Previous work has established that stellate cells of the medial entorhinal cortex produce prominent intrinsic subthreshold oscillations in the voltage response concentrated within the theta range (3-7 Hz). It has been speculated that these oscillations play an important role in vivo in establishing network behavior both in the entorhinal cortex and hippocampus. Consequently, it is important to investigate under what conditions theta oscillations in stellate cells can be generated and whether the spike-train power spectral density (PSD) also carries power at theta. We investigated the ability of stellate cells to generate theta oscillations in the presence of generic in vivo-like patterns of stimulation. Inputs were Poisson process-driven excitatory and inhibitory synaptic conductances or currents, introduced via dynamic clamp. We analyzed the subthreshold membrane oscillations and spike-train behavior in the presence of comparable synaptic conductance-or current-mediated membrane fluctuations. In the presence of conductance-based synapses, subthreshold oscillations are highly attenuated or entirely eliminated. Conversely, with current-based synapses stellate cells retain their ability to generate subthreshold oscillations in the theta band. These results also extend into the spiking regime, where only under current-based synapses does the PSD of the spike train show a prominent peak at theta. Furthermore, the peak in the spike-train PSD and spike clustering results from an increased probability of firing after a spike afterhyperpolarization and not directly from subthreshold oscillatory dynamics as has been previously suggested. Our results suggest that subthreshold oscillations may contribute less to in vivo response properties than has been hypothesized.

SUMMARY New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments.

The modification of first-spike latencies by low-threshold and inactivating K ϩ currents (I A ) have important implications in neuronal coding and synaptic integration. To date, cells in which first-spike latency characteristics have been analyzed have shown that increased hyperpolarization results in longer first-spike latencies, producing a monotonic relationship between first-spike latency and membrane voltage. Previous work has established that cerebellar stellate cells express members of the K v 4 potassium channel subfamily, which underlie I A in many central neurons. Spike timing in stellate cells could be particularly important to cerebellar output, because the discharge of even single spikes can significantly delay spike discharge in postsynaptic Purkinje cells. In the present work, we studied the first-spike latency characteristics of stellate cells. We show that first-spike latency is nonmonotonic, such that intermediate levels of prehyperpolarization produce the longest spike latencies, whereas greater hyperpolarization or depolarization reduces spike latency. Moreover, the range of first-spike latency values can be substantial in spanning 20 -128 ms with preceding membrane shifts of Ͻ10 mV. Using patch clamp and modeling, we illustrate that spike latency characteristics are the product of an interplay between I A and lowthreshold calcium current (I T ) that requires a steady-state difference in the inactivation parameters of the currents. Furthermore, we show that the unique first-spike latency characteristics of stellate cells have important implications for the integration of coincident IPSPs and EPSPs, such that inhibition can shift first-spike latency to differentially modulate the probability of firing.

Plasma membrane Na؉ /Ca 2؉ -exchangers play a predominant role in Ca 2؉ extrusion in brain. Neurons express several different Na ؉ /Ca 2؉ -exchangers belonging to both the K ؉ -independent NCX family and the K ؉ -dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca 2؉ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K ؉ -dependent Na ؉ /Ca 2؉ -exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca 2؉ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca 2؉ ] that is essential for the development of synaptic plasticity associated with learning and memory.

During a wide variety of behaviors, hippocampal field potentials show significant power in the theta (4–12 Hz) frequency range and individual neurons commonly phase-lock with the 4–12 Hz field potential. The underlying cellular and network mechanisms that generate the theta rhythm, however, are poorly understood. Oriens-lacunosum moleculare (O-LM) interneurons have been implicated as crucial contributors to generating theta in local hippocampal circuits because of their unique axonal projections, slow synaptic kinetics and the fact that spikes are phase locked to theta field potentials in vivo. We performed experiments in brain slice preparations from Long-Evans rats to investigate the ability of O-LM cells to generate phase-locked spike output in response to artificial synaptic inputs. We find that O-LM cells do not respond with any preference in spike output at theta frequencies when injected with broadband artificial synaptic inputs. However, when presented with frequency-modulated inputs, O-LM spike output shows the ability to phase-lock well to theta-modulated inputs, despite their strong low-pass profiles of subthreshold membrane impedance. This result was dependent on spike refractory dynamics and could be controlled by real-time manipulation of the post-spike afterhyperpolarization. Finally, we show that the ability of O-LM cells to phase-lock well to theta-rich inputs is independent of the h-current, a membrane mechanism often implicated in the generation of theta frequency activity.

Knowledge of intrinsic neuronal firing dynamics is a critical first step to establishing an accurate biophysical model of any neuron. In this study we examined cerebellar Purkinje cells to determine the bifurcations likely to underlie firing dynamics within a biophysically realistic and experimentally supported model. We show that Purkinje cell dynamics are consistent with a system undergoing a saddle-node bifurcation of fixed points in the transition from rest to firing and a saddle homoclinic bifurcation from firing to rest. Our analyses account for numerous observed Purkinje cell firing properties that include bistability, plateau potentials, specific aspects of the frequency-current (F-I) relationship, first spike latency, and the ability for climbing fiber input to induce state transitions in the bistable regime. We also experimentally confirm new properties predicted from our model and analysis that include the presence of a depolarizing afterpotential (DAP), the ability to fire at low frequencies (<50 Hz) and with a high gain in the F-I relationship, and a bistable region limited to low-frequency firing. Purkinje cell dynamics, including bistability, prove to arise from numerous biophysical factors that include the DAP, fast refractory dynamics, and a long membrane time constant. A hyperpolarizing activated cation current (I(H)) is shown not to be directly involved in establishing bistable dynamics but rather reduces the range for bistability. A combined electrophysiological and modeling approach thus accounts for several properties of Purkinje cells, providing a firm basis from which to assess Purkinje cell output patterns.

Gain modulation is an important feature of neural activity. Previous work has focused on the ability of background synaptic noise to modulate the slope (i.e., gain) of the frequency-current ( f-I) relationship in neurons. To date, demonstrations of gain control that are independent of synaptic noise have been limited. We investigated the effects of increasing somatic conductance in the form of tonic inhibition on the initial and steady-state f-I relationship of CA1 pyramidal cells. We find that increasing membrane conductance reduces the gain of the steady-state f-I relationship through a graded increase in the magnitude of spike frequency adaptation. Increased adaptation arises through a conductance-induced depolarization of spike voltage threshold. Thus, by increasing the magnitude of spike frequency adaptation, added conductance can reduce the gain of the steady-state f-I relationship in the absence of random background membrane fluctuations.

Neuromodulation at high spatial resolution poses great significance in advancing fundamental knowledge in the field of neuroscience and offering novel clinical treatments. Here, we developed a tapered fiber optoacoustic emitter (TFOE) generating an ultrasound field with a high spatial precision of 39.6 µm, enabling optoacoustic activation of single neurons or subcellular structures, such as axons and dendrites. Temporally, a single acoustic pulse of sub-microsecond converted by the TFOE from a single laser pulse of 3 ns is shown as the shortest acoustic stimuli so far for successful neuron activation. The precise ultrasound generated by the TFOE enabled the integration of the optoacoustic stimulation with highly stable patch-clamp recording on single neurons. Direct measurements of the electrical response of single neurons to acoustic stimulation, which is difficult for conventional ultrasound stimulation, have been demonstrated. By coupling TFOE with ex vivo brain slice electrophysiology, we unveil cell-type-specific responses of excitatory and inhibitory neurons to acoustic stimulation. These results demonstrate that TFOE is a non-genetic single-cell and sub-cellular modulation technology, which could shed new insights into the mechanism of ultrasound neurostimulation.