Neuronal Rhythms Research Articles

From Clock to Clock shops: Intra‐ and intercellular mechanisms regulating daily timekeeping

Biologists have long marveled at the endogenous pacemakers that dictate daily schedules in physiology and behavior. Genetics and recent advances in biological imaging and neural recording have revealed multiple “clock genes” and pacemaking cell types. In mammals, individual neurons of the suprachiasmatic nucleus (SCN) can express daily rhythms in vitro for months. To understand how SCN neurons synchronize and sustain their rhythms for coherent output, we recorded firing and clock-gene expression patterns while blocking candidate signaling pathways for at least 8 days. GABA A and B receptor antagonism increased circadian peak firing rates and rhythm precision of cultured SCN neurons, but did not impair synchrony or rhythmicity. In contrast, inhibiting Gi/o with pertussis toxin (PTX) abolished rhythms in most neurons and desynchronized the population, phenocopying the loss of vasoactive intestinal polypeptide (VIP). Daily VIP receptor agonist treatment restored synchrony and rhythmicity to VIP−/− SCN cultures during continuous GABA receptor antagonism, but not during Gi/o blockade. We conclude that endogenous GABA controls the amplitude of SCN neuronal rhythms by reducing daytime firing, while Gi/o signaling suppresses nighttime firing and is necessary for synchrony among SCN neurons. We propose that VIP and G-protein signaling, not GABA, maintain and coordinate rhythms among SCN neurons.

Synchronization-induced rhythmicity of circadian oscillators in the suprachiasmatic nucleus.

The suprachiasmatic nuclei (SCN) host a robust, self-sustained circadian pacemaker that coordinates physiological rhythms with the daily changes in the environment. Neuronal clocks within the SCN form a heterogeneous network that must synchronize to maintain timekeeping activity. Coherent circadian output of the SCN tissue is established by intercellular signaling factors, such as vasointestinal polypeptide. It was recently shown that besides coordinating cells, the synchronization factors play a crucial role in the sustenance of intrinsic cellular rhythmicity. Disruption of intercellular signaling abolishes sustained rhythmicity in a majority of neurons and desynchronizes the remaining rhythmic neurons. Based on these observations, the authors propose a model for the synchronization of circadian oscillators that combines intracellular and intercellular dynamics at the single-cell level. The model is a heterogeneous network of circadian neuronal oscillators where individual oscillators are damped rather than self-sustained. The authors simulated different experimental conditions and found that: (1) in normal, constant conditions, coupled circadian oscillators quickly synchronize and produce a coherent output; (2) in large populations, such oscillators either synchronize or gradually lose rhythmicity, but do not run out of phase, demonstrating that rhythmicity and synchrony are codependent; (3) the number of oscillators and connectivity are important for these synchronization properties; (4) slow oscillators have a higher impact on the period in mixed populations; and (5) coupled circadian oscillators can be efficiently entrained by light–dark cycles. Based on these results, it is predicted that: (1) a majority of SCN neurons needs periodic synchronization signal to be rhythmic; (2) a small number of neurons or a low connectivity results in desynchrony; and (3) amplitudes and phases of neurons are negatively correlated. The authors conclude that to understand the orchestration of timekeeping in the SCN, intracellular circadian clocks cannot be isolated from their intercellular communication components.

An electronic device for artefact suppression in human local field potential recordings during deep brain stimulation

The clinical efficacy of high-frequency deep brain stimulation (DBS) for Parkinson's disease and other neuropsychiatric disorders likely depends on the modulation of neuronal rhythms in the target nuclei. This modulation could be effectively measured with local field potential (LFP) recordings during DBS. However, a technical drawback that prevents LFPs from being recorded from the DBS target nuclei during stimulation is the stimulus artefact. To solve this problem, we designed and developed ‘FilterDBS’, an electronic amplification system for artefact-free LFP recordings (in the frequency range 2–40 Hz) during DBS. After defining the estimated system requirements for LFP amplification and DBS artefact suppression, we tested the FilterDBS system by conducting experiments in vitro and in vivo in patients with advanced Parkinson's disease undergoing DBS of the subthalamic nucleus (STN). Under both experimental conditions, in vitro and in vivo, the FilterDBS system completely suppressed the DBS artefact without inducing significant spectral distortion. The FilterDBS device pioneers the development of an adaptive DBS system retroacted by LFPs and can be used in novel closed-loop brain–machine interface applications in patients with neurological disorders.

VIP Activates and Couples Clock Cells. Focus on “Disrupted Neuronal Activity Rhythms in the Suprachiasmatic Nucleus of Vasoactive Intestinal Polypeptide-Deficient Mice”

Neurons of the suprachiasmatic nucleus (SCN), located in the ventromedial hypothalamus, comprise the central mammalian circadian pacemaker ([Reppert and Weaver 2002][1]). In dispersed culture, these neurons can generate autonomous circadian (ca. 24 h) oscillations in spontaneous firing rate ([Welsh

Molecular regulation of cognitive functions and developmental plasticity: impact of GABAAreceptors

By controlling spike timing and sculpting neuronal rhythms, inhibitory interneurons play a key role in regulating neuronal circuits and behavior. The pronounced diversity of GABAergic (gamma-aminobutyric acid) interneurons is paralleled by an extensive diversity of GABAA receptor subtypes. The region- and domain-specific location of these receptor subtypes offers the opportunity to gain functional insights into the role of defined neuronal circuits. These developments are reviewed with regard to the regulation of sleep, anxiety, memory, sensorimotor processing and post-natal developmental plasticity.

Nocturnal expression of phosphorylated‐ERK1/2 in gastrin‐releasing peptide neurons of the rat suprachiasmatic nucleus

Extracellular regulated kinase (ERK) signalling is believed to play roles in various aspects of circadian clock mechanisms. In this study, we show in rat that the nuclear versus cytoplasmic intracellular distribution of the phosphorylated forms of ERK1/2 (P-ERK1/2) in the central clock, namely the suprachiasmatic nucleus (SCN), is proportionally constant across the light/dark cycle while the spatial distribution and neurochemical phenotype of cells expressing these activated forms are time-regulated according to a daily rhythm and light-regulated. P-ERK1/2 was exclusively found in neuronal elements. At daytime, it was detected throughout the dorsoventral extent of the SCN, partly within neurons synthesizing either arginine-vasopressin or vasoactive intestinal peptide (VIP). At night time, it was segregated in the ventrolateral aspect of the nucleus, within a cluster of cells 45% of which were gastrin-releasing peptide (GRP) neurons with or without co-localization with VIP. After a light pulse at night, expression of P-ERK1/2 increased in GRP neurons but also appeared in a population of neurons that stained for VIP only. These data show that the GRP neurons are closely associated with ERK1/2 activation at night and point to the importance of ERK1/2 signalling not only in intra-SCN transmission of photic information but also in maintenance of neuronal rhythms in the SCN.

Juxtacellular/multiunit recordings from head-restraint rats performing forelimb movement task

Single neuronal juxtacellular recording with simultaneous cortical electroencephalogram (EEG) in whole-animal preparations in vivo has allowed the study of the behaviour of individual neurons in relation to whole brain activity. Data on single neuron firing, neural synchrony, network behaviour and their responses to pharmacological agents can be obtained with dual recordings. However, pharmacological effects on cellular and network activity during paired single-unit recordings have not been possible due to the difficulties in maintaining recordings of two cells for a prolonged period. Here, we describe a method of maintaining stable dual cell juxtacellular recordings from distinct brain regions, allowing the assessment of single unit activity before, during and after the intracerebroventricular (ICV) injection of drugs. Data collection using this technique allows correlation both between the two cells and with whole-brain EEG, and their responses to pharmacological interventions. This is particularly useful for the investigation of the effects of anti-epileptic drugs on animal models of epilepsy, where single unit activity of two cells from distinct regions can be correlated with each other and with whole-brain activity during pre-ictal, ictal and interictal states. We also describe standardised analytical methods of quantifying cell firing patterns, the rhythmicity of individual neurons and the synchronicity of firing between two neurons in ictal and interictal periods and their responses to drug exposure.

GABA and Gi/o differentially control circadian rhythms and synchrony in clock neurons.

Neurons in the mammalian suprachiasmatic nuclei (SCN) generate daily rhythms in physiology and behavior, but it is unclear how they maintain and synchronize these rhythms in vivo. We hypothesized that parallel signaling pathways in the SCN are required to synchronize rhythms in these neurons for coherent output. We recorded firing and clock-gene expression patterns while blocking candidate signaling pathways for at least 8 days. GABA(A) and GABA(B) antagonism increased circadian peak firing rates and rhythm precision of cultured SCN neurons, but G(i/o) did not impair synchrony or rhythmicity. In contrast, inhibiting G(i/o) with pertussis toxin abolished rhythms in most neurons and desynchronized the population, phenocopying the loss of vasoactive intestinal polypeptide (VIP). Daily VIP receptor agonist treatment restored synchrony and rhythmicity to VIP(-/-) SCN cultures during continuous GABA receptor antagonism but not during G(i/o) blockade. Pertussis toxin did not affect circadian cycling of the liver, suggesting that G(i/o) plays a specialized role in maintaining SCN rhythmicity. We conclude that endogenous GABA controls the amplitude of SCN neuronal rhythms by reducing daytime firing, whereas G(i/o) signaling suppresses nighttime firing, and it is necessary for synchrony among SCN neurons. We propose that G(i/o), not GABA activity, converges with VIP signaling to maintain and coordinate rhythms among SCN neurons.

Disrupted Neuronal Activity Rhythms in the Suprachiasmatic Nuclei of Vasoactive Intestinal Polypeptide-Deficient Mice

Vasoactive intestinal polypeptide (VIP), acting via the VPAC(2) receptor, is a key signaling pathway in the suprachiasmatic nuclei (SCN), the master clock controlling daily rhythms in mammals. Most mice lacking functional VPAC(2) receptors are unable to sustain behavioral rhythms and lack detectable SCN electrical rhythms in vitro. Adult mice that do not produce VIP (VIP/PHI(-/-)) exhibit less severe alterations in wheel-running rhythms, but the effects of this deficiency on the amplitude, phasing, or periodicity of their SCN cellular rhythms are unknown. To investigate this, we used suction electrodes to extracellularly record multiple- and single-unit electrical activity in SCN brain slices from mice with varying degrees of VIP deficiency, ranging from wild-type (VIP/PHI(+/+)) to heterozygous (VIP/PHI(+/-)) and VIP/PHI(-/-) animals. We found decreasing proportions of rhythmic cells in SCN slices from VIP/PHI(+/+) ( approximately 91%, n = 23) through VIP/PHI(-/+) ( approximately 71%, n = 28) to VIP/PHI(-/-) mice (62%; n = 37) and a parallel trend toward decreasing amplitude in the remaining rhythmic cells. SCN neurons from VIP/PHI(-/-) mice exhibited a broad range in the period and phasing of electrical rhythms, concordant with the known alterations in their behavioral rhythms. Further, treatment of VIP/PHI(-/-) slices with a VPAC(2) receptor antagonist significantly reduced the proportion of oscillating neurons, suggesting that VPAC(2) receptors still become activated in the SCN of these mice. The results establish that VIP is important for appropriate periodicity and phasing of SCN neuronal rhythms and suggest that residual VPAC(2) receptor signaling promotes rhythmicity in adult VIP/PHI(-/-) mice.

The pre-Bötzinger oscillator in the mouse embryo

Studies of the sites and mechanisms involved in mammalian respiratory rhythm generation point to two clusters of rhythmic neurons forming a coupled oscillator network within the brainstem. The location of these oscillators, the pre-Bötzinger complex (preBötC) at vagal level, and the para-facial respiratory group at facial level, probably result from regional patterning schemes specifying neural types in the hindbrain during embryogenesis. Here, we report evidence that the preBötC oscillator (i) is first active at embryonic stages, (ii) originates in the post-otic hindbrain neural tube and (iii) requires the glutamate vesicular transporter 2 for rhythm generation.

Unraveling cell processes: interference imaging interwoven with data analysis.

The paper presents results on the application of interference microscopy and wavelet-analysis for cell visualization and studies of cell dynamics. We demonstrate that interference imaging of erythrocytes can reveal reorganization of the cytoskeleton and inhomogenity in the distribution of hemoglobin, and that interference imaging of neurons can show intracellular compartmentalization and submembrane structures. We investigate temporal and spatial variations of the refractive index for different cell types: isolated neurons, mast cells and erythrocytes. We show that the refractive dynamical properties differ from cell type to cell type and depend on the cellular compartment. Our results suggest that low frequency variations (0.1-0.6 Hz) result from plasma membrane processes and that higher frequency variations (20-26 Hz) are related to the movement of vesicles. Using double-wavelet analysis, we study the modulation of the 1 Hz rhythm in neurons and reveal its changes under depolarization and hyperpolarization of the plasma membrane. We conclude that interference microscopy combined with wavelet analysis is a useful technique for non-invasive cell studies, cell visualization, and investigation of plasma membrane properties.

Imaging of respiratory-related population activity with single-cell resolution

The pre-Bötzinger complex (PBC) in the rostral ventrolateral medulla contains a kernel involved in respiratory rhythm generation. So far, its respiratory activity has been analyzed predominantly by electrophysiological approaches. Recent advances in fluorescence imaging now allow for the visualization of neuronal population activity in rhythmogenic networks. In the respiratory network, voltage-sensitive dyes have been used mainly, so far, but their low sensitivity prevents an analysis of activity patterns of single neurons during rhythmogenesis. We now have succeeded in using more sensitive Ca(2+) imaging to study respiratory neurons in rhythmically active brain stem slices of neonatal rats. For the visualization of neuronal activity, fluo-3 was suited best in terms of neuronal specificity, minimized background fluorescence, and response magnitude. The tissue penetration of fluo-3 was improved by hyperosmolar treatment (100 mM mannitol) during dye loading. Rhythmic population activity was imaged with single-cell resolution using a sensitive charge-coupled device camera and a x20 objective, and it was correlated with extracellularly recorded mass activity of the contralateral PBC. Correlated optical neuronal activity was obvious online in 29% of slices. Rhythmic neurons located deeper became detectable during offline image processing. Based on their activity patterns, 74% of rhythmic neurons were classified as inspiratory and 26% as expiratory neurons. Our approach is well suited to visualize and correlate the activity of several single cells with respiratory network activity. We demonstrate that neuronal synchronization and possibly even network configurations can be analyzed in a noninvasive approach with single-cell resolution and at frame rates currently not reached by most scanning-based imaging techniques.

Differential control of active and silent phases in relaxation models of neuronal rhythms

Rhythmic bursting activity, found in many biological systems, serves a variety of important functions. Such activity is composed of episodes, or bursts (the active phase, AP) that are separated by quiescent periods (the silent phase, SP). Here, we use mean field, firing rate models of excitatory neural network activity to study how AP and SP durations depend on two critical network parameters that control network connectivity and cellular excitability. In these models, the AP and SP correspond to the network's underlying bistability on a fast time scale due to rapid recurrent excitatory connectivity. Activity switches between the AP and SP because of two types of slow negative feedback: synaptic depression-which has a divisive effect on the network input/output function, or cellular adaptation-a subtractive effect on the input/output function. We show that if a model incorporates the divisive process (regardless of the presence of the subtractive process), then increasing cellular excitability will speed up the activity, mostly by decreasing the silent phase. Reciprocally, if the subtractive process is present, increasing the excitatory connectivity will slow down the activity, mostly by lengthening the active phase. We also show that the model incorporating both slow processes is less sensitive to parameter variations than the models with only one process. Finally, we note that these network models are formally analogous to a type of cellular pacemaker and thus similar results apply to these cellular pacemakers.

Neuronal Electrical Rhythms Described by Composite Mapped Clock Oscillators

The Mapped Clock Oscillator (MCO) model is a representation of omnipresent transmembrane voltage oscillations in excitable cells. We present a generalized version of the MCO that can model neuronal electrical oscillations, both labile and omnipresent, entirely within the framework of a system of ordinary differential equations. The previously described MCO was a second-order system, whereas the model presented here, which we call the composite MCO (cMCO) is a fourth-order system. Furthermore, we show how this cMCO can also be adapted to describe a pair of cells that forms a functional unit, as illustrated here by a model of the CA3 pyramidal cell and its basket cell interneuron feedback loop. The model was able to reproduce the high frequencies (super gamma) and possibly chaotic dynamics observed in the biological system.

Cytoarchitecture of Pneumotaxic Integration of Respiratory and Nonrespiratory Information in the Rat

The "pneumotaxic center" in the Kölliker-Fuse and medial parabrachial nuclei of dorsolateral pons (dl-pons) plays an important role in respiratory phase switching, modulation of respiratory reflex, and rhythmogenesis. Recent electrophysiological and neural tracing data implicate additional pneumotaxic nuclei in (and a broader role for) the dl-pons in integrating respiratory and nonrespiratory information. Here, we examined the cytoarchitecture of the greater pneumotaxic center and its integrating function by using combined extracellular recording and juxtacellular labeling of unit respiratory rhythmic neurons in dl-pons in urethane-anesthetized, vagotomized, paralyzed, and servo-ventilated adult Sprague Dawley rats. Perievent histogram analysis identified four major types of neuronal discharge patterns: inspiratory, expiratory (with three subdivisions), inspiratory-expiratory, and expiratory-inspiratory phase spanning, sometimes with mild tonic background activity. Most recorded neurons were localized in the Kölliker-Fuse and medial parabrachial nuclei, but some were also found in lateral parabrachial nucleus, intertrigeminal nucleus, principal trigeminal sensory nucleus, and supratrigeminal nucleus. The majority of labeled neurons had large and spatially extended dendritic trees that spanned several of these dl-pons subnuclei, often with terminal dendrites ending in the ventral spinocerebellar tract. The distal sections of the primary and higher-order dendrites exhibited rich varicosities, sometimes with dendritic spines. Axons of some labeled neurons were traced all the way to the ventrolateral pons (vl-pons). These findings extend and generalize the classical definition of the pneumotaxic center to include extensive somatic-axonal-dendritic integration of complex descending and ascending respiratory information as well as nociceptive and possibly musculoskeletal and trigeminal information in multiple dl-pons and vl-pons structures in the rat.

GABAA Receptors in Central Nervous System Disease: Anxiety, Epilepsy, and Insomnia

Brain function is based on an exquisite balance between excitatory and inhibitory neurotransmission. GABAergic neurons provide the major inhibitory control. By controlling spike timing and sculpting neuronal rhythms they play a key role in regulating behavior. GABAergic neurons are highly diverse and operate with a corresponding diversity of GABAA receptor subtypes. In this article, the contribution of GABAA receptor deficits to central nervous system disorders, in particular anxiety disorders, epilepsy, schizophrenia and insomnia, is reviewed.

Single-Cell Imaging

Single-Cell Imaging

Structural daily rhythms in GFP-labelled neurons in the visual system of Drosophila melanogaster

In the visual system of Drosophila melanogaster, two classes of interneurons in the first optic neuropil, or lamina, the monopolar cells L1 and L2, show rhythmic circadian changes in the shape and size of their axons. In the present study we have used the GAL4-UAS system to target the GFP expression to the L2 cells in D. melanogaster and to examine morphological changes in the cell body, nucleus, axon and dendritic spines. Our results showed that in addition to changes in the caliber of its axon, L2 also shows daily changes in the morphology of its dendritic spines, differences which are most pronounced at the beginning of the night. There are also changes in the sizes of the cells' nuclei in the lamina cortex, which are largest at the beginning and in the middle of day, in females and males, respectively. In contrast to the axon and dendrites, L2's soma does not change size significantly during the day or night. The observed changes clearly indicate the cyclical modulation of the structure of the L2 interneurons. These changes seem to be regulated by a circadian clock, which exhibits certain differences between the sexes.

Subthreshold membrane potential oscillations as a neuronal disease mechanism: implications from sensory receptors, nociceptive neurons and limbic neurons

Many neurons of the peripheral and central nervous system exhibit oscillations in membrane potential that operate close to the spike threshold. Such neurons are e.g. found in the amygdala, the entorhinal cortex, in nociceptice dorsal root ganglion (DRG) cells and peripheral sensory receptors. In the latter, the oscillations are used for sensitive and differential sensory stimulus encoding and in the cortex the oscillations are presumably used for collective neuronal rhythms (e.g. limbic theta rhythm) and neuronal synchronization and resonance behaviors. In nocieptive DRG cells, these oscillation occur as result to nerve injury and are discussed as a necessary mechanisms for ectopic spike generation and hence neuropathic pain. We were interested to assess the principal mechanisms underlying such oscillations and their resulting modulatory abilities from the perspective of a disease mechanism. In doing so we made use of existing computational models for subthreshold oscillatory neurons and simulated the responses in dependence on (i) synaptic depolarisation (ii) noise, (iii) ionic conductances and (iv) temperature. Our results agree very well with the experimental findings and point to a potential role of subthreshold oscillations in disease states such as neuropathic pain and also within context of pathology involving the amygdala or entorhinal cortex.

Neuroeffector Connections of Giant Multimodal Neurons in the African Snail Achatina Fulica

A new method of making preparations was used to analyse the neuroeffector connections of the paired giant neurons of the African snail Achatina fulica. These neurons were found to induce postsynaptic potentials in the muscles of the mantle, heart, the wall of the pulmonary cavity, and the muscular elements of the renal complex, the pericardium, the sexual apparatus, the walls of the cerebral arteries, the filaments of the columellar muscles, the wall of the abdomen, and the tentacle retractor muscles. Rhythmic neuron activity led to the development of marked facilitation and long-term potentiation of synaptic potentials. The possible significance of the multiple neuroeffector connections of giant neurons is discussed.