Riluzole-induced oscillations in spinal networks

We previously showed in dissociated cultures of fetal rat spinal cord that disinhibition-induced bursting is based on intrinsic spiking, network recruitment, and a network refractory period after the bursts. A persistent sodium current (I(NaP)) underlies intrinsic spiking, which, by recurrent excitation, generates the bursting activity. Although full blockade of I(NaP) with riluzole disrupts such bursting, the present study shows that partial blockade of I(NaP) with low doses of riluzole maintains bursting activity with unchanged burst rate and burst duration. More important, low doses of riluzole turned bursts composed of persistent activity into bursts composed of oscillatory activity at around 5 Hz. In a search for the mechanisms underlying the generation of such intraburst oscillations, we found that activity-dependent synaptic depression was not changed with low doses of riluzole. On the other hand, low doses of riluzole strongly increased spike-frequency adaptation and led to early depolarization block when bursts were simulated by injecting long current pulses into single neurons in the absence of fast synaptic transmission. Phenytoin is another I(NaP) blocker. When applied in doses that reduced intrinsic activity by 80-90%, as did low doses of riluzole, it had no effect either on spike-frequency adaptation or on depolarization block. Nor did phenytoin induce intraburst oscillations after disinhibition. A theoretical model incorporating a depolarization block mechanism could reproduce the generation of intraburst oscillations at the network level. From these findings we conclude that riluzole-induced intraburst oscillations are a network-driven phenomenon whose major accommodation mechanism is depolarization block arising from strong sodium channel inactivation.

Cellular and network mechanisms of rhythm generation in spinal cord circuits

The generation of rhythmic electrical activity is a prominent feature of spinal cord circuits that is used for locomotion and also for circuit refinement during development. The mechanisms involved in rhythm generation in spinal cord networks are not fully understood. It is for example not known whether spinal cord rhythms are driven by pacemaker neurons and if yes, which neurons are involved in this function. We studied the mechanisms involved in rhythm generation in slice cultures from fetal rats that were grown on multielectrode arrays (MEAs). We combined multisite extracellular recordings from the MEA electrodes with intracellular patch clamp recordings from single neurons. We found that spatially restricted oscillations of activity appeared in most of the cultures spontaneously. Such activity was based on intrinsic activity in a percentage of the neurons that could activate the spinal networks through recurrent excitation. The local oscillator networks critically involved NMDA, AMPA and GABA / glycine receptors at subsequent phases of the oscillation cycle. Intrinsic spiking in individual neurons (in the absence of functional synaptic coupling) was based on persistent sodium currents. Intrinsic firing as well as persistent sodium currents were increased by 5-HT through 5-HT2 receptors. Comparing neuronal activity to muscle activity in co-cultures of spinal cord slices with muscle fibers we found that a percentage of the intrinsically spiking neurons were motoneurons. These motoneurons were electrically coupled among each other and they could drive the spinal networks through cholinergic recurrent excitation. These findings open the possibility that during development rhythmic activity in motoneurons is not only involved in circuit refinement downstream at the neuromuscular endplates but also upstream at the level of spinal cord circuits.

Calcium indicator loading of neurons using single-cell electroporation

Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.

Multiple time scales of temporal response in pyramidal and fast spiking cortical neurons

Neural dynamic processes correlated over several time scales are found in vivo, in stimulus-evoked as well as spontaneous activity, and are thought to affect the way sensory stimulation is processed. Despite their potential computational consequences, a systematic description of the presence of multiple time scales in single cortical neurons is lacking. In this study, we injected fast spiking and pyramidal (PYR) neurons in vitro with long-lasting episodes of step-like and noisy, in-vivo-like current. Several processes shaped the time course of the instantaneous spike frequency, which could be reduced to a small number (1-4) of phenomenological mechanisms, either reducing (adapting) or increasing (facilitating) the neuron's firing rate over time. The different adaptation/facilitation processes cover a wide range of time scales, ranging from initial adaptation (<10 ms, PYR neurons only), to fast adaptation (<300 ms), early facilitation (0.5-1 s, PYR only), and slow (or late) adaptation (order of seconds). These processes are characterized by broad distributions of their magnitudes and time constants across cells, showing that multiple time scales are at play in cortical neurons, even in response to stationary stimuli and in the presence of input fluctuations. These processes might be part of a cascade of processes responsible for the power-law behavior of adaptation observed in several preparations, and may have far-reaching computational consequences that have been recently described.

Subcolumnar Dendritic and Axonal Organization of Spiny Stellate and Star Pyramid Neurons within a Barrel in Rat Somatosensory Cortex

Excitatory neurons at the level of cortical layer 4 in the rodent somatosensory barrel field often display a strong eccentricity in comparison with layer 4 neurons in other cortical regions. In rat, dendritic symmetry of the 2 main excitatory neuronal classes, spiny stellate and star pyramid neurons (SSNs and SPNs), was quantified by an asymmetry index, the dendrite-free angle. We carefully measured shrinkage and analyzed its influence on morphological parameters. SSNs had mostly eccentric morphology, whereas SPNs were nearly radially symmetric. Most asymmetric neurons were located near the barrel border. The axonal projections, analyzed at the level of layer 4, were mostly restricted to a single barrel except for those of 3 interbarrel projection neurons. Comparing voxel representations of dendrites and axon collaterals of the same neuron revealed a close overlap of dendritic and axonal fields, more pronounced in SSNs versus SPNs and considerably stronger in spiny L4 neurons versus extragranular pyramidal cells. These observations suggest that within a barrel dendrites and axons of individual excitatory cells are organized in subcolumns that may confer receptive field properties such as directional selectivity to higher layers, whereas the interbarrel projections challenge our view of barrels as completely independent processors of thalamic input.

Properties of basal dendrites of layer 5 pyramidal neurons: a direct patch-clamp recording study

Basal dendrites receive the majority of synapses that contact neocortical pyramidal neurons, yet our knowledge of synaptic processing in these dendrites has been hampered by their inaccessibility for electrical recordings. A new approach to patch-clamp recordings enabled us to characterize the integrative properties of these cells. Despite the short physical length of rat basal dendrites, synaptic inputs were electrotonically remote from the soma (>30-fold excitatory postsynaptic potential (EPSP) attenuation) and back-propagating action potentials were significantly attenuated. Unitary EPSPs were location dependent, reaching large amplitudes distally (>8 mV), yet their somatic contribution was relatively location independent. Basal dendrites support sodium and NMDA spikes, but not calcium spikes, for 75% of their length. This suggests that basal dendrites, despite their proximity to the site of action potential initiation, do not form a single basal-somatic region but rather should be considered as a separate integrative compartment favoring two integration modes: subthreshold, location-independent summation versus local amplification of incoming spatiotemporally clustered information.

Development of dendritic tonic GABAergic inhibition regulates excitability and plasticity in CA1 pyramidal neurons

Synaptic plasticity rules change during development: while hippocampal synapses can be potentiated by a single action potential pairing protocol in young neurons, mature neurons require burst firing to induce synaptic potentiation. An essential component for spike timing-dependent plasticity is the backpropagating action potential (BAP). BAP along the dendrites can be modulated by morphology and ion channel composition, both of which change during late postnatal development. However it is unclear whether these dendritic changes can explain the developmental changes in synaptic plasticity induction rules. Here, we show that tonic GABAergic inhibition regulates dendritic action potential backpropagation in adolescent but not pre-adolescent CA1 pyramidal neurons. These developmental changes in tonic inhibition also altered the induction threshold for spike timing-dependent plasticity in adolescent neurons. This GABAergic regulatory effect upon backpropagation is restricted to distal regions of apical dendrites (>200 μm) and mediated by α5-containing GABA(A) receptors. Direct dendritic recordings demonstrate α5-mediated tonic GABA(A) currents in adolescent neurons which can modulate backpropagating action potentials. These developmental modulations in dendritic excitability could not be explained by concurrent changes in dendritic morphology. To explain our data, model simulations propose a distally-increasing or localized distal expression of dendritic α5 tonic inhibition in mature neurons. Overall, our results demonstrate that dendritic integration and plasticity in more mature dendrites are significantly altered by tonic α5 inhibition in a dendritic region-specific and developmentally-regulated manner.

Control of ventricular ciliary beating by the melanin concentrating hormone-expressing neurons of the lateral hypothalamus: a functional imaging survey

The cyclic peptide Melanin Concentrating Hormone (MCH) is known to control a large number of brain functions in mammals such as food intake and metabolism, stress response, anxiety, sleep/wake cycle, memory, and reward. Based on neuro-anatomical and electrophysiological studies these functions were attributed to neuronal circuits expressing MCHR1, the single MCH receptor in rodents. In complement to our recently published work (1) we provided here new data regarding the action of MCH on ependymocytes in the mouse brain. First, we establish that MCHR1 mRNA is expressed in the ependymal cells of the third ventricle epithelium. Second, we demonstrated a tonic control of MCH-expressing neurons on ependymal cilia beat frequency using in vitro optogenics. Finally, we performed in vivo measurements of CSF flow using fluorescent micro-beads in wild-type and MCHR1-knockout mice. Collectively, our results demonstrated that MCH-expressing neurons modulate ciliary beating of ependymal cells at the third ventricle and could contribute to maintain cerebro-spinal fluid homeostasis.

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.