Fast synaptic transmission is important for rapid information processing. To explore the maximal rate of neuronal signaling and to analyze the presynaptic mechanisms, we focused on the input layer of the cerebellar cortex, where exceptionally high action potential (AP) frequencies have been reported in vivo. With paired recordings between presynaptic cerebellar mossy fiber boutons and postsynaptic granule cells, we demonstrate reliable neurotransmission up to ∼1 kHz. Presynaptic APs are ultrafast, with ∼100 μs half-duration. Both Kv1 and Kv3 potassium channels mediate the fast repolarization, rapidly inactivating sodium channels ensure metabolic efficiency, and little AP broadening occurs during bursts of up to 1.5 kHz. Presynaptic Cav2.1 (P/Q-type) calcium channels open efficiently during ultrafast APs. Furthermore, a subset of synaptic vesicles is tightly coupled to Ca(2+) channels, and vesicles are rapidly recruited to the release site. These data reveal mechanisms of presynaptic AP generation and transmitter release underlying neuronal kHz signaling.

Cerebellar granule cells (GCs) make up the majority of all neurons in the vertebrate brain, but heterogeneities among GCs and potential functional consequences are poorly understood. Here, we identified unexpected gradients in the biophysical properties of GCs in mice. GCs closer to the white matter (inner-zone GCs) had higher firing thresholds and could sustain firing with larger current inputs than GCs closer to the Purkinje cell layer (outer-zone GCs). Dynamic Clamp experiments showed that inner- and outer-zone GCs preferentially respond to high- and low-frequency mossy fiber inputs, respectively, enabling dispersion of the mossy fiber input into its frequency components as performed by a Fourier transformation. Furthermore, inner-zone GCs have faster axonal conduction velocity and elicit faster synaptic potentials in Purkinje cells. Neuronal network modeling revealed that these gradients improve spike-timing precision of Purkinje cells and decrease the number of GCs required to learn spike-sequences. Thus, our study uncovers biophysical gradients in the cerebellar cortex enabling a Fourier-like transformation of mossy fiber inputs.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels control electrical rhythmicity and excitability in the heart and brain, but the function of HCN channels at the subcellular level in axons remains poorly understood. Here, we show that the action potential conduction velocity in both myelinated and unmyelinated central axons can be bidirectionally modulated by a HCN channel blocker, cyclic adenosine monophosphate (cAMP), and neuromodulators. Recordings from mouse cerebellar mossy fiber boutons show that HCN channels ensure reliable high-frequency firing and are strongly modulated by cAMP (EC50 40 µM; estimated endogenous cAMP concentration 13 µM). In addition, immunogold-electron microscopy revealed HCN2 as the dominating subunit in cerebellar mossy fibers. Computational modeling indicated that HCN2 channels control conduction velocity primarily by altering the resting membrane potential and are associated with significant metabolic costs. These results suggest that the cAMP-HCN pathway provides neuromodulators with an opportunity to finely tune energy consumption and temporal delays across axons in the brain.

18Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels control electrical 19 rhythmicity and excitability in the heart and brain, but the function of HCN channels at 20 subcellular level in axons remains poorly understood. Here, we show that the action 21 potential conduction velocity in both myelinated and unmyelinated central axons can 22 bidirectionally be modulated by HCN channel blockers, cyclic adenosine 23 monophosphate (cAMP), and neuromodulators. Recordings from mice cerebellar mossy 24 fiber boutons show that HCN channels ensure reliable high-frequency firing and are 25 strongly modulated by cAMP (EC 50 40 µM; estimated endogenous cAMP concentration 26 13 µM). In accord, immunogold-electron microscopy revealed HCN2 as the dominating 27 subunit in cerebellar mossy fibers. Computational modeling indicated that HCN2 28 channels control conduction velocity primarily via altering the resting membrane 29 potential and was associated with significant metabolic costs. These results suggest that 30 the cAMP-HCN pathway provides neuromodulators an opportunity to finely tune 31 energy consumption and temporal delays across axons in the brain. 32 33 for statistical testing). Since some studies implied that ZD7288 might have some 102 unspecific side effects, such as blocking voltage-dependent Na + channels (Chevaleyre 103 and Castillo, 2002;Wu et al., 2012), we recorded Na + currents from 53 cMFBs and 104 found no change in amplitude or kinetics of voltage-dependent Na + currents after 105 ZD7288 application (Supplemental Fig. 1), suggesting that under our conditions and at 106 a concentration of 30 µM, ZD7288 did not affect the Na + currents. Because of the 107 modulation of HCN channels by intracellular cAMP, we measured conduction velocity 108 during application of 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP; 109 500 µM), a membrane-permeable cAMP-analogue. The conduction velocity increased 110 by 5.9 ± 2.8% in cerebellar mossy fibers (n = 17), by 3.7 ± 1.4% in parallel fibers (n = 111 10), and by 4.6 ± 0.6% in optic nerves (n = 5; see Fig. 1 and legend for statistical 112 testing). These results indicate that HCN channels control the conduction velocity both 113 Schweighofer et al., 2004), which decrease the cAMP concentration. Although we used 129 rather high concentrations of the agonists and off-target effects cannot be excluded (e.g., 130 NE activating dopamine receptors; Sánchez-Soto et al., 2016), these data nevertheless 131 indicate that physiological neuromodulators can both increase or decrease action 132 potential conduction velocity, depending on the type of neuromodulator and receptor. 133

In the central nervous system, the frequency at which reliable synaptic transmission can be maintained varies strongly between different types of synapses. Several pre- and postsynaptic processes must interact to enable high-frequency synaptic transmission. One of the mechanistically most challenging issues arises during repetitive neurotransmitter release, when synaptic vesicles fuse in rapid sequence with the presynaptic plasma membrane within the active zone (AZ), potentially interfering with the structural integrity of the AZ itself. Here we summarize potential mechanisms that help to maintain AZ integrity, including arrangement and mobility of release sites, calcium channel mobility, as well as release site clearance via lateral diffusion of vesicular proteins and via endocytotic membrane retrieval. We discuss how different types of synapses use these strategies to maintain high-frequency synaptic transmission.

Neuronal integration of high-frequency signals is important for rapid information processing. Cerebellar mossy fiber axons (MFs) can fire action potentials (APs) at frequencies of more than one kilohertz. However, it is unclear whether and how the postsynaptic cerebellar granule cells (GCs) are able to process these high-frequency MF inputs. Here, we measured AP firing in GCs during high-frequency MF stimulation and show that GC firing frequency increased non-linearly when MF stimulation frequency was increased from 100 to 750 Hz. To investigate the mechanisms enabling such high-frequency signaling, we analyzed the role of N-methyl-d-aspartate receptors (NMDARs), which have been implicated in synaptic signaling at lower frequencies. Application of D-2-amino-5-phosphonopentanoic acid (APV), a potent inhibitor of NMDARs, strongly impaired the GC firing frequency during high-frequency MF stimulation. APV had no significant effect on single excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) evoked at 1 Hz at resting membrane potentials. However, the time course of EPSCs evoked at 1 Hz at depolarized potentials or following high-frequency MF stimulation was accelerated by APV. Thus, our results show that NMDAR-mediated currents amplify high-frequency MF inputs by prolonging the time courses of synaptic inputs, thereby causing greater synaptic summation of inputs. Hence, NMDARs support the integration of MF synaptic input at frequencies up to at least 750 Hz. Synapse 70:269-276, 2016. © 2016 Wiley Periodicals, Inc.

No abstract

20Cerebellar granule cells (GCs) making up majority of all the neurons in the 21 vertebrate brain, but heterogeneities among GCs and potential functional 22 consequences are poorly understood. Here, we identified unexpected gradients 23 in the biophysical properties of GCs. GCs closer to the white matter (inner-zone 24 GCs) had higher firing thresholds and could sustain firing with larger current 25 inputs. Dynamic clamp experiments showed that inner-and outer-zone GCs 26 preferentially respond to high-and low-frequency mossy fiber inputs, 27 respectively, enabling to disperse the mossy fiber input into its frequency 28 components as performed by a Fourier transformation. Furthermore, inner-zone 29GCs have faster axonal conduction velocity and elicit faster synaptic potentials in 30 Purkinje cells. Neuronal network modeling revealed that these gradients improve 31 spike-timing precision of Purkinje cells and decrease the number of GCs required 32 to learn spike-sequences. Thus, our study uncovers biophysical gradients in the 33 cerebellar cortex enabling a Fourier-like transformation of mossy fiber inputs. 34Controlling the timing and precision of movements is considered to be one of the 52 main functions of the cerebellum. In the cerebellum, the firing frequency of 53Purkinje cells (PCs) (Heiney et al., 2014;Herzfeld et al., 2015; Hewitt et al., 54 2011;Medina and Lisberger, 2007;Payne et al., 2019; Sarnaik and Raman, 55 2018;Witter et al., 2013) or the timing of spikes (Brown and Raman, 2018; 56 Sarnaik and Raman, 2018) have been shown to be closely related to movement. 57Indeed, cerebellar pathology impairs precision in motor learning tasks (Gibo et 58 al., 2013;Martin et al., 1996) and timing of rhythmic learning tasks (Keele and 59 Ivry, 1990). These functions are executed by a remarkably simple neuronal 60 network architecture. Inputs from mossy fibers (MFs) are processed by GCs and 61 transmitted via their parallel fiber (PF) axons to PCs, which provide the sole 62 output from the cerebellar cortex. GCs represent the first stage in cerebellar 63 processing and have been proposed to provide pattern separation and 64 3 conversion into a sparser representation of the MF input (recently reviewed by 65 (Cayco-Gajic and Silver, 2019). These MF inputs show a wide variety of signaling 66 frequencies, ranging from slow modulating activity to kilohertz bursts of activity 67 (Arenz et al., 2008;Rancz et al., 2007; Ritzau-Jost et al., 2014; van Kan et al., 68 1993). Interestingly, in cellular models of the cerebellum, each MF is considered 69 to be either active or inactive with little consideration for this wide range of 70 frequencies (Albus, 1971;Marr, 1969). Furthermore, in these models, GCs are 71 generally considered as a uniform population of neurons. 72 Here we show that the biophysical properties of GCs differ according to their 73 vertical position in the GC layer. GCs located close to the white matter (inner-74 zone) selectively transmit high-frequency MF inputs, have shorter action 75 poten...