State-Dependent Firing Determines Intrinsic Dendritic Ca2+ Signaling in Thalamocortical Neurons

Activity-dependent dendritic Ca(2+) signals play a critical role in multiple forms of non-linear cellular output and plasticity. In thalamocortical neurons, despite the well-established spatial separation of sensory and cortical inputs onto proximal and distal dendrites respectively, little is known about the spatio-temporal dynamics of intrinsic dendritic Ca(2+) signalling during the different state-dependent firing patterns that are characteristic of these neurons. Here we demonstrate that T-type Ca(2+) channels are expressed throughout the entire dendritic tree of rat thalamocortical neurons and that they mediate regenerative propagation of low threshold spikes, typical of, but not exclusive to sleep states, resulting in global dendritic Ca(2+) influx. In contrast, actively backpropagating action potentials, typical of wakefulness, result in smaller Ca(2+) influxes that can temporally summate to produce dendritic Ca(2+) accumulations which are linearly related to firing frequency but spatially confined to proximal dendritic regions. Furthermore, dendritic Ca(2+) transients evoked by both action potentials and low threshold spikes are shaped by Ca(2+) uptake by sarco/endoplasmic reticulum Ca(2+) ATP-ases, but do not rely upon Ca(2+)-induced Ca(2+) release. Our data demonstrate that thalamocortical neurons are endowed with intrinsic dendritic Ca(2+) signalling properties that are spatially and temporally modified in a behavioural state-dependent manner, and suggest that backpropagating action potentials faithfully inform proximal sensory but not distal corticothalamic synapses of neuronal output whereas corticothalamic synapses only “detect” Ca(2+) signals associated with low threshold spikes

Iterative focused screening with biological fingerprints identifies selective Asc-1 inhibitors distinct from traditional high throughput screening

N-methyl-d-aspartate receptors (NMDARs) mediate glutamatergic signaling that is critical to cognitive processes in the central nervous system, and NMDAR hypofunction is thought to contribute to cognitive impairment observed in both schizophrenia and Alzheimer’s disease. One approach to enhance the function of NMDAR is to increase the concentration of an NMDAR coagonist, such as glycine or d-serine, in the synaptic cleft. Inhibition of alanine–serine–cysteine transporter-1 (Asc-1), the primary transporter of d-serine, is attractive because the transporter is localized to neurons in brain regions critical to cognitive function, including the hippocampus and cortical layers III and IV, and is colocalized with d-serine and NMDARs. To identify novel Asc-1 inhibitors, two different screening approaches were performed with whole-cell amino acid uptake in heterologous cells stably expressing human Asc-1: (1) a high-throughput screen (HTS) of 3 M compounds measuring 35S l-cysteine uptake into cells attached to scintillation proximity assay beads in a 1536 well format and (2) an iterative focused screen (IFS) of a 45 000 compound diversity set using a 3H d-serine uptake assay with a liquid scintillation plate reader in a 384 well format. Critically important for both screening approaches was the implementation of counter screens to remove nonspecific inhibitors of radioactive amino acid uptake. Furthermore, a 15 000 compound expansion step incorporating both on- and off-target data into chemical and biological fingerprint-based models for selection of additional hits enabled the identification of novel Asc-1-selective chemical matter from the IFS that was not identified in the full-collection HTS

Calcineurin signaling mediates activity-dependent relocation of the axon initial segment

The axon initial segment (AIS) is a specialised neuronal sub-compartment located at the beginning of the axon that is crucially involved in both the generation of action potentials and in the regulation of neuronal polarity. We recently showed that prolonged neuronal depolarisation produces a distal shift of the entire AIS structure away from the cell body, a change associated with a decrease in neuronal excitability. Here, we utilised dissociated rat hippocampal cultures, with a major focus on the dentate granule cell (DGC) population, to explore the signalling pathways underlying activity-dependent relocation of the AIS. Firstly, a pharmacological screen of voltage-gated calcium channels (VGCCs) showed that AIS relocation is triggered by activation of L-type Ca(v)1 VGCCs with negligible contribution from any other VGCC subtypes. Further pharmacological analysis revealed that downstream signalling events are mediated by the calcium-sensitive phosphatase calcineurin; inhibition of calcineurin with either FK506 or cyclosporin A totally abolished both depolarisation- and optogenetically-induced activity-dependent AIS relocation. Furthermore, calcineurin activation is sufficient for AIS plasticity, as expression of a constitutively active form of the phosphatase resulted in relocation of the AIS of DGCs without a depolarising stimulus. Finally, we assessed the role of calcineurin in other forms of depolarisation-induced plasticity. Neither membrane resistance changes nor spine density changes were affected by FK506 treatment, suggesting that calcineurin acts via a separate pathway to modulate AIS plasticity. Taken together, these results emphasise calcineurin as a vital player in the regulation of intrinsic plasticity as governed by the AIS

Essential Thalamic Contribution to Slow Waves of Natural Sleep

International audienceSlow waves represent one of the prominent EEG signatures of non-rapid eye movement (non-REM) sleep and are thought to play an important role in the cellular and network plasticity that occurs during this behavioral state. These slow waves of natural sleep are currently considered to be exclusively generated by intrinsic and synaptic mechanisms within neocortical territories, although a role for the thalamus in this key physiological rhythm has been suggested but never demonstrated. Combining neuronal ensemble recordings, microdialysis, and optogenetics, here we show that the block of the thalamic output to the neocortex markedly (up to 50%) decreases the frequency of slow waves recorded during non-REM sleep in freely moving, naturally sleeping-waking rats. A smaller volume of thalamic inactivation than during sleep is required for observing similar effects on EEG slow waves recorded during anesthesia, a condition in which both bursts and single action potentials of thalamocortical neurons are almost exclusively dependent on T-type calcium channels. Thalamic inactivation more strongly reduces spindles than slow waves during both anesthesia and natural sleep. Moreover, selective excitation of thalamocortical neurons strongly entrains EEG slow waves in a narrow frequency band (0.75-1.5 Hz) only when thalamic T-type calcium channels are functionally active. These results demonstrate that the thalamus finely tunes the frequency of slow waves during non-REM sleep and anesthesia, and thus provide the first conclusive evidence that a dynamic interplay of the neocortical and thalamic oscillators of slow waves is required for the full expression of this key physiological EEG rhythm

T-type calcium channels regulate cortical plasticity in-vivo NR-D-08-7049

T-type voltage-dependent calcium channels may play an important role in synaptic plasticity, but lack of specific antagonists has hampered investigation into this possible function. We investigated the role of the T-type channel in a canonical model of in-vivo cortical plasticity triggered by monocular deprivation. We identified a compound (TTA-I1) with subnanomolar potency in standard voltage clamp assays and high selectivity for the T-type channel. When infused intracortically, TTA-I1 reduced cortical plasticity triggered by monocular deprivation while preserving normal visual response properties. These results show that the T-type calcium channel plays a central role in cortical plasticity

Antagonism of T-type calcium channels inhibits high-fat diet–induced weight gain in mice

The epidemics of obesity and metabolic disorders have well-recognized health and economic burdens. Pharmacologic treatments for these diseases remain unsatisfactory with respect to both efficacy and side-effect profiles. Here, we have identified a potential central role for T-type calcium channels in regulating body weight maintenance and sleep. Previously, it was shown that mice lacking CaV3.1 T-type calcium channels have altered sleep/wake activity. We found that these mice were also resistant to high-fat diet–induced weight gain, without changes in food intake or sensitivity to high-fat diet–induced disruptions of diurnal rhythm. Administration of a potent and selective antagonist of T-type calcium channels, TTA-A2, to normal-weight animals prior to the inactive phase acutely increased sleep, decreased body core temperature, and prevented high-fat diet–induced weight gain. Administration of TTA-A2 to obese rodents reduced body weight and fat mass while concurrently increasing lean muscle mass. These effects likely result from better alignment of diurnal feeding patterns with daily changes in circadian physiology and potentially an increased metabolic rate during the active phase. Together, these studies reveal what we believe to be a previously unknown role for T-type calcium channels in the regulation of sleep and weight maintenance and suggest the potential for a novel therapeutic approach to treating obesity