Mechanistic insights into cAMP-mediated presynaptic potentiation at hippocampal mossy fiber synapses.

Presynaptic plasticity is an activity-dependent change in the neurotransmitter release and plays a key role in dynamic modulation of synaptic strength. Particularly, presynaptic potentiation mediated by cyclic adenosine monophosphate (cAMP) is widely seen across the animals and thought to contribute to learning and memory. Hippocampal mossy fiber-CA3 pyramidal cell synapses have been used as a model because of robust presynaptic potentiation in short- and long-term forms. Moreover, direct presynaptic recordings from large mossy fiber terminals allow one to dissect the potentiation mechanisms. Recently, super-resolution microscopy and flash-and-freeze electron microscopy have revealed the localizations of release site molecules and synaptic vesicles during the potentiation at a nanoscale, identifying the molecular mechanisms of the potentiation. Incorporating these growing knowledges, we try to present plausible mechanisms underlying the cAMP-mediated presynaptic potentiation.

Increased vesicle fusion competence underlies long-term potentiation at hippocampal mossy fiber synapses.

Presynaptic long-term potentiation (LTP) is thought to play an important role in learning and memory. However, the underlying mechanism remains elusive because of the difficulty of direct recording during LTP. Hippocampal mossy fiber synapses exhibit pronounced LTP of transmitter release after tetanic stimulation and have been used as a model of presynaptic LTP. Here, we induced LTP by optogenetic tools and applied direct presynaptic patch-clamp recordings. The action potential waveform and evoked presynaptic Ca2+ currents remained unchanged after LTP induction. Membrane capacitance measurements suggested higher release probability of synaptic vesicles without changing the number of release-ready vesicles after LTP induction. Synaptic vesicle replenishment was also enhanced. Furthermore, stimulated emission depletion microscopy suggested an increase in the numbers of Munc13-1 and RIM1 molecules within active zones. We propose that dynamic changes in the active zone components may be relevant for the increased fusion competence and synaptic vesicle replenishment during LTP.

Rapid Ca2+ channel accumulation contributes to cAMP-mediated increase in transmission at hippocampal mossy fiber synapses.

The cyclic adenosine monophosphate (cAMP)-dependent potentiation of neurotransmitter release is important for higher brain functions such as learning and memory. To reveal the underlying mechanisms, we applied paired pre- and postsynaptic recordings from hippocampal mossy fiber-CA3 synapses. Ca2+ uncaging experiments did not reveal changes in the intracellular Ca2+ sensitivity for transmitter release by cAMP, but suggested an increase in the local Ca2+ concentration at the release site, which was much lower than that of other synapses before potentiation. Total internal reflection fluorescence (TIRF) microscopy indicated a clear increase in the local Ca2+ concentration at the release site within 5 to 10 min, suggesting that the increase in local Ca2+ is explained by the simple mechanism of rapid Ca2+ channel accumulation. Consistently, two-dimensional time-gated stimulated emission depletion microscopy (gSTED) microscopy showed an increase in the P/Q-type Ca2+ channel cluster size near the release sites. Taken together, this study suggests a potential mechanism for the cAMP-dependent increase in transmission at hippocampal mossy fiber-CA3 synapses, namely an accumulation of active zone Ca2+ channels.

Structural and Functional Refinement of the Axon Initial Segment in Avian Cochlear Nucleus during Development.

The axon initial segment (AIS) is involved in action potential initiation. Structural and biophysical characteristics of the AIS differ among cell types and/or brain regions, but the underlying mechanisms remain elusive. Using immunofluorescence and electrophysiological methods, combined with super-resolution imaging, we show in the developing nucleus magnocellularis of the chicken in both sexes that the AIS is refined in a tonotopic region-dependent manner. This process of AIS refinement differs among cells tuned to different frequencies. At hearing onset, the AIS was ∼50 µm long with few voltage-gated sodium channels regardless of tonotopic region. However, after hatching, the AIS matured and displayed an ∼20-µm-long structure with a significant enrichment of sodium channels responsible for an increase in sodium current and a decrease in spike threshold. Moreover, the shortening was more pronounced, while the accumulation of channels was not, in neurons tuned to higher frequency, creating tonotopic differences in the AIS. We conclude that AIS shortening is mediated by disassembly of the cytoskeleton at the distal end of the AIS, despite intact periodicity of the submembranous cytoskeleton across the AIS. Importantly, deprivation of afferent input diminished the shortening in neurons tuned to a higher frequency to a larger extent in posthatch animals, with little effect on the accumulation of sodium channels. Thus, cytoskeletal reorganization and sodium channel enrichment at the AIS are differentially regulated depending on tonotopic region, but work synergistically to optimize neuronal output in the auditory nucleus.SIGNIFICANCE STATEMENT The axon initial segment (AIS) plays fundamental roles in determining neuronal output. The AIS varies structurally and molecularly across tonotopic regions in avian cochlear nucleus. However, the mechanism underlying these variations remains unclear. The AIS is immature around hearing onset, but becomes shorter and accumulates more sodium channels during maturation, with a pronounced shortening and a moderate channel accumulation at higher tonotopic regions. Afferent input adjusts sodium conductance at the AIS by augmenting AIS shortening (via disassembly of cytoskeletons at its distal end) specifically at higher-frequency regions. However, this had little effect on channel accumulation. Thus, cytoskeletal structure and sodium channel accumulation at the AIS are regulated differentially but work synergistically to optimize the neuronal output.

Auditory Input Shapes Tonotopic Differentiation of Kv1.1 Expression in Avian Cochlear Nucleus during Late Development.

Tonotopic differentiation is fundamental for signal processing in the auditory system. However, when and how this differentiation arises remain elusive. We addressed this issue using electrophysiology and immunohistochemistry in nucleus magnocellularis of chickens of both sexes, which is known to differ in the expression of Kv1.1 channels depending on characteristic frequency (CF). Just after hearing onset (embryonic day 12-14), Kv1 current gradually increased to a slightly larger extent in neurons with higher CF, causing a tonotopic difference of Kv1 current before hatch. However, after hatch, a much larger increase of Kv1 current occurred, particularly in higher-CF neurons, due to an augmentation of Kv1.1 expression at the plasma membrane. This later change in expression led to the large tonotopic difference of Kv1 current characteristic of mature animals. Attenuation of auditory input by inducing conductive or sensorineural hearing loss around hatch suppressed the differentiation in a level-dependent manner. Moreover, elevation of auditory input during embryonic periods could not reproduce the differentiation, suggesting that the capacity of neurons to drive Kv1.1 expression via auditory input develops in a cell-specific manner, thus underlying the frequency-specific expression of the channel within the nucleus. The results indicated that the tonotopic differentiation of Kv1.1 in nucleus magnocellularis is partially determined before hatch, but largely driven by afferent input after hatch. Our results highlight the importance of neuronal capacity for sound to drive ion channel expression as well as the level of auditory experience in the frequency tuning of brainstem auditory circuits.SIGNIFICANCE STATEMENT Tuning-frequency-specific expression of ion channels is a prerequisite for auditory system function, but its underlying mechanisms remain unclear. Here, we revealed in avian cochlear nucleus that the expression of Kv1.1 became more dependent on auditory input at a late period of maturation in neurons tuned to higher-frequency sound, leading to frequency-specific Kv1.1 expression. Attenuation of auditory input during this period suppressed the differentiation in a level-dependent manner, whereas elevation of input in earlier periods could not reproduce the differentiation. Thus, the capacity of neurons to drive Kv1.1 expression via auditory input develops in a cell-specific manner and directs differentiation, highlighting the importance of neuronal character as well as the level of input in the frequency tuning of auditory circuits.

Tonotopic Variation of the T-Type Ca2+ Current in Avian Auditory Coincidence Detector Neurons.

Neurons in avian nucleus laminaris (NL) are binaural coincidence detectors for sound localization and are characterized by striking structural variations in dendrites and axon initial segment (AIS) according to their acoustic tuning [characteristic frequency (CF)]. T-type Ca2+ (CaT) channels regulate synaptic integration and firing behavior at these neuronal structures. However, whether or how CaT channels contribute to the signal processing in NL neurons is not known. In this study, we addressed this issue with whole-cell recording and two-photon Ca2+ imaging in brain slices of posthatch chicks of both sexes. We found that the CaT current was prominent in low-CF neurons, whereas it was almost absent in higher-CF neurons. In addition, a large Ca2+ transient occurred at the dendrites and the AIS of low-CF neurons, indicating a localization of CaT channels at these structures in the neurons. Because low-CF neurons have long dendrites, dendritic CaT channels may compensate for the attenuation of EPSPs at dendrites. Furthermore, the short distance of AIS from the soma may accelerate activation of axonal CaT current in the neurons and help EPSPs reach spike threshold. Indeed, the CaT current was activated by EPSPs and augmented the synaptic response and spike generation of the neurons. Notably, the CaT current was inactivated during repetitive inputs, and these augmenting effects predominated at the initial phase of synaptic activity. These results suggested that dendritic and axonal CaT channels increase the sensitivity to sound at its onset, which may expand the dynamic range for binaural computation in low-CF NL neurons.SIGNIFICANCE STATEMENT Neurons in nucleus laminaris are binaural coincidence detectors for sound localization. We report that T-type Ca2+ (CaT) current was prominent at dendrites and the axonal trigger zone in neurons tuned to low-frequency sound. Because these neurons have long dendrites and a closer trigger zone compared with those tuned to higher-frequency sound, the CaT current augmented EPSPs at dendrites and accelerated spike triggers in the neurons, implying a strategic arrangement of the current within the nucleus. This effect was limited to the onset of repetitive inputs due to progressive inactivation of CaT current. The results suggested that the CaT current increases the sensitivity to sound at its onset, which may expand the dynamic range for binaural computation of low-frequency sound.