Fernando R. Fernandez

Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in Motor Learning and Memory

Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-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 Ca2+ 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+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ 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 [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.

Artificial Synaptic Conductances Reduce Subthreshold Oscillations and Periodic Firing in Stellate Cells of the Entorhinal Cortex

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.

A-Type and T-Type Currents Interact to Produce a Novel Spike Latency-Voltage Relationship in Cerebellar Stellate Cells

The modification of first-spike latencies by low-threshold and inactivating K+ currents (IA) 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 Kv4 potassium channel subfamily, which underlie IA 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 <10 mV. Using patch clamp and modeling, we illustrate that spike latency characteristics are the product of an interplay between IA and low-threshold calcium current (IT) 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.

Imaging Activity in Neurons and Glia with a Polr2a-Based and Cre-Dependent GCaMP5G-IRES-tdTomato Reporter Mouse

UNLABELLEDnNew 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 processesxa0of 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.nnnVIDEO ABSTRACT

Inactivation of Kv3.3 potassium channels in heterologous expression systems.

Kv3.3 K+ channels are believed to incorporate an NH2-terminal domain to produce an intermediate rate of inactivation relative to the fast inactivating K+ channels Kv3.4 and Kv1.4. The rate of Kv3.3 inactivation has, however, been difficult to establish given problems in obtaining consistent rates of inactivation in expression systems. This study characterized the properties of AptKv3.3, the teleost homologue of Kv3.3, when expressed in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. We show that the properties of AptKv3.3 differ significantly between CHO and HEK cells, with the largest difference occurring in the rate and voltage dependence of inactivation. While AptKv3.3 in CHO cells showed a fast and voltage-dependent rate of inactivation consistent with N-type inactivation, currents in HEK cells showed rates of inactivation that were voltage-independent and more consistent with a slower C-type inactivation. Examination of the mRNA sequence revealed that the first methionine start site had a weak Kozak consensus sequence, suggesting that the lack of inactivation in HEK cells could be due to translation at a second methionine start site downstream of the NH2-terminal coding region. Mutating the nucleotide sequence surrounding the first methionine start site to one more closely resembling a Kozak consensus sequence produced currents that inactivated with a fast and voltage-dependent rate of inactivation in both CHO and HEK cells. These results indicate that under the appropriate conditions Kv3.3 channels can exhibit fast and reliable inactivation that approaches that more typically expected of “A”-type K+ currents.

Gain control in CA1 pyramidal cells using changes in somatic conductance

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.

Spike Resonance Properties in Hippocampal O-LM Cells Are Dependent on Refractory Dynamics

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 postspike 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.

High-Threshold K+ Current Increases Gain by Offsetting a Frequency-Dependent Increase in Low-Threshold K+ Current

High-frequency firing neurons are found in numerous central systems, including the auditory brainstem, thalamus, hippocampus, and neocortex. The kinetics of high-threshold K+ currents (IKHT) from the Kv3 subfamily has led to the proposal that these channels offset cumulative Na+ current inactivation and stabilize tonic high-frequency firing. However, all high-frequency firing neurons, examined to date, also express low-threshold K+ currents (IKLT) that have slower kinetics and play an important role in setting the subthreshold and filtering properties of the neuron. IKLT has also been shown to dampen excitability and is therefore likely to oppose high-frequency firing. In this study, we examined the role of IKHT in pyramidal cells of the electrosensory lobe of weakly electric fish, which are characterized by high-frequency firing, a very wide frequency range, and high levels of IKHT. In particular, we examined the mechanisms that allow IKHT to set the gain of the F-I relationship by interacting with another low-threshold K+ current. We found that IKHT increases the gain of the F-I relationship and influences spike waveform almost exclusively in the high-frequency firing range. The frequency dependence arises from IKHT influencing both the IKLT and Na+ currents. IKHT thus plays a significant role in stabilizing high-frequency firing by preventing a steady-state accumulation of IKLT that is as important as preventing Na+ current inactivation.

A C-terminal domain directs Kv3.3 channels to dendrites.

Pyramidal neurons of the electrosensory lateral line lobe (ELL) of Apteronotus leptorhynchus express Kv3-type voltage-gated potassium channels that give rise to high-threshold currents at the somatic and dendritic levels. Two members of the Kv3 channel family, AptKv3.1 and AptKv3.3, are coexpressed in these neurons. AptKv3.3 channels are expressed at uniformly high levels in each of four ELL segments, whereas AptKv3.1 channels appear to be expressed in a graded manner with higher levels of expression in segments that process high-frequency electrosensory signals. Immunohistochemical and recombinant channel expression studies show a differential distribution of these two channels in the dendrites of ELL pyramidal neurons. AptKv3.1 is concentrated in somas and proximal dendrites, whereas AptKv3.3 is distributed throughout the full extent of the large dendritic tree. Recombinant channel expression of AptKv3 channels through in vivo viral injections allowed directed retargeting of AptKv3 subtypes over the somadendritic axis, revealing that the sequence responsible for targeting channels to distal dendrites lies within the C-terminal domain of the AptKv3.3 protein. The targeting domain includes a consensus sequence predicted to bind to a PDZ (postsynaptic density-95/Discs large/zona occludens-1)-type protein–protein interaction motif. These findings reveal that different functional roles for Kv3 potassium channels at the somatic and dendritic level of a sensory neuron are attained through specific targeting that selectively distributes Kv3.3 channels to the dendritic compartment.

Membrane Voltage Fluctuations Reduce Spike Frequency Adaptation and Preserve Output Gain in CA1 Pyramidal Neurons in a High-Conductance State

Modulating the gain of the input–output function of neurons is critical for processing of stimuli and network dynamics. Previous gain control mechanisms have suggested that voltage fluctuations play a key role in determining neuronal gain in vivo. Here we show that, under increased membrane conductance, voltage fluctuations restore Na+ current and reduce spike frequency adaptation in rat hippocampal CA1 pyramidal neurons in vitro. As a consequence, membrane voltage fluctuations produce a leftward shift in the frequency–current relationship without a change in gain, relative to an increase in conductance alone. Furthermore, we show that these changes have important implications for the integration of inhibitory inputs. Due to the ability to restore Na+ current, hyperpolarizing membrane voltage fluctuations mediated by GABAA-like inputs can increase firing rate in a high-conductance state. Finally, our data show that the effects on gain and synaptic integration are mediated by voltage fluctuations within a physiologically relevant range of frequencies (10–40 Hz).