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Ca(2+)-activated chloride channels (CaCCs) regulate numerous physiological processes including epithelial transport, smooth muscle contraction and sensory processing. Anoctamin-1 (ANO1, TMEM16A) is a principal CaCC subunit in many cell types, yet our understanding of the mechanisms of ANO1 activation and regulation are only beginning to emerge. Ca(2+) sensitivity of ANO1 is rather low and at negative membrane potentials the channel requires several micromoles of intracellular Ca(2+) for activation. However, global Ca(2+) levels in cells rarely reach such levels and, therefore, there must be mechanisms that focus intracellular Ca(2+) transients towards the ANO1 channels. Recent findings indeed indicate that ANO1 channels often co-localize with sources of intracellular Ca(2+) signals. Interestingly, it appears that in many cell types ANO1 is particularly tightly coupled to the Ca(2+) release sites of the intracellular Ca(2+) stores. Such preferential coupling may represent a general mechanism of ANO1 activation in native tissues.

Many excitatory synapses in the brain release glutamate with both synchronous and asynchronous components. Immediately following an action potential, neurons display a reduced excitability due to the post-spike afterhyperpolarization (AHP). This gives rise to a relative refractory period. When an action potential is evoked by glutamate synaptic input possessing asynchronous release, the delayed glutamate release events act to depolarize the neuron during the AHP and overcome the relative refractory period. These results demonstrate a new role for asynchronous release in regulating post-spike excitability and the relative refractory period in central neurons.Post-spike afterhyperpolarizations (AHPs) functionally inhibit neuronal excitability for tens to hundreds of milliseconds following each action potential. This imposes a relative refractory period during which synaptic excitation is less effective at evoking spikes. Here we asked whether some synapses have mechanisms in place that allow them to overcome the AHP and drive spiking in target cells during this period of reduced excitability. We examined glutamate synapses onto oxytocin and vasopressin neurons in the paraventricular nucleus of the hypothalamus. These synapses can display pronounced asynchronous glutamate release following a single presynaptic spike, with the time course of release being similar to that of the post-spike AHP. To test whether asynchronous release is more effective at overcoming the relative refractory period, we evoked a single action potential with either a brief synchronous depolarization or an asynchronous potential and then assessed excitability at multiple time points following the spike. Neurons receiving asynchronous depolarizing synaptic inputs had a shorter relative refractory period than those receiving synchronous depolarizations. Our data demonstrate that synapses releasing glutamate in an asynchronous and delayed manner are ideally adapted to counter the AHP. By effectively overcoming the relative refractory period, the kinetics of excitatory synaptic input can play an important role in controlling post-spike excitability.

In neurons, the Ca(2+) signal associated with the dendritic back-propagating action potential codes a chemical message to the different dendritic sites, playing a crucial role in electrical signalling, synaptic transmission and synaptic plasticity. The study of the underlying Ca(2+) current, mediated by different types of voltage-gated Ca(2+) channels, cannot be achieved by using the patch clamp technique. In this article, we used a recently developed cutting-edge optical technique to investigate the physiological behaviour of local Ca(2+) currents along the apical dendrite of CA1 hippocampal pyramidal neurons. We directly measure, for the first time, the synergistic activation and deactivation of the diverse dendritic voltage-gated Ca(2+) channels operating during bursts of back-propagating action potentials to precisely control the Ca(2+) signal. We demonstrate that the Ca(2+) loss via high-voltage-activated channels is compensated by the Ca(2+) entry via the other channels translating in high fidelity of Ca(2+) signalling.In CA1 hippocampal pyramidal neurons, the dendritic Ca(2+) signal associated with somatic firing represents a fundamental activation code for several proteins. This signal, mediated by voltage-gated Ca(2+) channels (VGCCs), varies along the dendrites. In this study, using a recent optical technique based on the low-affinity indicator Oregon Green 488 BAPTA-5N, we analysed how activation and deactivation of VGCCs produced by back-propagating action potentials (bAPs) along the apical dendrite shape the Ca(2+) signal at different locations in CA1 hippocampal pyramidal neurons of the mouse. We measured, at multiple dendritic sites, the Ca(2+) transients and the changes in membrane potential associated with bAPs at 50 μs temporal resolution and we estimated the kinetics of the Ca(2+) current. We found that during somatic bursts, the bAPs decrease in amplitude along the apical dendrite but the amplitude of the associated Ca(2+) signal in the initial 200 μm dendritic segment does not change. Using a detailed pharmacological analysis, we demonstrate that this effect is due to the perfect compensation of the loss of Ca(2+) via high-voltage-activated (HVA) VGCCs by a larger Ca(2+) component via low-voltage-activated (LVA) VGCCs, revealing a mechanism coupling the two VGCC families of K(+) channels. More distally, where the bAP does not activate HVA-VGCCs, the Ca(2+) signal is variable during the burst. Thus, we demonstrate that HVA- and LVA-VGCCs operate synergistically to stabilise Ca(2+) signals associated with bAPs in the most proximal 200 μm dendritic segment.

Cerebellar Purkinje cells project GABAergic inhibitory input to neurons of the deep cerebellar nuclei (DCN) that generate a rebound increase in firing, but the specific patterns of input that might elicit a rebound response have not been established. We used recordings of Purkinje cell firing obtained during perioral whisker stimulation in vivo to create a physiological stimulus template to activate Purkinje cell afferents in vitro. DCN cell bursts were evoked by the stimulus pattern but not in relation to the perioral whisker stimulus, complex spikes or regular patterns within the Purkinje cell record. Reverse correlation revealed that bursts were triggered by an elevation-pause pattern of Purkinje cell firing, with pause duration a key factor in burst generation. Our data identify for the first time a physiological pattern of Purkinje cell input that can be encoded by the generation of rebound bursts in DCN cells.The end result of signal processing in cerebellar cortex is encoded in the output of Purkinje cells that project inhibitory input to deep cerebellar nuclear (DCN) neurons. DCN cells can respond to a period of inhibition in vitro with a rebound burst of firing, yet the optimal physiological pattern of Purkinje cell input that might evoke a rebound burst is unknown. The current study used spike trains recorded from rat Purkinje cells in response to perioral stimuli in vivo to create a physiological pattern to stimulate Purkinje cell axons in vitro. The perioral stimulus-evoked Purkinje cell firing pattern proved to be virtually ineffective in evoking a rebound burst despite the ability to reliably evoke rebounds using a traditional brief 100 Hz stimulus. Similarly, neither complex spike firing nor Purkinje cell patterns identified by CV2 analysis were reliably associated with rebound bursts. Reverse correlation revealed that the optimal Purkinje cell input to evoke a rebound burst was a sequential increase in mean firing rate of at least 30 Hz above baseline over 250 ms followed by a reduction of 40-60 Hz below baseline for up to 500 ms. The most important factor was the duration of a pause in Purkinje cell firing that allowed DCN cells to recover from a state of net inhibitory influence. These data indicate that physiological patterns of Purkinje cell firing can elicit rebound bursts in DCN cells in vitro, with pauses in Purkinje cell firing rate acting as a key stimulus for DCN cell rebound responses.

In the dorsal raphe nucleus, it is known that serotonin release activates metabotropic 5-HT1A autoreceptors located on serotonin neurons that leads to an inhibition of firing through the activation of G-protein-coupled inwardly rectifying potassium channels. We found that in mouse brain slices evoked serotonin release produced a 5-HT1A receptor-mediated inhibitory postsynaptic current (IPSC) that resulted in only a transient pause in firing. While spillover activation of receptors contributed to evoked IPSCs, serotonin reuptake transporters prevented pooling of serotonin in the extrasynaptic space from activating 5-HT1A -IPSCs. As a result, the decay of 5-HT1A -IPSCs was independent of the intensity of stimulation or the probability of transmitter release. These results indicate that evoked serotonin transmission in the dorsal raphe nucleus mediated by metabotropic 5-HT1A autoreceptors may occur via point-to-point synapses rather than by paracrine mechanisms.In the dorsal raphe nucleus (DRN), feedback activation by Gαi/o -coupled 5-HT1A autoreceptors reduces the excitability of serotoninergic neurons, which decreases serotonin release both locally within the DRN and in projection regions. Serotonin transmission within the DRN is thought to occur via transmitter spillover and paracrine activation of extrasynaptic receptors. Here, we tested the volume transmission hypothesis in mouse DRN brain slices by recording 5-HT1A receptor-mediated inhibitory postsynaptic currents (5-HT1A -IPSCs) generated by the activation of G-protein-coupled inwardly rectifying potassium channels (GIRKs). We found that in the DRN of ePET1-EYFP mice, which selectively express enhanced yellow fluorescent protein in serontonergic neurons, the local release of serotonin generated 5-HT1A -IPSCs in serotonin neurons that rose and fell within a second. The transient activation of 5-HT1A autoreceptors resulted in brief pauses in neuron firing that did not alter the overall firing rate. The duration of 5-HT1A -IPSCs was primarily shaped by receptor deactivation due to clearance via serotonin reuptake transporters. Slowing diffusion with dextran prolonged the rise and reduced the amplitude the IPSCs and the effects were potentiated when uptake was inhibited. By examining the decay kinetics of IPSCs, we found that while spillover may allow for the activation of extrasynaptic receptors, efficient uptake by serotonin reuptake transporters (SERTs) prevented the pooling of serotonin from prolonging the duration of transmission when multiple inputs were active. Together the results suggest that the activation of 5-HT1A receptors in the DRN results from the local release of serotonin rather than the extended diffusion throughout the extracellular space.

At the parallel fibre-Purkinje cell glutamatergic synapse, little or no Ca(2+) entry takes place through postsynaptic neurotransmitter receptors, although postsynaptic calcium increases are clearly involved in the synaptic plasticity. Postsynaptic voltage-gated Ca(2+) channels therefore constitute the sole rapid postsynaptic Ca(2+) signalling mechanism, making it essential to understand how they contribute to the synaptic signalling. Using a selective T-type calcium channel antagonist, we describe a T-type component of the EPSC that is activated by the AMPA receptor-mediated depolarization of the spine and thus will contribute to the local calcium dynamics. This component can amount up to 20% of the EPSC, and this fraction is maintained even at the high frequencies sometimes encountered in sensory processing. Modelling based on our biophysical characterization of T-type calcium channels in Purkinje cells suggests that the brief spine EPSCs cause the activated T-type channels to deactivate rather than inactivate, enabling repetitive activation.In the cerebellum, sensory information is conveyed to Purkinje cells (PC) via the granule cell/parallel fibre (PF) pathway. Plasticity at the PF-PC synapse is considered to be a mechanism of information storage in motor learning. The induction of synaptic plasticity in the cerebellum and elsewhere usually involves intracellular Ca(2+) signals. Unusually, postsynaptic Ca(2+) signalling in PF-PC spines does not involve ionotropic glutamatergic receptors because postsynaptic NMDA receptors are absent and the AMPA receptors are Ca(2+) -impermeable; postsynaptic voltage-gated Ca(2+) channels therefore constitute the sole rapid Ca(2+) signalling mechanism. Low-threshold activated T-type calcium channels are present at the synapse, although their contribution to PF-PC synaptic responses is unknown. Taking advantage of 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide, a selective T-type channel antagonist, we show in the mouse that inhibition of these channels reduces PF-PC excitatory postsynaptic currents and excitatory postsynaptic potentials by 15-20%. This contribution was preserved during sparse input and repetitive activity. We characterized the biophysical properties of native T-type channels in young animals and modelled their activation during simulated dendritic excitatory postsynaptic potential waveforms. The comparison of modelled and observed synaptic responses suggests that T-type channels only activate in spines that are strongly depolarized by their synaptic input, a process requiring a high spine neck resistance. This brief and local activation ensures that T-type channels rapidly deactivate, thereby limiting inactivation during repetitive synaptic activity. T-type channels are therefore ideally situated to provide synaptic Ca(2+) entry at PF-PC spines.

How the brain extracellular fluid influences the activity of GABAergic interneurons in vivo is not known. This issue is examined in the hippocampal brain slice by comparing GABAergic interneuron activity in human versus artificial cerebrospinal fluid. Human cerebrospinal fluid (hCSF) substantially increases the excitability of fast-spiking and non-fast-spiking CA1 interneurons. CA1 pyramidal cells are even more strongly excited by hCSF. The tonic excitation of pyramidal cells, in combination with an increased responsiveness of interneurons to excitatory input, is likely to promote the generation of synchronized network activity in the hippocampus.GABAergic interneurons intricately regulate the activity of hippocampal and neocortical networks. Their function in vivo is likely to be tuned by neuromodulatory substances in the brain extracellular fluid. However, in vitro investigations of GABAergic interneuron function do not account for such effects, as neurons are kept in artificial extracellular fluid. To examine the neuromodulatory influence of brain extracellular fluid on GABAergic activity, we recorded from fast-spiking and non-fast-spiking CA1 interneurons, as well as from pyramidal cells, in the presence of human cerebrospinal fluid (hCSF), using a matched artificial cerebrospinal fluid (aCSF) as control. We found that hCSF increased the frequency of spontaneous firing more than twofold in the two groups of interneurons, and more than fourfold in CA1 pyramidal cells. hCSF did not affect the resting membrane potential of CA1 interneurons but caused depolarization in pyramidal cells. The increased excitability of interneurons and pyramidal cells was accompanied by reductions in after-hyperpolarization amplitudes and a left-shift in the frequency-current relationships. Our results suggest that ambient concentrations of neuromodulators in the brain extracellular fluid powerfully influence the excitability of neuronal networks.

Using whole-cell clamp methods, we characterized the temporal coding in each type of OFF bipolar cell. We found that type 2 and 3a cells are transient, type 1 and 4 cells are sustained, and type 3b cells are intermediate. The light-evoked excitatory postsynaptic potentials in some types were rectified, suggesting that they provide inputs to the non-linear ganglion cells. Visual signalling from the photoreceptors was mediated exclusively through the kainate receptors in the transient OFF bipolar cells, whereas both kainate and AMPA receptors contributed in the other cells. This study demonstrates, for the first time, that parallel visual encoding starts at the OFF bipolar cells in a type-specific manner.The retina is the entrance to the visual system, which receives various kinds of image signals and forms multiple encoding pathways. The second-order retinal neurons, the bipolar cells, are thought to initiate multiple neural streams by encoding various visual signals in different types of cells. However, the functions of each bipolar cell type have not been fully understood. We investigated whether OFF bipolar cells encode visual signals in a type-dependent manner. We recorded the changes in the bipolar cell voltage in response to two input functions: step and sinusoidal light stimuli. Type 1 and 4 OFF bipolar cells were sustained cells and responded to sinusoidal stimuli over a broad range of frequencies. Type 2 and 3a cells were transient and exhibited band-pass filtering. Type 3b cells were in the middle of these two groups. The distinct temporal responses might be attributed to different types of glutamate receptors. We examined the AMPA and kainate glutamate receptor composition in each bipolar cell type. The light responses in the transient OFF bipolar cells were exclusively mediated by kainate receptors. Although the kainate receptors mediated the light responses in the sustained cells, the AMPA receptors also mediated a portion of the responses in sustained cells. Furthermore, we found that some types of cells were rectified more than other types. Taken together, we found that the OFF bipolar cells encode diverse temporal image signals in a type-dependent manner, confirming that each type of OFF bipolar cell initiates diverse temporal visual processing in parallel.

The zebrafish pinball wizard (pwi) mutant is deaf and blind. The pwi phenotype includes a reduced auditory startle response and reduced visual evoked potentials, suggesting fatigue of synaptic release at ribbon synapses in hair cells and photoreceptors. The gene defective in the pwi mutant is WRB, a protein homologous to the yeast protein Get1, which is involved in the insertion of 'tail-anchored' membrane proteins. Many tail-anchored proteins are associated with synaptic vesicles, and both vesicles and synaptic ribbons are reduced in synaptic regions of hair cells in pwi. Abnormal processing of synaptic vesicle proteins important for ribbon synapses can explain the pwi phenotype.In a large-scale zebrafish insertional mutagenesis screen, we identified the pinball wizard (pwi) line, which displays a deafness and blindness phenotype. Although the gross morphology and structure of the pwi larval inner ear was near normal, acoustic startle stimuli evoked smaller postsynaptic responses in afferent neurons, which rapidly fatigued. In the retina, similarly, an abnormal electroretinogram suggested reduced transmission at the photoreceptor ribbon synapse. A functional deficit in these specialized synapses was further supported by a reduction of synaptic marker proteins Rab3 and cysteine-string protein (CSP/Dnajc5) in hair cells and photoreceptors, as well as by a reduction of the number of both ribbons and vesicles surrounding the ribbons in hair cells. The pwi gene encodes a homologue of the yeast Get1 and human tryptophan-rich basic (WRB) proteins, which are receptors for membrane insertion of tail-anchored (TA) proteins. We identified more than 100 TA proteins expressed in hair cells, including many synaptic proteins. The expression of synaptobrevin and syntaxin 3, TA proteins essential for vesicle fusion, was reduced in the synaptic layers of mutant retina, consistent with a role for the pwi/WRB protein in TA-protein processing. The WRB protein was located near the apical domain and the ribbons in hair cells, and in the inner segment and the axon of the photoreceptor, consistent with a role in vesicle biogenesis or trafficking. Taken together, our results suggest that WRB plays a critical role in synaptic functions in these two sensory cells, and that disrupted processing of synaptic vesicle TA proteins explains much of the mutant phenotype.