For sounds of a given frequency, spiral ganglion neurons (SGNs) with different thresholds and dynamic ranges collectively encode the wide range of audible sound pressures. Heterogeneity of synapses between inner hair cells (IHCs) and SGNs is an attractive candidate mechanism for generating complementary neural codes covering the entire dynamic range. Here, we quantified active zone (AZ) properties as a function of AZ position within mouse IHCs by combining patch clamp and imaging of presynaptic Ca 2+ influx and by immunohistochemistry. We report substantial AZ heterogeneity whereby the voltage of half-maximal activation of Ca 2+ influx ranged over ∼20 mV. Ca 2+ influx at AZs facing away from the ganglion activated at weaker depolarizations. Estimates of AZ size and Ca 2+ channel number were correlated and larger when AZs faced the ganglion. Disruption of the deafness gene GIPC3 in mice shifted the activation of presynaptic Ca 2+ influx to more hyperpolarized potentials and increased the spontaneous SGN discharge. Moreover, Gipc3 disruption enhanced Ca 2+ influx and exocytosis in IHCs, reversed the spatial gradient of maximal Ca 2+ influx in IHCs, and increased the maximal firing rate of SGNs at sound onset. We propose that IHCs diversify Ca 2+ channel properties among AZs and thereby contribute to decomposing auditory information into complementary representations in SGNs.auditory system | spiral ganglion neuron | dynamic range | synaptic strength | presynaptic heterogeneity T he auditory system enables us to perceive sound pressures that vary over six orders of magnitude. This is achieved by active amplification of cochlear vibrations at low sound pressures and compression at high sound pressures. The receptor potential of inner hair cells (IHCs) represents the full range (1), whereas each postsynaptic type I spiral ganglion neuron (hereafter termed SGN) encodes only a fraction (2-6). SGNs with comparable frequency tuning but different spontaneous spike rates and sound responses are thought to emanate from neighboring, if not the same, IHC at a given tonotopic position of the organ of Corti (2,5,7,8). Even in silence, IHC active zones (AZs) release glutamate at varying rates, evoking "spontaneous" spiking in SGNs. SGNs with greater spontaneous spike rates respond to softer sounds (highspontaneous rate, low-threshold SGNs), than those with lower spontaneous spike rates (low-spontaneous rate, high-threshold SGNs) (2, 9). This diversity likely underlies the representation of sounds across all audible sound pressure levels in the auditory nerve, to which neural adaptation also contributes (10).How SGN diversity arises is poorly understood. Candidate mechanisms include the heterogeneity of ribbon synapses that differ in pre-and/or postsynaptic properties even within individual IHCs (7,(11)(12)(13)(14). IHC AZs vary in the number (11, 15) and voltage dependence of gating (11) of their Ca 2+ channels regardless of tonotopic position (16). Lateral olivocochlear efferent projections to the SGNs regulate postsynaptic exc...

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes.

Extinction-based exposure therapy is used to treat anxiety- and trauma-related disorders; however, there is the need to improve its limited efficacy in individuals with impaired fear extinction learning and to promote greater protection against return-of-fear phenomena. Here, using 129S1/SvImJ mice, which display impaired fear extinction acquisition and extinction consolidation, we revealed that persistent and context-independent rescue of deficient fear extinction in these mice was associated with enhanced expression of dopamine-related genes, such as dopamine D1 (Drd1a) and -D2 (Drd2) receptor genes in the medial prefrontal cortex (mPFC) and amygdala, but not hippocampus. Moreover, enhanced histone acetylation was observed in the promoter of the extinction-regulated Drd2 gene in the mPFC, revealing a potential gene-regulatory mechanism. Although enhancing histone acetylation, via administering the histone deacetylase (HDAC) inhibitor MS-275, does not induce fear reduction during extinction training, it promoted enduring and context-independent rescue of deficient fear extinction consolidation/retrieval once extinction learning was initiated as shown following a mild conditioning protocol. This was associated with enhanced histone acetylation in neurons of the mPFC and amygdala. Finally, as a proof-of-principle, mimicking enhanced dopaminergic signaling by L-dopa treatment rescued deficient fear extinction and co-administration of MS-275 rendered this effect enduring and context-independent. In summary, current data reveal that combining dopaminergic and epigenetic mechanisms is a promising strategy to improve exposure-based behavior therapy in extinction-impaired individuals by initiating the formation of an enduring and context-independent fear-inhibitory memory.

Cav1.3 L-type Ca2+-channel function is regulated by a C-terminal automodulatory domain (CTM). It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca2+- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca2+-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRDHA/HA). Using these mice we provide biochemical evidence for the existence of long (CTM-containing) and short (CTM-deficient) Cav1.3 α1-subunits in brain. The long (HA-labeled) Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca2+-dependent inactivation of Ca2+-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It stabilizes gating properties of Cav1.3 channels required for normal electrical excitability.

A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Cav1.3 L-type Ca2+ channels, in particular the voltage dependence of activation (V0.5) and Ca2+ dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Cav1.3 (Cav1.3L), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCav1.3scg). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCav1.3scg with a rat cDNA isolated from rat pancreas (rCav1.3L). When expressed in tsA-201 cells under identical experimental conditions rCav1.3L exhibited Ca2+ current properties indistinguishable from human and mouse Cav1.3L, compatible with the presence of a functional CTM. In contrast, rCav1.3scg showed gating properties similar to human short splice variants lacking a CTM. rCav1.3scg differs from rCav1.3L at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-terminal polymethionine stretch with two additional lysines. Two aa (S244, A2075) in rCav1.3scg explained most of the functional differences to rCav1.3L. Their mutation to the corresponding residues in rCav1.3L (G244, V2075) revealed that both contributed to the more negative V0.5, but caused opposite effects on CDI. A2075 (located within a region forming the CTM) additionally permitted higher channel open probability. The cooperative action in the double-mutant restored gating properties similar to rCav1.3L. We found no evidence for transcripts containing one of the single rCav1.3scg mutations in rat superior cervical ganglion preparations. However, the rCav1.3scg variant provided interesting insight into the structural machinery involved in Cav1.3 gating.