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Journal of Physiology
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Oscillatory common inputs of cortical or peripheral origin can be identified from the motor neuron output with coherence analysis. Linear transmission is possible despite the motor neuron non-linearity because the same input is sent commonly to several neurons. Because of the linear transmission, common input components to motor neurons can be investigated from the surface EMG, for example by EEG–EMG or EMG–EMG coherence. In these studies, there is an open debate on the utility and appropriateness of EMG rectification. The present study addresses this issue using an analytical, simulation and experimental approach. The main novel theoretical contribution that we report is that the spectra of both the rectified and the raw EMG contain input spectral components to motor neurons. However, they differ by the contribution of amplitude cancellation which influences the rectified EMG spectrum when extracting common oscillatory inputs. Therefore, the degree of amplitude cancellation has an impact on the effectiveness of EMG rectification in extracting input spectral peaks. The theoretical predictions were exactly confirmed by realistic simulations of a pool of motor neurons innervating a muscle in a cylindrical volume conductor of EMG generation and by experiments conducted on the first dorsal interosseous and the abductor pollicis brevis muscles of seven healthy subjects during pinching. It was concluded that when the contraction level is relatively low, EMG rectification may be preferable for identifying common inputs to motor neurons, especially when the energy of the action potentials in the low frequency range is low. Nonetheless, different levels of cancellation across conditions influence the relative estimates of the degree of linear transmission of oscillatory inputs to motor neurons when using the rectified EMG.
Dipeptidyl-peptidase-like-proteins confer high sensitivity to the scorpion toxin AmmTX3 to Kv4-mediated A-type K+ channels
K+ channels containing Kv4.2 and Kv4.3 pore-forming subunits mediate most of the subthreshold-operating somatodendritic A-type K+ current in CNS neurons. These channels are believed to be important in regulating the frequency of repetitive firing, the backpropagation of action potential into dendrites, and dendritic integration and plasticity. Moreover, they have been implicated in several diseases from pain to epilepsy and autism spectrum disorders. The lack of toxins that specifically and efficiently block these channels has hampered studies aimed at confirming their functional role and their involvement in disease. AmmTX3 and other related members of the -KTX15 family of scorpion toxins have been shown to block the A-type K+ current in cultured neurons, but their specificity has been questioned because the toxins do not efficiently block the currents mediated by Kv4.2 or Kv4.3 subunits expressed in heterologous cells. Here we show that the high-affinity blockade of Kv4.2 and Kv4.3 channels by AmmTX3 depends on the presence of the auxiliary subunits DPP6 and DPP10. These proteins are thought to be components of the Kv4 channel complex in neurons and to be important for channel expression in dendrites. These studies validate the use of AmmTX3 as a blocker of the Kv4-mediated A-type K+ current in neurons.
A functional role for both -aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus
Tonic -aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K+-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased.
Mitochondria and plasma membrane Ca2+-ATPase control presynaptic Ca2+ clearance in capsaicin-sensitive rat sensory neurons
The central processes of primary nociceptors form synaptic connections with the second-order nociceptive neurons located in the dorsal horn of the spinal cord. These synapses gate the flow of nociceptive information from the periphery to the CNS, and plasticity at these synapses contributes to centrally mediated hyperalgesia and allodynia. Although exocytosis and synaptic plasticity are controlled by Ca2+ at the release sites, the mechanisms underlying presynaptic Ca2+ signalling at the nociceptive synapses are not well characterized. We examined the presynaptic mechanisms regulating Ca2+ clearance following electrical stimulation in capsaicin-sensitive nociceptors using a dorsal root ganglion (DRG)/spinal cord neuron co-culture system. Cytosolic Ca2+ concentration ([Ca2+]i) recovery following electrical stimulation was well approximated by a monoexponential function with a ~2 s. Inhibition of sarco-endoplasmic reticulum Ca2+-ATPase did not affect presynaptic [Ca2+]i recovery, and blocking plasmalemmal Na+/Ca2+ exchange produced only a small reduction in the rate of [Ca2+]i recovery (~12%) that was independent of intracellular K+. However, [Ca2+]i recovery in presynaptic boutons strongly depended on the plasma membrane Ca2+-ATPase (PMCA) and mitochondria that accounted for ~47 and 40%, respectively, of presynaptic Ca2+ clearance. Measurements using a mitochondria-targeted Ca2+ indicator, mtPericam, demonstrated that presynaptic mitochondria accumulated Ca2+ in response to electrical stimulation. Quantitative analysis revealed that the mitochondrial Ca2+ uptake is highly sensitive to presynaptic [Ca2+]i elevations, and occurs at [Ca2+]i levels as low as ~200–300 nm. Using RT-PCR, we detected expression of several putative mitochondrial Ca2+ transporters in DRG, such as MCU, Letm1 and NCLX. Collectively, this work identifies PMCA and mitochondria as the major regulators of presynaptic Ca2+ signalling at the first sensory synapse, and underlines the high sensitivity of the mitochondrial Ca2+ uniporter in neurons to cytosolic Ca2+.
Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.
GABAB receptor-mediated frequency-dependent and circadian changes in synaptic plasticity modulate retinal input to the suprachiasmatic nucleus
Light is the most important environmental signal that entrains the circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN). The retinohypothalamic tract (RHT) was stimulated to simulate the light intensity-dependent discharges of intrinsically photosensitive retinal ganglion cells projecting axons to the hypothalamus. EPSCs were evoked by paired-pulse stimulation or by application of stimulus trains, and recorded from SCN neurons in rat brain slices. Initial release probability (Pr) and synaptic plasticity changes depended on the strength of GABAB receptor (GABABR)-mediated presynaptic inhibition and could be different at the same GABABR agonist concentration. Facilitation caused by frequency-dependent relief of GABABR-mediated inhibition was observed when the initial Pr was decreased to less than 15% of control during strong activation of presynaptic GABAB receptors by (±)baclofen (10 m), GABA (2 mm) or by GABA uptake inhibitor nipecotic acid (5 mm). In contrast, short-term synaptic depression appeared during baclofen (10 m) application when initial Pr was greater than 30% of control. Block of 4-aminopyridine-sensitive K+ currents increased the amplitude and time constant of decay of evoked EPSCs (eEPSCs), and decreased the GABABR-mediated presynaptic inhibition. The GABAB receptor antagonist CGP55845 (3 m) increased the eEPSCs amplitude 30% throughout the light–dark cycle. During light and dark phases the RHT inputs to 55% and 33% of recorded neurons, respectively, were under GABAB inhibitory control indicating that the tonic inhibition induced by local changes of endogenous GABA concentration contributes to the circadian variation of RHT transmitter release. We conclude that GABABR-mediated presynaptic inhibition decreased with increasing frequency and broadening of presynaptic action potentials, and depended on the sensitivity of RHT terminals to GABABR agonists, and diurnal changes of the extracellular GABA concentration around RHT axon terminals in the SCN.
Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites
Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.
Calcium dynamics during NMDA-induced membrane potential oscillations in lamprey spinal neurons - contribution of L-type calcium channels (CaV1.3)
NMDA receptor-dependent, intrinsic membrane potential oscillations are an important element in the operation of the lamprey locomotor network. They involve a cyclic influx of calcium, leading to an activation of calcium-activated potassium (KCa) channels that in turn contributes to the termination of the depolarized plateau and membrane repolarization. In this study, we have investigated the calcium dynamics in different regions of lamprey spinal neurons during membrane potential oscillations, using confocal calcium imaging in combination with intracellular recordings. Calcium fluctuations were observed in both soma and dendrites, timed to the oscillations. The calcium level increased sharply at the onset of membrane depolarization, to reach its maximum by the end of the plateau. The calcium peak in distal dendrites typically occurred earlier than in the soma during the oscillatory cycle. The L-type calcium channel blocker nimodipine increased the duration of the depolarized plateau phase in most cells tested, whereas the agonist Bay K 8644 decreased plateau duration. Bay K 8644 increased the amplitude of calcium fluctuations, particularly in distal dendrites, whereas nimodipine caused a decrease, suggesting that L-type low-voltage-activated calcium channels are mainly localized in these regions. Our results thus indicate that dendritic CaV1.3-like calcium channels are activated during NMDA-mediated membrane potential oscillations. This calcium influx activates KCa channels involved in plateau termination.
Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles
Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending we have described previously. Both types of endings are formed by the peripheral sensory terminals of primary mechanosensory dorsal root ganglion cells, so the presence of such a system in the lanceolate ending would provide support for our hypothesis that it is a general property of fundamental importance to the regulation of the responsiveness of the broad class of primary mechanosensory endings. We show not only that an SLV-based system is present in lanceolate endings, but also that there are clear parallels between its operation in the two types of mechanosensory endings. In particular, we demonstrate that, as in the muscle spindle: (i) FM1-43 labels the sensory terminals of the lanceolate ending, rather than the closely associated accessory (glial) cells; (ii) the dye enters and leaves the terminals primarily by SLV recycling; (iii) the dye does not block the electrical response to mechanical stimulation, in contrast to its effect on the hair cell and dorsal root ganglion cells in culture; (iv) SLV recycling is Ca2+ sensitive; and (v) the sensory terminals are enriched in glutamate. Thus, in the lanceolate sensory ending SLV recycling is itself regulated, at least in part, by glutamate acting through a phospholipase D-coupled metabotropic glutamate receptor.
Glucose sensitivity of mouse olfactory bulb neurons is conveyed by a voltage-gated potassium channel
The olfactory bulb has recently been proposed to serve as a metabolic sensor of internal chemistry, particularly that modified by metabolism. Because the voltage-dependent potassium channel Kv1.3 regulates a large proportion of the outward current in olfactory bulb neurons and gene-targeted deletion of the protein produces a phenotype of resistance to diet-induced obesity in mice, we hypothesized that this channel may play a role in translating energy availability into a metabolic signal. Here we explored the ability of extracellular glucose concentration to modify evoked excitability of the mitral neurons that principally regulate olfactory coding and processing of olfactory information. Using voltage-clamp electrophysiology of heterologously expressed Kv1.3 channels in HEK 293 cells, we found that Kv1.3 macroscopic currents responded to metabolically active (d-) rather than inactive (l-) glucose with a response profile that followed a bell-shaped curve. Olfactory bulb slices stimulated with varying glucose concentrations showed glucose-dependent mitral cell excitability as evaluated by current-clamp electrophysiology. While glucose could be either excitatory or inhibitory, the majority of the sampled neurons displayed a decreased firing frequency in response to elevated glucose concentration that was linked to increased latency to first spike and decreased action potential cluster length. Unlike modulation attributed to phosphorylation, glucose modulation of mitral cells was rapid, less than one minute, and was reversible within the time course of a patch recording. Moreover, we report that modulation targets properties of spike firing rather than action potential shape, involves synaptic activity of glutamate or GABA signalling circuits, and is dependent upon Kv1.3 expression. Given the rising incidence of metabolic disorders attributed to weight gain, changes in neuronal excitability in brain regions regulating sensory perception of food are of consequence.
Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique to modulate cortical excitability. Although increased/decreased excitability under the anode/cathode electrode is nominally associated with membrane depolarization/hyperpolarization, which cellular compartments (somas, dendrites, axons and their terminals) mediate changes in cortical excitability remains unaddressed. Here we consider the acute effects of DCS on excitatory synaptic efficacy. Using multi-scale computational models and rat cortical brain slices, we show the following. (1) Typical tDCS montages produce predominantly tangential (relative to the cortical surface) direction currents (4–12 times radial direction currents), even directly under electrodes. (2) Radial current flow (parallel to the somatodendritic axis) modulates synaptic efficacy consistent with somatic polarization, with depolarization facilitating synaptic efficacy. (3) Tangential current flow (perpendicular to the somatodendritic axis) modulates synaptic efficacy acutely (during stimulation) in an afferent pathway-specific manner that is consistent with terminal polarization, with hyperpolarization facilitating synaptic efficacy. (4) Maximal polarization during uniform DCS is expected at distal (the branch length is more than three times the membrane length constant) synaptic terminals, independent of and two–three times more susceptible than pyramidal neuron somas. We conclude that during acute DCS the cellular targets responsible for modulation of synaptic efficacy are concurrently somata and axon terminals, with the direction of cortical current flow determining the relative influence.
Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs, voltage-gated delayed rectifier K+ (KDR) channels are dominantly expressed. However, the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1, a high voltage-gated KDR channel, was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties, such as resting membrane potential, of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation, not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation.
Single KATP channel opening in response to stimulation of AMPA/kainate receptors is mediated by Na+ accumulation and submembrane ATP and ADP changes
Excessive stimulation of glutamatergic receptors (GluRs) can overexcite neurons. This can be dampened by KATP channels linking metabolic and neuronal activities, but the cross-talk has not yet been examined on the single channel level. In the brainstem and hippocampal neurons, GluR agonists augmented the open state probability (Popen) of KATP channels with relative efficacy: kainate AMPA > NMDA > t-ACPD. Inhibition of calcium influx and chelation of intracellular calcium did not modify the effects. Kainate did not augment production of reactive oxygen species measured with roGFP1. H2O2 slightly increased Popen, but GluR effects were not modified. GluR actions were abolished in Na+-free solutions and after blockade of Na+-K+-ATPase. KATP channels in open-cell patch-clamp measurements were inhibited by ATP, stimulated by ADP, and kainate was effective only in the presence of ATP. GluR stimulation enhanced ATP consumption that decreased submembrane ATP levels, whereas metabolic poisoning diminished bulk ATP. Modelling showed strong ATP depletion and ADP accumulation near the membrane, and both effects contributed to Popen increases after GluR stimulation. Kainate and hypoxia activated KATP channels in the functional brainstem slices. Inhibition of aerobic ATP production and GluR stimulation were about equally effective in KATP channel opening during hypoxia. Induction of seizure-like activity in hippocampal slices with Mg2+-free solutions was accompanied by ATP decrease and KATP channel opening. We propose that KATP channels and GluRs are functionally coupled that can regulate long-lasting changes of neuronal activity in the CNS neurons.
Extensive in vitro data and modeling studies suggest that intrinsic properties of medial entorhinal cortex (MEC) neurons contribute to the spiking behaviour of functional cell types of MEC neurons, such as grid cells, recorded in behaving animals. It remains unclear, however, how intrinsic properties of MEC neurons influence cellular dynamics in intact networks in vivo. In order to begin to bridge the gap between electrophysiological data sets from brain slices and behaving animals, in the present study we performed intracellular recordings using sharp electrodes in urethane-anaesthetized rats to elucidate the cellular dynamics of MEC neurons in vivo. We focused on the h-current-dependent sag potential during hyperpolarizing current steps, subthreshold resonance in response to oscillatory frequency sweeps (chirp stimuli), persistent spiking in response to brief depolarizing inputs and the relationship between firing frequency and input (f–I curve), each of which is sensitive to cholinergic modulation in vitro. Consistent with data from in vitro studies, cholinergic activation by systemic application of the acetylcholinesterase inhibitor, physostigmine, resulted in decreased sag amplitude, increased sag time constant and a decrease of the peak resonance frequency. The f–I curve was also modulated by physostigmine in many neurons, but persistent spiking was not observed in any of our recordings, even when picrotoxin, a GABAA blocker, was included in the internal solution of the recording pipette to reduce possible effects of network inhibition. These results suggest that intrinsic oscillatory and rate-coding mechanisms, but not intrinsic bistability, are significantly modulated by acetylcholine in the intact entorhinal network.
Differential presynaptic control of the synaptic effectiveness of cutaneous afferents evidenced by effects produced by acute nerve section
In the anaesthetized cat, the acute section of the saphenous (Saph) and/or the superficial peroneal (SP) nerves was found to produce a long-lasting increase of the field potentials generated in the dorsal horn by stimulation of the medial branch of the sural (mSU) nerve. This facilitation was associated with changes in the level of the tonic primary afferent depolarization (PAD) of the mSU intraspinal terminals. The mSU afferent fibres projecting into Rexed's laminae III–IV were subjected to a tonic PAD that was reduced by the acute section of the SP and/or the Saph nerves. The mSU afferents projecting deeper into the dorsal horn (Rexed's laminae V–VI) were instead subjected to a tonic PAD that was increased after Saph and SP acute nerve section. A differential control of the synaptic effectiveness of the low-threshold cutaneous afferents according to their sites of termination within the dorsal horn is envisaged as a mechanism that allows selective processing of sensory information in response to tactile and nociceptive stimulation or during the execution of different motor tasks.
Differential responses of fast- and slow-conducting pyramidal tract neurons to changes in accuracy demands during locomotion
Most movements need to be accurate. The neuronal mechanisms controlling accuracy during movements are poorly understood. In this study we compare the activity of fast- and slow-conducting pyramidal tract neurons (PTNs) of the motor cortex in cats as they walk over both a flat surface, a task that does not require accurate stepping and can be accomplished without the motor cortex, as well as along a horizontal ladder, a task that requires accuracy and the activity of the motor cortex to be successful. Fast- and slow-conducting PTNs are known to have distinct biophysical properties as well as different afferent and efferent connections. We found that while the activity of all PTNs changes substantially upon transition from simple locomotion to accurate stepping on the ladder, slow-conducting PTNs respond in a much more concerted manner than fast-conducting ones. As a group, slow-conducting PTNs increase discharge rate, especially during the late stance and early swing phases, decrease discharge variability, have a tendency to shift their preferred phase of the discharge into the swing phase, and almost always produce a single peak of activity per stride during ladder locomotion. In contrast, the fast-conducting PTNs do not display such concerted changes to their activity. In addition, upon transfer from simple locomotion to accurate stepping on the ladder slow-conducting PTNs more profoundly increase the magnitude of their stride-related frequency modulation compared with fast-conducting PTNs. We suggest that slow-conducting PTNs are involved in control of accuracy of locomotor movements to a greater degree than fast-conducting PTNs.
The hippocampus is a pivotal structure for episodic memory function. This ability relies on the possibility of integrating different features of sensory stimuli with the spatio-temporal context in which they occur. While recent studies now suggest that somatosensory information is already processed by the hippocampus, the basic mechanisms still remain unexplored. Here, we used electrical stimulation of the paws, the whisker pad or the medial lemniscus to probe the somatosensory pathway to the hippocampus in the anaesthetized rat, and multisite electrodes, in combination with tetrode and intracellular recordings, to look at the properties of somatosensory hippocampal responses. We found that peripheral and lemniscal stimulation elicited small local field potential responses in the dorsal hippocampus about 35–40 ms post-stimulus. Current source density analysis established the local nature of these responses, revealing associated synaptic sinks that were consistently confined to the molecular layer (ML) of the dentate gyrus (DG), with less regular activation of the CA1 stratum lacunosum moleculare (SLM). A delayed (40–45 ms), potentially active, current source that outlasted the SLM sink was present in about 50% cases around the CA1 pyramidal cell layer. Somatosensory stimulation resulted in multi-unit firing increases in the majority of DG responses (79%), whereas multi-unit firing suppression was observed in the majority of CA1 responses (62%). Tetrode and intracellular recordings of individual cells confirmed different firing modulation in the DG and the CA1 region, and verified the active nature of both the early ML sink and delayed somatic CA1 source. Hippocampal responses to somatosensory stimuli were dependent on fluctuations in the strength and composition of synaptic inputs due to changes of the ongoing local (hippocampal) and distant (cortical) state. We conclude that somatosensory signals reach the hippocampus mainly from layer II entorhinal cortex to directly discharge DG granule cells, while a different predominantly inhibitory process takes place in CA1, further controlling the hippocampal output. Therefore, our data reveal a distinct organization of somatosensory-related extra-hippocampal inputs converging onto DG and CA1.
Physiological and morphological properties of Dbx1-derived respiratory neurons in the pre-Botzinger complex of neonatal mice
Breathing in mammals depends on an inspiratory-related rhythm that is generated by glutamatergic neurons in the pre-Bötzinger complex (preBötC) of the lower brainstem. A substantial subset of putative rhythm-generating preBötC neurons derive from a single genetic line that expresses the transcription factor Dbx1, but the cellular mechanisms of rhythmogenesis remain incompletely understood. To elucidate these mechanisms, we carried out a comparative analysis of Dbx1-expressing neurons (Dbx1+) and non-Dbx1-derived (Dbx1–) neurons in the preBötC. Whole-cell recordings in rhythmically active newborn mouse slice preparations showed that Dbx1+ neurons activate earlier in the respiratory cycle and discharge greater magnitude inspiratory bursts compared with Dbx1– neurons. Furthermore, Dbx1+ neurons required less input current to discharge spikes (rheobase) in the context of network activity. The expression of intrinsic membrane properties indicative of A-current (IA) and hyperpolarization-activated current (Ih) tended to be mutually exclusive in Dbx1+ neurons. In contrast, there was no such relationship in the expression of currents IA and Ih in Dbx1– neurons. Confocal imaging and digital morphological reconstruction of recorded neurons revealed dendritic spines on Dbx1– neurons, but Dbx1+ neurons were spineless. The morphology of Dbx1+ neurons was largely confined to the transverse plane, whereas Dbx1– neurons projected dendrites to a greater extent in the parasagittal plane. The putative rhythmogenic nature of Dbx1+ neurons may be attributable, in part, to a higher level of intrinsic excitability in the context of network synaptic activity. Furthermore, Dbx1+ neuronal morphology may facilitate temporal summation and integration of local synaptic inputs from other Dbx1+ neurons, taking place largely in the dendrites, which could be important for initiating and maintaining bursts and synchronizing activity during the inspiratory phase.
In the search for cochlear correlates of auditory masking by noise stimuli, we recorded basilar membrane (BM) vibrations evoked by either tone or click signals in the presence of varying levels of background noise. The BM vibrations were recorded from basal regions in healthy cochleae of anaesthetized chinchilla and gerbil. Non-linear interactions that could underpin various aspects of psychophysical masking data, including both compression and suppression at the BM level, were observed. The suppression effects, whereby the amplitude of the responses to each stimulus component could be reduced, depended on the relative intensities of the noise and the tones or clicks. Only stimulus components whose frequencies fell inside the non-linear region of the recording site, i.e. around its characteristic frequency (CF), were affected by presentation of the ‘suppressing' stimulus (which could be either the tone or the noise). Mutual suppression, the simultaneous reduction of the responses to both tones and noise components, was observed under some conditions, but overall reductions of BM vibration were rarely observed. Moderate- to high-intensity tones suppressed BM responses to low-intensity Gaussian stimuli, including both broadband and narrowband noise. Suppression effects were larger for spectral components of the noise response that were closer to the CF. In this regime, the tone and noise stimuli became the suppressor and probe signals, respectively. This study provides the first detailed observations of cochlear mechanical correlates of the masking effects of noise. Mechanical detection thresholds for tone signals, which were arbitrarily defined using three criteria, are shown to increase in almost direct proportion to the noise level for low and moderately high noise levels, in a manner that resembles the findings of numerous psychophysical observations.
Selective mitochondrial Ca2+ uptake deficit in disease endstage vulnerable motoneurons of the SOD1G93A mouse model of amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that targets some somatic motoneuron populations, while others, e.g. those of the oculomotor system, are spared. The pathophysiological basis of this pattern of differential vulnerability, which is preserved in a transgenic mouse model of amyotrophic lateral sclerosis (SOD1G93A), and the mechanism of neurodegeneration in general are unknown. Hyperexcitability and calcium dysregulation have been proposed by others on the basis of data from juvenile mice that are, however, asymptomatic. No studies have been done with symptomatic mice following disease progression to the disease endstage. Here, we developed a new brainstem slice preparation for whole-cell patch-clamp recordings and single cell fura-2 calcium imaging to study motoneurons in adult wild-type and SOD1G93A mice up to disease endstage. We analysed disease-stage-dependent electrophysiological properties and intracellular Ca2+ handling of vulnerable hypoglossal motoneurons in comparison to resistant oculomotor neurons. Thereby, we identified a transient hyperexcitability in presymptomatic but not in endstage vulnerable motoneurons. Additionally, we revealed a remodelling of intracellular Ca2+ clearance within vulnerable but not resistant motoneurons at disease endstage characterised by a reduction of uniporter-dependent mitochondrial Ca2+ uptake and enhanced Ca2+ extrusion across the plasma membrane. Our study challenged the notion that hyperexcitability is a direct cause of neurodegeneration in SOD1G93A mice, but molecularly identified a Ca2+ clearance deficit in motoneurons and an adaptive Ca2+ handling strategy that might be targeted by future therapeutic strategies.