The potassium channel subunit Kvβ1 serves as a major control point for synaptic facilitation

Analysis of the presynaptic action potential’s (AP_syn) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca²⁺ in cultured hippocampal neurons. These recordings revealed a critical and selective role for K_v1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic K_v1 channel inactivation was mediated by the K_vβ1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of K_vβ1 blocked all broadening of the AP_syn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic K_v channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery.

The action potential (AP) firing pattern or “spike code” as typically measured from the soma is a gold standard for neural excitability within circuits. However, at presynaptic terminals the quantitative relationship between the input AP spike code and the magnitude of exocytosis, or vesicle fusion events per AP, can change dynamically as a result of stimulation frequency or firing pattern. Increased firing frequency can significantly increase the number of vesicles that fuse from an identical number of APs. This phenomenon is known as short-term synaptic facilitation, which can significantly enhance information transfer at synapses influencing several aspects of learning and memory (1). Thus, it is important to completely understand the underlying molecular and cellular mechanisms of synaptic facilitation.A critical initial step in exocytosis is the arrival of AP_syn at boutons, whose waveform can exhibit plasticity based on firing frequency. Repetitive firing may cause inactivation of axonal voltage-gated sodium (Na_v) channels and voltage-gated potassium (K_v) channels that control the depolarization and hyperpolarization of the waveform, respectively. K_v inactivation primarily leads to an increase in AP width or broadening (2–9). The width of the AP_syn controls the fraction of time that Ca²⁺ channels open and the driving force of Ca²⁺ entry (10). These changes in voltage kinetics during the AP_syn will also impact the shape or profile of the Ca²⁺ microdomain envelope that builds locally around open Ca²⁺ channels in the terminal (11, 12). The highly nonlinear influence of Ca²⁺ on exocytosis (13, 14) thus dictates that modest AP_syn broadening has the potential to critically impact synaptic facilitation (15–17). Indeed, AP_syn broadening during repetitive firing has been demonstrated to cause the facilitation of exocytosis in the pituitary nerve (3), dorsal root ganglion (18), and mossy fiber bouton (2), all due to K_v channel inactivation. However, the AP_syn waveform in Purkinje cells has also been shown to undergo frequency-dependent decreases in amplitude that substantially contribute to synaptic depression (19). Thus, it is best to consider the AP_syn as a plastic signal that can powerfully modulate exocytosis bidirectionally, rather than as a digital spike acting solely as an initiation signal. We therefore reason that the somatic AP has proven to be a poor predictor of exocytosis magnitude as a result of a failure to resolve the AP_syn waveform and its molecular regulators in the majority of brain regions.As opposed to the majority of larger synapses, en passant terminals are most commonly involved in brain regions associated with synaptic plasticity. Investigating the molecular regulation of AP_syn in the common en passant nerve terminals of the cortex and hippocampus remains elusive due to the small size of these structures (<1 µm), which makes them inaccessible for whole-cell patch clamp recording. An innovative initial strategy to overcome these size restrictions was the use of voltage dyes, which failed to detect use-dependent changes in the AP_syn in hippocampal slices (20). However, these dyes were limited by very low voltage sensitivity (<0.5% change in fluorescence for an AP) requiring population averaging, and these dyes were unable to report a stable resting voltage during stimulation (20). Moreover, it was found only later that this class of voltage dyes were phototoxic and altered membrane physiology, limiting their usefulness in small axons (21). As a result of these complications, the question of AP_syn plasticity as a modulator of synaptic facilitation remains unanswered for hippocampal neurons. Our group has overcome the size barrier of hippocampal axons, while also avoiding cell population averaging and dye toxicity by pioneering the use of genetically encoded rhodopsin-based voltage indicators. Here, we measure the AP_syn of individual en passant terminals from both inhibitory and excitatory hippocampal neurons. These measurements demonstrate a striking contrast between facilitating excitatory and depressing inhibitory nerve terminals in the hippocampus. We discovered that excitatory axons and terminals are uniquely enriched with a combination of K_v1.1/1.2 heteromers and K_vβ1 subunits. This combination of K_v subunits causes rapid AP_syn broadening during brief periods of high-frequency firing. This broadening was essential for enabling synaptic facilitation without altering initial exocytosis. We also found that simply overexpressing this K_vβ1 subunit made inhibitory neurons switch from depressing during high-frequency stimulation to facilitation. Taken together, these results suggest that the molecular control of presynaptic K_v channel inactivation is an important modulator of synaptic facilitation upstream of vesicle release machinery.     

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na⁺- and Ca²⁺-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.Although homeostatic mechanisms generally maintain the brain’s extracellular pH within narrow limits, neural activity can induce transient and localized pH fluctuations. For example, acidification may occur when synaptic vesicles, which have a pH of ∼5.2–5.7 (1–3), release their contents into the synapse. Studies of mammalian cone photoreceptors showed that synaptic vesicle exocytosis rapidly reduced synaptic cleft pH by an estimated 0.2–0.6 units (4–6). Transient synaptic cleft acidification also occurred with GABAergic transmission (7). Some, but not all, studies also reported that high-frequency stimulation (HFS) transiently acidified hippocampal brain slices, likely as a result of the release of synaptic vesicle contents (8, 9). Neurotransmission also induces a slower, more prolonged alkalinization (10, 11). In addition to release of synaptic vesicle protons, neuronal and glial H⁺ and HCO₃⁻ transporters, channels, H⁺-ATPases, and metabolism might influence extracellular pH (10–12).ASICs are potential targets of reduced extracellular pH. ASICs are Na⁺-permeable and, to a lesser extent, Ca²⁺-permeable channels that are activated by extracellular acidosis (13–19). In the brain, ASICs consist of homotrimeric and heterotrimeric complexes of ASIC1a, ASIC2a, and ASIC2b. The ASIC1a subunit is required for acid-activation in the physiological range (>pH 5.0) (20, 21). Several observations indicate that ASIC are located postsynaptically. ASICs are located on dendritic spines. Although similar to glutamate receptors, they are also present on dendrites and cell bodies (20, 22–24). ASIC subunits interact with postsynaptic scaffolding proteins, including postsynaptic density protein 95 and protein interacting with C-kinase-1 (20, 24–29). In addition, ASICs are enriched in synaptosome-containing brain fractions (20, 24, 30).Although these observations raised the possibility that protons might be a neurotransmitter, postsynaptic ASIC currents have not been detected in cultured hippocampal neurons (31, 32), and whether localized pH transients might play a signaling role in neuronal communication remains unclear. In previous studies of hippocampal brain slices, extracellular field potential recordings suggested impaired hippocampal long-term potentiation (LTP) in ASIC1a^−/− mice (20), although another study did not detect an effect of ASIC1a (33). Another study using microisland cultures of hippocampal neurons suggested that the probability of neurotransmitter release increased in ASIC1a^−/− mice (32).Here, we tested the hypothesis that protons are a neurotransmitter and that ASICs are the receptor. Criteria to identify substances as neurotransmitters have been proposed (34). Beg and colleagues (35) used these criteria to conclude that protons are a transmitter released from Caenorhabditis elegans intestine to cause muscle contraction. Key questions about whether protons meet criteria for a neurotransmitter are: Does presynaptic stimulation increase the extracellular proton concentration? Do protons activate currents in postsynaptic cells? Can exogenously applied protons reproduce effects of endogenous protons? What is the postsynaptic proton receptor? We studied lateral amygdala brain slices because amygdala-dependent fear-related behavior depends on a pH reduction (36). In addition, ASICs are abundantly expressed there, and ASIC1a^−/− mice have impaired fear-like behavior (36–38).     

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.