facial whisker, termed its principal whisker (PW). Trimming or plucking a subset of whiskers in young adults weakens spiking responses to deprived PWs in layer 2/3 (L2/3) of deprived columns (19, 20). This process is mediated by Hebbian synaptic weakening at L4–L2/3 and L2/3–L2/3 excitatory synapses (21–23). No homeostatic restoration or strengthening of deprived whisker responses is known. However, PW response weakening is often preceded by an unexplained initial delay of ∼7 d, in which deprived whisker-evoked spiking responses remain stable (24, 25). We hypothesized that this initial delay reflects not a lack of plasticity but rapid homeostasis that (i) masks initial Hebbian weakening of L2/3 excitatory input and (ii) is mediated by loss of inhibition and/or increased intrinsic excitability in L2/3 neurons. Such rapid homeostasis would be a unique component of whisker map plasticity.     

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to short-term synaptic plasticity in hippocampal neurons

Short-term synaptic plasticity is induced by calcium (Ca²⁺) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated Ca_V2.1 Ca²⁺ channels by Ca²⁺ sensor proteins induces facilitation of Ca²⁺ currents and synaptic facilitation in cultured neurons expressing exogenous Ca_V2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca²⁺ sensor binding site. This mutation does not alter voltage dependence or kinetics of Ca_V2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by ∼50%. In the presence of EGTA-AM to prevent global increases in free Ca²⁺, the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca²⁺ is dependent upon regulation of Ca_V2.1 channels by Ca²⁺ sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10–20 Hz. Evidently, regulation of Ca_V2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.Modification of synaptic strength in central synapses is highly dependent upon presynaptic activity. The frequency and pattern of presynaptic action potentials regulates the postsynaptic response through diverse forms of short- and long-term plasticity that are specific to individual synapses and depend upon accumulation of intracellular Ca²⁺ (1–4). Presynaptic plasticity regulates neurotransmission by varying the amount of neurotransmitter released by each presynaptic action potential (1–5). P/Q-type Ca²⁺ currents conducted by voltage-gated Ca_V2.1 Ca²⁺ channels initiate neurotransmitter release at fast excitatory glutamatergic synapses in the brain (6–9) and regulate short-term presynaptic plasticity (3, 10). These channels exhibit Ca²⁺-dependent facilitation and inactivation that is mediated by the Ca²⁺ sensor (CaS) protein calmodulin (CaM) bound to a bipartite site in their C-terminal domain composed of an IQ-like motif (IM) and a CaM binding domain (CBD) (11–14). Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents correlate with facilitation and rapid depression of synaptic transmission at the Calyx of Held (15–18). Elimination of Ca_V2.1 channels by gene deletion prevents facilitation of synaptic transmission at the Calyx of Held (19, 20). Cultured sympathetic ganglion neurons with presynaptic expression of exogenous Ca_V2.1 channels harboring mutations in their CaS regulatory site have reduced facilitation and slowed depression of postsynaptic responses because of reduced Ca²⁺-dependent facilitation and Ca²⁺-dependent inactivation of Ca_V2.1 currents (21). The CaS proteins Ca²⁺-binding protein 1 (CaBP-1), visinin-like protein-2 (VILIP-2), and neuronal Ca²⁺ sensor-1 (NCS-1) induce different degrees of Ca²⁺-dependent facilitation and inactivation of channel activity (22–26). Expression of these different CaS proteins with Ca_V2.1 channels in cultured sympathetic ganglion neurons results in corresponding bidirectional changes in facilitation and depression of the postsynaptic response (25, 26). Therefore, binding of CaS proteins to Ca_V2.1 channels at specific synapses can change the balance of CaS-dependent facilitation and inactivation of Ca_V2.1 channels, and determine the outcome of synaptic plasticity (27). Currently, it is not known whether such molecular regulation of Ca_V2.1 by CaS proteins induces or modulates synaptic plasticity in native hippocampal synapses.To understand the functional role of regulation of Ca_V2.1 channels by CaS proteins in synaptic plasticity in vivo, we generated knock-in mice with paired alanine substitutions for the isoleucine and methionine residues in the IM motif (IM-AA) in their C-terminal domain. Here we investigated the effects of mutating this CaS regulatory site on hippocampal neurotransmission and synaptic plasticity. This mutation had no effect on basal Ca²⁺ channel function or on basal synaptic transmission. However, we found reduced short-term facilitation in response to paired stimuli in autaptic synapses in hippocampal cultures and in Schaffer collateral (SC)-CA1 synapses in acutely prepared hippocampal slices. Moreover, synaptic facilitation in mutant SC-CA1 synapses developed and decayed more slowly during trains of stimuli. These results identify a critical role for modulation of Ca_V2.1 channels by CaS proteins in short-term synaptic plasticity, which is likely to have important consequences for encoding and transmitting information in the hippocampus.     

GABAB receptor deficiency causes failure of neuronal homeostasis in hippocampal networks

Stabilization of neuronal activity by homeostatic control systems is fundamental for proper functioning of neural circuits. Failure in neuronal homeostasis has been hypothesized to underlie common pathophysiological mechanisms in a variety of brain disorders. However, the key molecules regulating homeostasis in central mammalian neural circuits remain obscure. Here, we show that selective inactivation of GABA_B, but not GABA_A, receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA_B receptor (GABA_BR) blockade or genetic deletion of the GB_1a receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA_BRs mediate adaptive presynaptic enhancement to neuronal inactivity by two principle mechanisms: First, neuronal silencing promotes syntaxin-1 switch from a closed to an open conformation to accelerate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and second, it boosts spike-evoked presynaptic calcium flux. In both cases, neuronal inactivity removes tonic block imposed by the presynaptic, GB_1a-containing receptors on syntaxin-1 opening and calcium entry to enhance probability of vesicle fusion. We identified the GB_1a intracellular domain essential for the presynaptic homeostatic response by tuning intermolecular interactions among the receptor, syntaxin-1, and the Ca_V2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB_1b-containing receptors. Thus, GABA_BRs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA_BR as a key regulator of population firing stability and propose that disruption of homeostatic synaptic plasticity may underlie seizure''s persistence in the absence of functional GABA_BRs.Neural circuits achieve an ongoing balance between plasticity and stability to enable adaptations to constantly changing environments while maintaining neuronal activity within a stable regime. Hebbian-like plasticity, reflected by persistent changes in synaptic and intrinsic properties, is crucial for refinement of neural circuits and information storage; however, alone it is unlikely to account for the stable functioning of neural networks (1). In the last 2 decades, major progress has been made toward understanding the homeostatic negative feedback systems underlying restoration of a baseline neuronal function after prolonged activity perturbations (2–4). Homeostatic processes may counteract the instability by adjusting intrinsic neuronal excitability, inhibition-to-excitation balance, and synaptic strength via postsynaptic or presynaptic modifications (5, 6) through a profound molecular reorganization of synaptic proteins (7, 8). These stabilizing mechanisms have been collectively termed homeostatic plasticity. Homeostatic mechanisms enable invariant firing rates and patterns of neural networks composed from intrinsically unstable activity patterns of individual neurons (9).However, nervous systems are not always capable of maintaining constant output. Although some mutations, genetic knockouts, or pharmacologic perturbations induce a compensatory response that restores network firing properties around a predefined “set point” (10), the others remain uncompensated, or their compensation leads to pathological function (11). The inability of neural networks to compensate for a perturbation may result in epilepsy and various types of psychiatric disorders (12). Therefore, determining under which conditions activity-dependent regulation fails to compensate for a perturbation and identifying the key regulatory molecules of neuronal homeostasis is critical for understanding the function and malfunction of central neural circuits.In this work, we explored the mechanisms underlying the failure in stabilizing hippocampal network activity by combining long-term extracellular spike recordings by multielectrode arrays (MEAs), intracellular patch-clamp recordings of synaptic responses, imaging of synaptic vesicle exocytosis, and calcium dynamics, together with FRET-based analysis of intermolecular interactions at individual synapses. Our results demonstrate that metabotropic, G protein-coupled receptors for GABA, GABA_BRs, are essential for firing rate homeostasis in hippocampal networks. We explored the mechanisms by which GABA_BRs gate homeostatic synaptic plasticity. Our study raises the possibility that persistence of epileptic seizures in GABA_BR-deficient mice (13–15) is directly linked to impairments in a homeostatic control system.     

NMDA receptor–BK channel coupling regulates synaptic plasticity in the barrel cortex

Postsynaptic N-methyl-D-aspartate receptors (NMDARs) are crucial mediators of synaptic plasticity due to their ability to act as coincidence detectors of presynaptic and postsynaptic neuronal activity. However, NMDARs exist within the molecular context of a variety of postsynaptic signaling proteins, which can fine-tune their function. Here, we describe a form of NMDAR suppression by large-conductance Ca²⁺- and voltage-gated K⁺ (BK) channels in the basal dendrites of a subset of barrel cortex layer 5 pyramidal neurons. We show that NMDAR activation increases intracellular Ca²⁺ in the vicinity of BK channels, thus activating K⁺ efflux and strong negative feedback inhibition. We further show that neurons exhibiting such NMDAR–BK coupling serve as high-pass filters for incoming synaptic inputs, precluding the induction of spike timing–dependent plasticity. Together, these data suggest that NMDAR-localized BK channels regulate synaptic integration and provide input-specific synaptic diversity to a thalamocortical circuit.

Glutamate is the primary excitatory chemical transmitter in the mammalian central nervous system (CNS), where it is essential for neuronal viability, network function, and behavioral responses (1). Glutamate activates a variety of pre- and postsynaptic receptors, including ionotropic receptors (iGluRs) that form ligand-gated cation-permeable ion channels. The iGluR superfamily includes α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), kainate receptors, and N-methyl-D-aspartate receptors (NMDARs), all of which form tetrameric assemblies that are expressed throughout the CNS (2).NMDARs exhibit high sensitivity to glutamate (apparent half maximal effective concentration in the micromolar range) and a voltage-dependent block by Mg²⁺ (3, 4), slow gating kinetics (5), and high permeability to Ca²⁺ (6, 7) (for a review, see ref. 8). Together, these characteristics confer postsynaptic NMDARs with the ability to detect and decode coincidental activity of pre- and postsynaptic neurons: presynaptic glutamate release brings about the occupation of the agonist-binding site and AMPAR-driven postsynaptic depolarization, removing the voltage-dependent Mg²⁺ block. The coincidence of these two events leads to NMDAR activation and a Ca²⁺ influx through the channel (8, 9), which initiates several forms of synaptic plasticity (10, 11).Large-conductance Ca²⁺- and voltage-gated K⁺ (BK) channels are opened by a combination of membrane depolarization and relatively high levels of intracellular Ca²⁺ (12, 13). In CNS neurons, such micromolar Ca²⁺ increases are usually restricted to the immediate vicinity of Ca²⁺ sources, including voltage-gated Ca²⁺ channels (VGCCs) (14–16) and ryanodine receptors (RyRs) (17, 18). In addition, Ca²⁺ influx through nonselective cation-permeable channels, including NMDARs, has also been shown to activate BK channels in granule cells from the olfactory bulb and dentate gyrus (19–21). In these neurons, Ca²⁺ entry through NMDARs opens BK channels in somatic and perisomatic regions, causing the repolarization of the surrounding plasma membrane and subsequent closure of NMDARs. Because BK channel activation blunts NMDAR-mediated excitatory responses, it provides a negative feedback mechanism that modulates the excitability of these neurons (19, 20). Thus, the same characteristics that make NMDARs key components in excitatory synaptic transmission and plasticity can paradoxically give rise to an inhibitory response when NMDARs are located in the proximity of BK channels. However, it is unclear whether functional NMDAR–BK coupling is relevant at dendrites and dendritic spines.The barrel field area in the primary somatosensory cortex, also known as the barrel cortex (BC), processes information from peripheral sensory receptors for onward transmission to cortical and subcortical brain regions (22, 23). Sensory information is received in the BC from different nuclei of the thalamus. Among these nuclei, the ventral posterior medial nucleus, ventrobasal nucleus, and posterior medial nucleus are known to directly innervate layer 5 pyramidal neurons (BC-L5PNs) (24–27). In basal dendrites of BC-L5PN, the coactivation of neighboring dendritic inputs can initiate NMDAR-mediated dendritically restricted spikes characterized by large Ca²⁺ transients and long-lasting depolarizations (28–30), providing the appropriate environment for BK activation.To determine whether functional NMDAR–BK coupling plays a role in synaptic transmission, and potentially synaptic plasticity, we investigated the thalamocortical synapses at basal dendrites of BC-L5PNs. We found that the suppression of NMDAR activity by BK channels occurs in the basal dendrites of about 40% of BC-L5PNs, where NMDAR activation triggers strong negative feedback inhibition by delivering Ca²⁺ to nearby BK channels. This inhibition regulates the amplitude of postsynaptic responses and increases the threshold for the induction of synaptic plasticity. Our findings thus unveil a calibration mechanism that can decode the amount and frequency of afferent synaptic inputs by selectively attenuating synaptic plasticity and providing input-specific synaptic diversity to a thalamocortical circuit.     

AMPA receptor inhibition by synaptically released zinc

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.The development, function, and experience-dependent plasticity of the mammalian brain depend on the refined neuronal interactions that occur in synapses. In the majority of excitatory synapses, the release of the neurotransmitter glutamate from presynaptic neurons opens transmembrane ion channels in postsynaptic neurons, the ionotropic glutamate receptors, thereby generating the flow of excitatory signaling in the brain. As a result, these receptors play a fundamental role in normal function and development of the brain, and they are also involved in many brain disorders (1).The ionotropic glutamate receptor family consists of AMPA, kainate, and NMDA receptors (NMDARs). Although kainate receptor-mediated excitatory postsynaptic responses occur in a few central synapses (2), AMPA receptors (AMPARs) and NMDARs are localized in the postsynaptic density of the vast majority of glutamatergic synapses in the brain, mediating most of excitatory neurotransmission (1). NMDAR function is regulated by a wide spectrum of endogenous allosteric neuromodulators that fine-tune synaptic responses (3–5); however, much less is known about endogenous AMPAR neuromodulators [(1, 5), but see refs. 6 and 7]. Recent structural studies revealed that the amino terminal domain (ATD) and ligand-binding domain (LBD) are tightly packed in NMDARs but not AMPARs (8–10). These structural differences explain some of the functional differences in allosteric modulation between AMPARs and NMDARs, such as why the ATD of NMDARs, unlike that of AMPARs, modulates function and contains numerous binding sites for allosteric regulators. Nonetheless, given the importance of fine-tuning both synaptic AMPAR and NMDAR responses for brain function, it is puzzling that there is not much evidence for endogenous, extracellular AMPAR modulation. The discovery and establishment of endogenous AMPAR modulators is crucial both for understanding ionotropic glutamate receptor signaling and for developing therapeutic agents for the treatment of AMPAR-related disorders, such as depression, cognitive dysfunctions associated with Alzheimer’s disease, and schizophrenia (1, 11).Free, or readily chelatable, zinc is an endogenous modulator of synaptic and extrasynaptic NMDARs (12–15). Free zinc is stored in glutamatergic vesicles in many excitatory synapses in the cerebral cortex, limbic, and brainstem nuclei (16). In some brain areas, such as in the hippocampus, 50% of boutons synapsing onto CA1 neurons and all mossy fibers synapsing onto CA3 neurons contain synaptic free zinc (17). Whereas earlier studies demonstrated that exogenous zinc inhibits AMPARs (18–21), more recent work suggests that endogenously released synaptic zinc does not modulate AMPARs in central synapses (14, 22). This conclusion was derived from the inability to efficiently chelate and quantify synaptic zinc with the zinc-selective chelators and probes used (15), in apparent support of the hypothesized low levels of released zinc during synaptic stimulation (14).Recent work in our laboratories used new chemical tools that allowed us to intercept and visualize mobile zinc efficiently (15). These studies revealed modulation of extrasynaptic NMDARs by zinc and led us to reinvestigate whether synaptically released zinc might be an endogenous modulator of AMPARs as well. In the present study, we applied these same tools in electrophysiological, laser-based glutamate uncaging and in imaging experiments using wild type and genetically modified mice that lack synaptic zinc.     

A second S4 movement opens hyperpolarization-activated HCN channels

Rhythmic activity in pacemaker cells, as in the sino-atrial node in the heart, depends on the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. As in depolarization-activated K⁺ channels, the fourth transmembrane segment S4 functions as the voltage sensor in hyperpolarization-activated HCN channels. But how the inward movement of S4 in HCN channels at hyperpolarized voltages couples to channel opening is not understood. Using voltage clamp fluorometry, we found here that S4 in HCN channels moves in two steps in response to hyperpolarizations and that the second S4 step correlates with gate opening. We found a mutation in sea urchin HCN channels that separate the two S4 steps in voltage dependence. The E356A mutation in S4 shifts the main S4 movement to positive voltages, but channel opening remains at negative voltages. In addition, E356A reveals a second S4 movement at negative voltages that correlates with gate opening. Cysteine accessibility and molecular models suggest that the second S4 movement opens up an intracellular crevice between S4 and S5 that would allow radial movement of the intracellular ends of S5 and S6 to open HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are important for generating pacemaker activity in cardiomyocytes and neurons (1, 2). The rhythmic and autonomous activity of these cells is triggered by the activation of a depolarizing Na⁺ current through HCN channels at hyperpolarized voltages after an action potential that drives the membrane potential back to the threshold for firing another action potential. Studies in patients and animal models have found that impaired function of HCN channels is associated with diseases like neuropathic pain (3, 4), epilepsies (5, 6), and cardiac arrhythmias (7–9). HCN channels belong to the same superfamily as voltage-gated K⁺ (Kv) channels. But, in contrast to most Kv channels, which are opened by depolarization, HCN channels are opened by hyperpolarization of the cell membrane (10–12). In addition to voltage, HCN channels are further opened by cyclic nucleotides that bind to a cyclic nucleotide-binding domain in the cytoplasmic C-terminal region (13–15). Both HCN and Kv channels are tetramers and each of their subunits has six transmembrane segments, where segments S1 through S4 form the voltage-sensing domain (VSD) and segments S5 through S6 form the pore domain (PD) (14, 16). Similar to Kv channels, the gate of HCN channels is formed by the most C-terminal region of the four S6 helices (14, 17–19) and their pore selectivity filter contains the typical GYG of the potassium signature sequence, but not the complete TVGYG signature sequence, which might explain why HCN channels are only slightly more selective for K⁺ than for Na⁺ (1, 14, 20). In addition, both HCN and Kv channels sense changes in the membrane potential through the positively charged S4 segment that moves across the membrane in response to changes in the membrane potential (21–24). However, in HCN channels the gate opens when S4 moves inward at hyperpolarized voltages, whereas Kv channels open when S4 moves outwards at depolarized voltages (22). Despite many studies into the voltage-gating mechanism of HCN channels (22, 23, 25–28), it is still not completely understood how inward S4 movement in response to a hyperpolarization opens HCN channels. We here study S4 movement in channels carrying mutations at the interface between the VSD and the PD to understand how hyperpolarization opens HCN channels.In contrast to most Kv channels, HCN channels have a nondomain swapped architecture, which means that the VSD interacts with the PD from the same subunit (14, 16). In domain-swapped Kv channels (Kv1 to -7), in which the VSD is next to the PD from the neighboring subunit, it is assumed that the long S4-S5 linker transduces the S4 movement to the opening of the gate by pulling on the lower S6 (16, 29). In rEAG (Kv10.1), a nondomain swapped depolarization-activated Kv channel, and spHCN (Strongylocentrotus purpuratus HCN, sea urchin HCN), a nondomain swapped hyperpolarization-activated HCN channel, cutting the short S4-S5 linker does not prevent gating (25, 30), which shows that the voltage-gating mechanism in these nondomain-swapped channels does not require a covalent link between the S4 and S5 segments. Instead, several studies have suggested that noncovalent interactions between S4 and S5 are required for the opening of HCN channels by hyperpolarization (25, 26, 28, 31, 32). In addition, the S4 segment of HCN channels is two helical turns longer than the S4 of depolarization-activated Kv channels. Recently, three different groups showed that in HCN channels S4 moves inward by around 10 Å in response to hyperpolarization, and that there is a kink in the most C-terminal part of S4 when S4 moves inwards (27, 33, 34). But, how the inward movement of voltage sensor S4 causes channel opening is not clear from these studies.At the S4-S5 interface of HCN channels, there are several residues that are absolutely conserved in HCN channels but that are different in depolarization-activated Kv channels. We mutated these conserved residues, one at a time, in the sea urchin HCN channel (spHCN) and used voltage clamp fluorometry (VCF) (21) to test their role in HCN gating. The VCF technique allows us to simultaneously detect gate movement, by recording the currents through the channels, and S4 movement, by recording the fluorescence emitted by fluorophores attached to the extracellular end of S4. We found that S4 moves in two steps and that the second S4 movement correlates with channel opening. We also show that the second S4 movement changes the intracellular accessibility of residues L340C in S4 and N370C in S5, as if an internal crevice opens between the voltage sensors and the pore, thereby allowing the intracellular pore to expand and open the channel.     

Separation of presynaptic Cav2 and Cav1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca2+ pump PMCA

Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca²⁺ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)–triggered presynaptic Ca²⁺ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Ca_v2 homolog, Dmca1A or cacophony, and the Ca_v1 homolog, Dmca1D) but with spatial and functional separation. Ca_v2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Ca_v1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca²⁺ influx but has a small impact on release. Instead, L-type calcium currents through Ca_v1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca²⁺ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Ca_v1-mediated Ca²⁺ influx. We show that the plasma membrane Ca²⁺ ATPase (PMCA) resides in between active zones and isolates Ca_v2-triggered release from Ca_v1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Ca_v2 may also contribute to. As L-type Ca_v1 channels also localize next to PQ-type Ca_v2 channels within axon terminals of some central mammalian synapses, we propose that Ca_v2, Ca_v1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.

Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca²⁺-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca²⁺ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca²⁺ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood.SV release probability depends on the sensitivity of the vesicular Ca²⁺ sensor and the positioning of VGCCs inside active zones (AZs) (1). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered (2–4). Irrespective of such modulation, efficient Ca²⁺-triggered SV release through presynaptic VGCCs (mainly Ca_v2.1 and Ca_v2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca²⁺ buffers (5, 6). It is thus conceivable that Ca²⁺ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity.Apart from the need for fast activating and inactivating Ca_v2 channels for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of Ca_v1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression (7). In hippocampal CA3 mossy fiber boutons (8–10) or in synapses of the lateral amygdala (11), Ca_V2.3 and Ca_V1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (9, 11).Differential functions of Ca_v2 and Ca_v1 channels in neurotransmitter release versus other Ca²⁺-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant (4, 10). Moreover, compared with Ca_v2.1 and Ca_v2.2, Ca_v1 channels display higher conductances (12), suggesting that additional mechanisms are required to allocate Ca_V1-related Ca²⁺ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca²⁺ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca²⁺-triggered SV release (13–15). We hypothesize that activity-dependent regulation of SV recycling employs Ca_v1-dependent Ca²⁺ entry and that active mechanisms exist to regulate the relative contributions of Ca_v2 and Ca_v1 channels to SV release versus recycling. We address these hypotheses at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (16–18).     

Mossy fiber-evoked subthreshold responses induce timing-dependent plasticity at hippocampal CA3 recurrent synapses

Dentate granule cells exhibit exceptionally low levels of activity and rarely elicit action potentials in targeted CA3 pyramidal cells. It is thus unclear how such weak input from the granule cells sustains adequate levels of synaptic plasticity in the targeted CA3 network. We report that subthreshold potentials evoked by mossy fibers are sufficient to induce synaptic plasticity between CA3 pyramidal cells, thereby complementing the sparse action potential discharge. Repetitive pairing of a CA3–CA3 recurrent synaptic response with a subsequent subthreshold mossy fiber response induced long-term potentiation at CA3 recurrent synapses in rat hippocampus in vitro. Reversing the timing of the inputs induced long-term depression. The underlying mechanism depends on a passively conducted giant excitatory postsynaptic potential evoked by a mossy fiber that enhances NMDA receptor-mediated current at active CA3 recurrent synapses by relieving magnesium block. The resulting NMDA spike generates a supralinear depolarization that contributes to synaptic plasticity in hippocampal neuronal ensembles implicated in memory.The CA3 area of the hippocampus exhibits a distinctive, highly recurrent circuitry proposed to support autoassociative memory representation (1, 2). This prediction has been confirmed by experimental work demonstrating the pattern completion capabilities of CA3 networks (3), as well as their roles in the spatial tuning of CA1 pyramidal cells, in one-trial contextual learning (4) and in certain forms of memory consolidation (5). CA3 pyramidal cells receive, via the mossy fibers, information processed by granule cells important for both pattern separation (6, 7) and pattern completion functions (7). The faithful transmission of mossy fiber input appears to be ensured by giant synapses composed of presynaptic boutons with up to 45 release sites (8) that target massive spines, the thorny excrescences, on the apical dendrite of CA3 pyramidal cells. Thus, the mossy fiber synapse is often referred to as a detonator synapse (9). In fact, mossy fiber signaling is more compatible with a gatekeeper function than a high-throughput data relay. Although high-frequency bursts of action potentials in a hippocampal granule cell can discharge a targeted CA3 pyramidal cell, the majority of responses evoked by granule cells in CA3 pyramidal cells do not attain the firing threshold (10). Nevertheless, mossy fibers generate powerful signals evoking subthreshold responses that are much larger than typical synaptic events in the brain, with excitatory postsynaptic potentials (EPSPs) and excitatory postsynaptic currents (EPSCs) reaching amplitudes of 10 mV and 1 nA, respectively (11). Here we examined in rat slice cultures how EPSPs generated at mossy fiber synapses are processed in CA3 pyramidal cell dendrites, and evaluated whether subthreshold synaptic responses evoked by mossy fiber stimulation can act as instructive signals to induce plasticity at the pyramidal cell synapses forming the CA3 recurrent network.     

Activity-dependent endoplasmic reticulum Ca2+ uptake depends on Kv2.1-mediated endoplasmic reticulum/plasma membrane junctions to promote synaptic transmission

The endoplasmic reticulum (ER) forms a continuous and dynamic network throughout a neuron, extending from dendrites to axon terminals, and axonal ER dysfunction is implicated in several neurological disorders. In addition, tight junctions between the ER and plasma membrane (PM) are formed by several molecules including Kv2 channels, but the cellular functions of many ER-PM junctions remain unknown. Recently, dynamic Ca²⁺ uptake into the ER during electrical activity was shown to play an essential role in synaptic transmission. Our experiments demonstrate that Kv2.1 channels are necessary for enabling ER Ca²⁺ uptake during electrical activity, as knockdown (KD) of Kv2.1 rendered both the somatic and axonal ER unable to accumulate Ca²⁺ during electrical stimulation. Moreover, our experiments demonstrate that the loss of Kv2.1 in the axon impairs synaptic vesicle fusion during stimulation via a mechanism unrelated to voltage. Thus, our data demonstrate that a nonconducting role of Kv2.1 exists through its binding to the ER protein VAMP-associated protein (VAP), which couples ER Ca²⁺ uptake with electrical activity. Our results further suggest that Kv2.1 has a critical function in neuronal cell biology for Ca²⁺ handling independent of voltage and reveals a critical pathway for maintaining ER lumen Ca²⁺ levels and efficient neurotransmitter release. Taken together, these findings reveal an essential nonclassical role for both Kv2.1 and the ER-PM junctions in synaptic transmission.

The members of the Kv2 family of voltage-gated K⁺ (Kv) channels, Kv2.1 and Kv2.2, are widely expressed in neurons within the mammalian brain, with Kv2.1 dominating in hippocampal neurons (1–3). These channels play an important classical role in repolarizing somatic membrane potential during high-frequency stimulation (4). However, Kv2 channels also form micrometer-sized clusters on the cell membrane, where they are largely nonconductive (5). When clustered, these nonconductive channels act as molecular hubs directing protein insertion and localization, including during the fusion of dense-core vesicles (6–9). Clusters are also sites for the enrichment of voltage-gated Ca²⁺ channels (10). The Kv2 clustering mechanism is due to the formation of stable tethers between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM) through a noncanonical FFAT motif located on the Kv2 C terminus, which interacts with VAMP-associated protein (VAP) embedded in the ER membrane (11). These Kv2.1-mediated junctions between the ER and PM are in close (∼15 nm) proximity (12), forming critical Ca²⁺-signaling domains that have been conserved from yeast to mammals (12–14) and are necessary to cluster Kv2.1 channels. ER-PM junctions are formed by many types of proteins, although most are ER proteins that transiently interact with specific lipids on the PM (reviewed previously in ref. 15). The purpose of the Kv2.1-VAP–mediated ER-PM junction is not functionally understood in neurons to date.Cytosolic Ca²⁺ is essential for initiating multiple cell functions, including secretion, muscle contraction, proliferation, apoptosis, and gene expression (reviewed previously in ref. 16). However, Ca²⁺ is also strongly buffered, especially in most neurons, and often requires local Ca²⁺ exchange between channels and pumps localized to organelles and the PM. The ER plays a central role in both Ca²⁺ signaling and storage (17), and dysfunction of ER morphology and Ca²⁺ handling has been linked to several unique neurological pathologies, including hereditary spastic paraplegia (18), Alzheimer’s disease (19), and amyotrophic lateral sclerosis (20). Currently, the only known cellular mechanism used to replenish ER Ca²⁺ stores is through activation of store-operated Ca²⁺ entry (SOCE). Depletion of the ER’s luminal Ca²⁺ is sensed by stromal interaction molecule 1 (STIM1), which aggregates and concentrates Orai proteins on the PM to initiate Ca²⁺ influx through Ca²⁺ release–activated Ca²⁺ (CRAC) channels. Recent studies, however, have revealed that a second frequently accessed pathway exists in neurons where stimulation-evoked Ca²⁺ influx is rapidly taken up by the ER through sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps during neuronal activity, rather than in reaction to severe depletion of ER lumen Ca²⁺. Failure to quickly increase luminal Ca²⁺ during action potential (AP) firing leads to ER Ca²⁺ depletion and impaired synaptic vesicle fusion (21). Thus, luminal ER Ca²⁺ plays an essential role in maintaining synaptic transmission in active healthy neurons, suggesting that a mechanism other than SOCE must be important for neuronal communication.Taken together, Kv2.1 clusters have been shown to localize L-type voltage-gated Ca²⁺ channels at the PM while also anchoring the ER in close proximity to the PM (10). We hypothesized that these Kv2.1-mediated ER-PM junctions are uniquely positioned to serve a critical role as dynamic signaling domains for rapid ER Ca²⁺ uptake during electrical activity in neurons. We measured Kv2.1’s role in ER Ca²⁺ handling using ER-GCaMP (a genetically encoded calcium indicator) and found that AP-evoked Ca²⁺ entry into the somatic ER was absent with short hairpin RNA (shRNA) knockdown (KD) of Kv2.1 channels. This nonconducting role of Kv2.1 which enables ER-Ca²⁺ filling also requires SERCA pumps. Moreover, we demonstrate a nonconducting role for Kv2.1 in the axon that is essential for enabling ER-Ca²⁺ uptake during electrical activity. We go on to show that KD of Kv2.1 impaired overall synaptic physiology through decreased presynaptic Ca²⁺ entry and synaptic vesicle exocytosis. Finally, we demonstrate that this role requires Kv2.1’s C-terminal VAP-binding domain to restore synaptic transmission.     

NMDARs in granule cells contribute to parallel fiber–Purkinje cell synaptic plasticity and motor learning

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

The ability of an organism to adjust its behavior to environmental demands depends on its capacity to learn and execute coordinated movements. The cerebellum plays a central role in this process by optimizing motor programs through trial-and-error learning (1). Within the cerebellum, the synaptic output from granule cells (GCs) to Purkinje cells (PCs) shapes computational operations during basal motor function and serves as a substrate for motor learning (2). Several forms of motor learning depend on changes in the strength of the parallel fiber (PF), the axon of GCs, to the PC synapse (3, 4).In the mammalian forebrain, synaptic plasticity typically relies on postsynaptic N-methyl-D-aspartate receptor (NMDAR) activation, which alters AMPA receptor (AMPAR) turnover at the postsynaptic site (5). However, this may not extend to the cerebellar synapse between GCs and PCs, since no functional postsynaptic NMDARs have been identified in young or adult rodents (6, 7). Pharmacological approaches, however, have shown that both long-term depression (LTD) and long-term potentiation (LTP) induction depend on NMDAR activation at the PF-PC synapse in young rodents (8–12). Hence, the alternative mechanisms for NMDAR-dependent synaptic modulation may involve presynaptic NMDARs activation [(12–15); for review: refs. 16 and 17]. Indeed, cell-specific deletion of NMDARs in GCs abolishes LTP in young rodents (12). In addition to NMDARs, PF-PC synaptic plasticity also requires nitric-oxide (NO) signaling (18–20). As nitric-oxide synthase (NOS) is expressed in GCs, but not in PCs (21), the activation of presynaptic NMDARs might allow Ca²⁺ influx that activates NO synthesis, which in turn may act upon the PCs. However, in the mature cerebellum, the existence of presynaptic NMDARs on PFs and the role of NO in PF-PC plasticity remains a matter of debate. Previously, we have proposed that the activation of putatively presynaptic NMDARs in young rodents is necessary for inducing PF-PC synaptic plasticity without affecting transmitter release (8, 9, 11, 12). More recently, it has been shown that a subset of PFs express presynaptic NMDARs containing GluN2A subunits and that these receptors are functional (11, 12). Thus, in contrast to their role at other synapses, at least in young rodent, presynaptic NMDARs as part of the PF-PC synapses might act via the production of NO to induce postsynaptic plasticity, without altering neurotransmitter release (9, 11, 12, 18–22). However, a causal link between NMDARs activation in PFs, NO synthesis, and synaptic plasticity induction is still missing.In the cerebral cortex, the expression of presynaptic NMDARs is developmentally regulated (23, 24). However, little is known about the presence and function of presynaptic NMDARs in adult tissue. In the adult cerebellum, PCs only express postsynaptic NMDARs at their synapse with climbing fibers (CFs) (25). It has been proposed that the activation of these receptors could have heterosynaptic effects during PF-PC LTD. This mechanism would explain why LTD in adults depends on NMDARs. According to this model, presynaptic NMDARs would be a transient feature of developing tissue and not necessary for induction of synaptic plasticity and motor learning in adult animals (25).Here, we combine electron microscopy, two-photon calcium imaging, synaptic plasticity experiments, and behavioral measurements to show that presynaptic NMDARs are not developmentally regulated but are required for cerebellar motor learning in adults. We demonstrate that presynaptic NMDARs are present and functional in PFs of mature rodents. By specifically deleting the NMDAR subunit GluN1 either in the post- (PC) or the presynaptic cells (GCs), we demonstrate that NMDAR activation in GCs plays a key role in bidirectional synaptic plasticity and in vestibulo-ocular reflex (VOR) adaptation, an important paradigm for testing cerebellar motor learning (26–28). In contrast, NMDARs in PCs are neither involved in PF-PC synaptic plasticity nor required for cerebellar motor learning.     

HCN channels enhance spike phase coherence and regulate the phase of spikes and LFPs in the theta-frequency range

What are the implications for the existence of subthreshold ion channels, their localization profiles, and plasticity on local field potentials (LFPs)? Here, we assessed the role of hyperpolarization-activated cyclic-nucleotide–gated (HCN) channels in altering hippocampal theta-frequency LFPs and the associated spike phase. We presented spatiotemporally randomized, balanced theta-modulated excitatory and inhibitory inputs to somatically aligned, morphologically realistic pyramidal neuron models spread across a cylindrical neuropil. We computed LFPs from seven electrode sites and found that the insertion of an experimentally constrained HCN-conductance gradient into these neurons introduced a location-dependent lead in the LFP phase without significantly altering its amplitude. Further, neurons fired action potentials at a specific theta phase of the LFP, and the insertion of HCN channels introduced large lags in this spike phase and a striking enhancement in neuronal spike-phase coherence. Importantly, graded changes in either HCN conductance or its half-maximal activation voltage resulted in graded changes in LFP and spike phases. Our conclusions on the impact of HCN channels on LFPs and spike phase were invariant to changes in neuropil size, to morphological heterogeneity, to excitatory or inhibitory synaptic scaling, and to shifts in the onset phase of inhibitory inputs. Finally, we selectively abolished the inductive lead in the impedance phase introduced by HCN channels without altering neuronal excitability and found that this inductive phase lead contributed significantly to changes in LFP and spike phase. Our results uncover specific roles for HCN channels and their plasticity in phase-coding schemas and in the formation and dynamic reconfiguration of neuronal cell assemblies.Local field potentials (LFPs) have been largely believed to be a reflection of the synaptic drive that impinges on a neuron. In recent experimental and modeling studies, there has been a lot of debate on the source and spatial extent of LFPs (1–9). However, most of these studies have used neurons with passive dendrites in their models and/or have largely focused on the contribution of spike-generating conductances to LFPs (7, 8, 10, 11). Despite the widely acknowledged regulatory roles of subthreshold-activated ion channels and their somatodendritic gradients in the physiology and pathophysiology of synapses and neurons (12–17), the implications for their existence on LFPs and neuronal spike phase have surprisingly remained unexplored. This lacuna in LFP analysis is especially striking because local and widespread plasticity of these channels has been observed across several physiological and pathological conditions, translating to putative roles for these channels in neural coding, homeostasis, disease etiology and remedies, learning, and memory (16, 18–23).In this study, we focus on the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that mediate the h current (I_h) in regulating LFPs and theta-frequency spike phase. From a single-neuron perspective, HCN channels in CA1 pyramidal neurons play a critical role in regulating neuronal integration and excitability (14, 24–27) and importantly introduce an inductive phase lead in the voltage response to theta-frequency oscillatory inputs (28), thereby enabling intraneuronal synchrony of incoming theta-frequency inputs (29). Given these and their predominant dendritic expression (25), we hypothesized HCN channels as regulators of LFPs through their ability to alter the amplitude and phase of the intracellular voltage response, thereby altering several somatodendritic transmembrane currents that contribute to LFPs. The CA1 region of the hippocampus offers an ideal setup to test this hypothesis, given the regular, open-field organization (4, 6, 7) of the pyramidal neurons endowed with well-established somatodendritic gradients in ion channel densities (16). As this organization enables us to assess the role of location-dependent channel expression profiles on LFPs across different strata, we tested our hypothesis, using a computational scheme involving morphologically realistic, physiologically constrained conductance-based model neurons. Our results positively test our hypothesis and provide specific evidence for novel roles for HCN channels and their inductive component in regulating LFP and spike phases, apart from enhancing spike-phase coherence. These results identify definite roles for HCN channels in phase-coding schemas and in the formation and dynamic reconfiguration of neuronal cell assemblies and argue for the incorporation of subthreshold-activated ion channels, their gradients, and their plasticity into the computation of LFPs.     

Dystroglycan mediates homeostatic synaptic plasticity at GABAergic synapses

Dystroglycan (DG), a cell adhesion molecule well known to be essential for skeletal muscle integrity and formation of neuromuscular synapses, is also present at inhibitory synapses in the central nervous system. Mutations that affect DG function not only result in muscular dystrophies, but also in severe cognitive deficits and epilepsy. Here we demonstrate a role of DG during activity-dependent homeostatic regulation of hippocampal inhibitory synapses. Prolonged elevation of neuronal activity up-regulates DG expression and glycosylation, and its localization to inhibitory synapses. Inhibition of protein synthesis prevents the activity-dependent increase in synaptic DG and GABA_A receptors (GABA_ARs), as well as the homeostatic scaling up of GABAergic synaptic transmission. RNAi-mediated knockdown of DG blocks homeostatic scaling up of inhibitory synaptic strength, as does knockdown of like-acetylglucosaminyltransferase (LARGE)—a glycosyltransferase critical for DG function. In contrast, DG is not required for the bicuculline-induced scaling down of excitatory synaptic strength or the tetrodotoxin-induced scaling down of inhibitory synaptic strength. The DG ligand agrin increases GABAergic synaptic strength in a DG-dependent manner that mimics homeostatic scaling up induced by increased activity, indicating that activation of this pathway alone is sufficient to regulate GABA_AR trafficking. These data demonstrate that DG is regulated in a physiologically relevant manner in neurons and that DG and its glycosylation are essential for homeostatic plasticity at inhibitory synapses.Muscular dystrophies are often associated with mild to severe cognitive deficits, epilepsy, and other neurological deficits (1–3). This is particularly evident in muscular dystrophies caused by mutations that affect glycosylation of the membrane glycoprotein α-dystroglycan (α-DG) (4). α-DG docks with transmembrane β-DG to form the functional core of the dystrophin-associated glycoprotein complex (DGC) that links adhesive proteins in the extracellular matrix to dystrophin (5). α-DG is heavily glycosylated and interacts via its carbohydrate side chains with laminin and laminin G-like domains in a variety of proteins including agrin, perlecan, slit, neurexin, and pikachurin (6–10). Key carbohydrate residues are added onto α-DG by several glycosyltransferases, most notably like-acetylglucosaminyltransferase (LARGE) (11). LARGE is necessary for functional glycosylation of α-DG (12), and is mutated in muscular dystrophies associated with severe cognitive deficits (4).DG was first identified in the nervous system (13), where it is important during development for neuroblast migration (14), axon guidance (7), and ribbon synapse formation (8). At neuromuscular synapses, DG is required for the stabilization of acetylcholine receptors in the postsynaptic density and contributes to the accumulation of acetylcholinesterase (10, 15). However, the function of DG at central synapses remains essentially unknown. In the mature central nervous system (CNS), neuronal DGC components are exclusively colocalized with GABA_A receptors (GABA_ARs) in multiple brain regions (16–18), raising the possibility for a role in GABA_AR regulation. However, DG is dispensable for GABAergic synapse formation in hippocampal cultures (17), although adult mice lacking full-length dystrophin show reduced clustering of GABA_ARs in the hippocampus and other brain regions (16, 19, 20). Because dystrophin localization at GABAergic synapses depends on DG (17), these findings suggest that DG may regulate the plasticity of mature GABAergic synapses. Homeostatic synaptic plasticity is widely thought to be essential for brain function and involves the reciprocal regulation of glutamatergic and GABAergic synapses to stabilize neuronal activity (21). Chronic elevation of neuronal activity is associated with an increase in synaptic GABA_ARs (22, 23), but the mechanistic details are incompletely understood.Here, we assess the roles of DG and α-DG glycosylation in regulating the expression of homeostatic synaptic plasticity at GABAergic synapses. We find that in mature hippocampal cultures, prolonged elevation of neuronal activity up-regulates DG expression and the coclustering of α-DG and GABA_ARs. Inhibition of protein synthesis or knockdown of DG blocks homeostatic scaling up of GABAergic synaptic strength. Knockdown of the selective α-DG glycosyltransferase LARGE also blocks homeostatic scaling up, suggesting a role for ligand binding. Furthermore, exogenous application of agrin—a ligand for glycosylated α-DG—is sufficient to scale up GABAergic synaptic strength in a DG-dependent fashion. These data identify a mechanism whereby expression of glycosylated α-DG is linked to neuronal activity level and is essential for homeostatic scaling up of GABAergic synaptic strength by regulating GABA_AR abundance at the synapse.     

Postsynaptic activity reverses the sign of the acetylcholine-induced long-term plasticity of GABAA inhibition

Acetylcholine (ACh) regulates forms of plasticity that control cognitive functions but the underlying mechanisms remain largely unknown. ACh controls the intrinsic excitability, as well as the synaptic excitation and inhibition of CA1 hippocampal pyramidal cells (PCs), cells known to participate in circuits involved in cognition and spatial navigation. However, how ACh regulates inhibition in function of postsynaptic activity has not been well studied. Here we show that in rat PCs, a brief pulse of ACh or a brief stimulation of cholinergic septal fibers combined with repeated depolarization induces strong long-term enhancement of GABA_A inhibition (GABA_A-LTP). Indeed, this enhanced inhibition is due to the increased activation of α₅βγ₂ subunit-containing GABA_A receptors by the GABA released. GABA_A-LTP requires the activation of M1-muscarinic receptors and an increase in cytosolic Ca²⁺. In the absence of PC depolarization ACh triggered a presynaptic depolarization-induced suppression of inhibition (DSI), revealing that postsynaptic activity gates the effects of ACh from presynaptic DSI to postsynaptic LTP. These results provide key insights into mechanisms potentially linked with cognitive functions, spatial navigation, and the homeostatic control of abnormal hyperexcitable states.Long-term potentiation (LTP) at excitatory synapses is thought to be the cellular substrate of learning of the brain. Less is known about LTP at inhibitory synapses, a vital process given that inhibition regulates network behavior and LTP at excitatory synapses (1–3). Cholinergic activity can influence intrinsic excitability, as well as both excitatory (4, 5) and inhibitory synaptic plasticity (6, 7). However, less is known about the postsynaptic cholinergic-mediated control of synaptic inhibition and specifically of its regulation by postsynaptic activity. The CA1 region of the hippocampus receives a significant cholinergic projection from the medial septal nuclei (8). These act primarily through acetylcholine (ACh) muscarinic receptors (mAChRs) on CA1 pyramidal cells (PCs) (9), as well as through mAChRs and nicotinic cholinergic receptors (nAChRs) on interneurons (10). In addition, the retrograde modulation of γ-aminobutyric acid (GABA)-mediated inhibition by endocannabinoids (eCBs) (11) and its regulation by ACh and postsynaptic activity have been analyzed (12).We analyzed the modifications induced in PCs in the CA1 of rat hippocampal slices by repeated postsynaptic depolarization, applied in combination with a single brief ACh pulse delivered to the apical dendritic shaft. The postsynaptic depolarization reproduced either the rhythmic bursting that typifies the hippocampal theta rhythm [i.e., theta burst stimulation (TBS)] or that of prolonged repeated depolarization. Indeed, these protocols induced a robust long-term enhancement of inhibition because of the increased activation of α₅βγ₂ subunit-containing GABA_A receptors (GABA_ARs) by the released GABA, with no involvement of GABA_BRs. We termed this long-term enhancement of inhibition GABA_A-LTP. GABA_A-LTP was also evoked by a physiological relevant stimulation of cholinergic septal fibers of the oriens/alveus (O/A), combined with repeated depolarization or TBS stimulation. This GABA_A-LTP required activation of the M1 subtype mAChRs (M1-mAChRs) and an increased cytosolic Ca²⁺. In the absence of postsynaptic depolarization, ACh generated a type 1 eCB receptor (CB₁R)-dependent depolarization-induced suppression of inhibition (DSI) (13), indicating that the effects of ACh on synaptic inhibition depend on the active or quiescent state of the postsynaptic PC. Therefore, ACh triggers a state-dependent gating that transfers the dominant effects of postsynaptic activity from presynaptic DSI to postsynaptic LTP. Such a relocation may be essential to regulate the network activity that may be linked to the information-processing capacity of the system in terms of spatial and cognitive functions (14) and of the homeostatic control of abnormal hyperexcitable states.     

Polyunsaturated fatty acid analogs act antiarrhythmically on the cardiac IKs channel

Polyunsaturated fatty acids (PUFAs) affect cardiac excitability. Kv7.1 and the β-subunit KCNE1 form the cardiac I_Ks channel that is central for cardiac repolarization. In this study, we explore the prospects of PUFAs as I_Ks channel modulators. We report that PUFAs open Kv7.1 via an electrostatic mechanism. Both the polyunsaturated acyl tail and the negatively charged carboxyl head group are required for PUFAs to open Kv7.1. We further show that KCNE1 coexpression abolishes the PUFA effect on Kv7.1 by promoting PUFA protonation. PUFA analogs with a decreased pK_a value, to preserve their negative charge at neutral pH, restore the sensitivity to open I_Ks channels. PUFA analogs with a positively charged head group inhibit I_Ks channels. These different PUFA analogs could be developed into drugs to treat cardiac arrhythmias. In support of this possibility, we show that PUFA analogs act antiarrhythmically in embryonic rat cardiomyocytes and in isolated perfused hearts from guinea pig.The cardiac action potential is initiated and maintained by inward sodium and calcium currents and terminated by outward potassium currents (1). The I_Ks channel, formed by four α subunits (voltage-gated potassium channel subunit Kv7.1, originally called KCNQ1 or KvLQT1) and two to four auxiliary β subunits (Kv channel beta subunit KCNE1, originally called minK) (1, 2), contributes a major component of the repolarizing potassium current. More than 300 mutations in the genes encoding Kv7.1 and KCNE1 have been identified in patients with cardiac arrhythmia (1). Loss-of-function mutations of the I_Ks channel prolong the QT interval as observed in long QT syndrome, leading to ventricular arrhythmias, ventricular fibrillation, and sudden death (1). Gain-of-function mutations of the I_Ks channel shorten the QT interval, possibly leading to arrhythmia such as short QT syndrome or atrial fibrillation (1). Pharmacological augmentation (in the case of long QT syndrome) or inhibition (in the case of short QT syndrome) of I_Ks channel activity is a logical pharmacological strategy to treat these forms of cardiac arrhythmias.Kv7.1 is a tetrameric voltage-gated K (Kv) channel with six transmembrane segments (called S1–S6) per subunit (3). S5 and S6 from all four subunits together form the pore domain with the central ion-conducting pore. In Kv channels, S6 has been shown to function as the activation gate, shutting off the intracellular access to the pore for K⁺ ions in the closed state of the channel (3–5). Most reported activators or inhibitors of Kv7.1 channels target the ion-conducting pore domain of the channel, opening or blocking the ionic pathway (6–10). S1–S4 of each subunit form a voltage-sensor domain (VSD). In Kv channels, each S4 segment has several positively charged residues and has been shown to move in response to changes in the transmembrane voltage (3, 11). In response to membrane depolarization, the S4 segments move outward with respect to the membrane, which causes channel opening. Although four Kv7.1 subunits per se form a functional channel, Kv7.1 needs to coassemble with the auxiliary β-subunit KCNE1 to recapitulate the biophysical properties of the native cardiac I_Ks channel (12, 13). KCNE1, a single transmembrane helix protein, has been proposed to associate with Kv7.1 in the lipid cleft between adjacent VSDs, making contact with VSD transmembrane segments S1 and S4 and pore transmembrane segment S6 (14–16).In this study, we explore the prospects of polyunsaturated fatty acids (PUFAs) and PUFA analogs as small molecules enhancing or inhibiting the activity of the cardiac I_Ks channel by changing I_Ks channel voltage dependence. We previously suggested that PUFAs facilitate opening of the related Shaker Kv channel via electrostatic attraction of S4 (17–20). The pharmacological sensitivity of I_Ks to small-molecule activators has been shown to depend on the Kv7.1:KCNE1 stoichiometry (21–23). We therefore also determine the impact of Kv7.1:KCNE1 stoichiometry on PUFA sensitivity.Below we show that PUFAs affect the Kv7.1 channel by an electrostatic effect on the voltage sensor movement. We also show that KCNE1 abolishes the PUFA sensitivity of the Kv7.1 channel at physiological pH, suggesting that physiologically occurring PUFAs do not act on I_Ks channels in vivo. Furthermore, we identify PUFA analogs that have effects on the I_Ks channel at physiological pH, increase I_Ks in cardiomyocytes, restore rhythmic firing in arrhythmic cardiomyocytes, and shorten the QT interval in isolated perfused guinea pig hearts. These results may form the basis for development of pharmacological drugs that target the I_Ks channel to prevent cardiac arrhythmias.