A Model for the Fast Synchronous Oscillations of Firing Rate in Rat Suprachiasmatic Nucleus Neurons Cultured in a Multielectrode Array Dish

When dispersed and cultured in a multielectrode dish (MED), suprachiasmatic nucleus (SCN) neurons express fast oscillations of firing rate (FOFR; fast relative to the circadian cycle), with burst duration ∼10 min, and interburst interval varying from 20 to 60 min in different cells but remaining nevertheless rather regular in individual cells. In many cases, separate neurons in distant parts of the 1 mm recording area of a MED exhibited correlated FOFR. Neither the mechanism of FOFR nor the mechanism of their synchronization among neurons is known. Based on recent data implicating vasoactive intestinal polypeptide (VIP) as a key intercellular synchronizing agent, we built a model in which VIP acts as both a feedback regulator to generate FOFR in individual neurons, and a diffusible synchronizing agent to produce coherent electrical output of a neuronal network. In our model, VIP binding to its (VPAC₂) receptors acts through G_s G-proteins to activate adenylyl cyclase (AC), increase intracellular cAMP, and open cyclic-nucleotide-gated (CNG) cation channels, thus depolarizing the cell and generating neuronal firing to release VIP. In parallel, slowly developing homologous desensitization and internalization of VPAC₂ receptors terminates elevation of cAMP and thereby provides an interpulse silent interval. Through mathematical modeling, we show that this VIP/VPAC₂/AC/cAMP/CNG-channel mechanism is sufficient for generating reliable FOFR in single neurons. When our model for FOFR is combined with a published model of synchronization of circadian rhythms based on VIP/VPAC₂ and Per gene regulation synchronization of circadian rhythms is significantly accelerated. These results suggest that (a) auto/paracrine regulation by VIP/VPAC₂ and intracellular AC/cAMP/CNG-channels are sufficient to provide robust FOFR and synchrony among neurons in a heterogeneous network, and (b) this system may also participate in synchronization of circadian rhythms.     

MULTIPLE OSCILLATORS IN THE SUPRACHIASMATIC NUCLEUS

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371–387, 2001)     

Fibroblast Circadian Rhythms of PER2 Expression Depend on Membrane Potential and Intracellular Calcium

The suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes circadian rhythms of cells and tissues throughout the body. In SCN neurons, rhythms of clock gene expression are suppressed by manipulations that hyperpolarize the plasma membrane or lower intracellular Ca²⁺. However, whether clocks in other cells also depend on membrane potential and calcium is unknown. In this study, the authors investigate the effects of membrane potential and intracellular calcium on circadian rhythms in mouse primary fibroblasts. Rhythms of clock gene expression were monitored using a PER2::LUC knockin reporter. Rhythms were lost or delayed at lower (hyperpolarizing) K⁺ concentrations. Bioluminescence imaging revealed that this loss of rhythmicity in cultures was due to loss of rhythmicity of single cells rather than loss of synchrony among cells. In lower Ca²⁺ concentrations, rhythms were advanced or had shorter periods. Buffering intracellular Ca²⁺ by the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) or manipulation of inositol triphosphate (IP₃)-sensitive intracellular calcium stores by thapsigargin delayed rhythms. These results suggest that the circadian clock in fibroblasts, as in SCN neurons, is regulated by membrane potential and Ca²⁺. Changes in intracellular Ca²⁺ may mediate the effects of membrane potential observed in this study. (Author correspondence: welshdk@ucsd.edu)     

LACK OF CALBINDIN-D28K ALTERS RESPONSE OF THE MURINE CIRCADIAN CLOCK TO LIGHT

A strong stimulus adjusting the circadian clock to the prevailing light-dark cycle is light. However, the circadian clock is reset by light only at specific times of the day. The mechanisms mediating such gating of light input to the CNS are not well understood. There is evidence that Ca²⁺ ions play an important role in intracellular signaling mechanisms, including signaling cascades stimulated by light. Therefore, Ca²⁺ is hypothesized to play a role in the light-mediated resetting of the circadian clock. Calbindin-D28k (CB; gene symbol: Calb1) is a Ca²⁺ binding protein implicated in Ca²⁺ homeostasis and sensing. The absence of this protein influences Ca²⁺ buffering capacity of a cell, alters spatio-temporal aspects of intracellular Ca²⁺ signaling, and hence might alter transmission of light information to the circadian clock in neurons of the suprachiasmatic nuclei (SCN). We tested mice lacking a functional Calb1 gene (Calb1^?/?) and found an increased phase-delay response to light applied at circadian time (CT) 14 in these animals. This is accompanied by elevated induction of Per2 gene expression in the SCN. Period length and circadian rhythmicity were comparable between Calb1^?/? and wild-type animals. Our findings indicate an involvement of CB in the signaling pathway that modulates the behavioral and molecular response to light. (Author correspondence: urs.albrecht@unifr.ch)     

Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca2+ Oscillations in Striatum

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson''s disease. The group I mGluRs are known to modulate the intracellular Ca²⁺ signaling. To characterize Ca²⁺ signaling in striatal cells, spontaneous cytoplasmic Ca²⁺ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca²⁺ transients (referred to as slow Ca²⁺ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca²⁺ oscillation. Depletion of the intracellular Ca²⁺ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca²⁺ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. The slow Ca²⁺ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca²⁺ oscillation may involve in the neuron-glia interaction in the striatum.     

Duck Pancreatic Acinar Cell as a Unique Model for Independent Cholinergic Stimulation–Secretion Coupling

This paper investigated the role of acetylcholine (ACh) in physiological regulation of amylase secretion in avian exocrine pancreas. In the isolated duck pancreatic acini, ACh dose dependently stimulated amylase secretion, with a maximal effective concentration at 10 μM. The cAMP-mobilizing compounds forskolin, vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 had no effect on the dose–response curve. ACh dose dependently induced increases in cytosolic Ca²⁺ concentration ([Ca²⁺] _c ), with increasing concentrations transforming oscillations into plateau increases. Forskolin (10 μM), PACAP-38 (1 nM), PACAP-27 (1 nM), or VIP (10 nM) alone did not stimulate [Ca²⁺] _c increase; neither did they modulate ACh-induced oscillations, nor made ACh low concentration effective. These data indicate that ACh-stimulated zymogen secretion in duck pancreatic acinar cells is not subject to modulation from the cAMP signaling pathway; whereas it has been widely reported in the rodents that ACh-stimulated exocrine pancreatic secretion is significantly enhanced by cAMP-mobilizing agents. This makes the duck exocrine pancreas unique in that cholinergic stimulus-secretion coupling is not subject to cAMP regulation.     

Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF''s importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca²⁺ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca²⁺ baseline concentration and frequency of oscillating Ca²⁺ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca²⁺ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca²⁺ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K⁺ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K⁺ and Na⁺ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.     

GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in intracellular Ca2+ modulation

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca²⁺ ([Ca²⁺]_i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABA_AR and GABA_BR) with the specific agonists muscimol and baclofen, respectively induces [Ca²⁺]_i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca²⁺. Bicuculline did not change the GABA-evoked Ca²⁺ responses in Ca²-containing buffers, but suppressed them significantly in Ca²⁺-free buffers suggesting the two receptors couple to convergent Ca²⁺ mobilization mechanisms with different extracellular Ca²⁺ sensitivity. Prolonged activation of GABA_BR induced wave propagation of the Ca²⁺ signal and persistent oscillations. The number of cells reacting to GABA or GABA + bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABA_AR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca²⁺ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca²⁺ signaling may be involved in a variety of Ca²⁺-dependent cellular processes during lens growth and epithelial cell differentiation.     

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.     

Multiple oscillators in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of the pacemaker that controls circadian rhythms of a variety of physiological functions. Data strongly indicate the majority of the SCN neurons express self-sustaining oscillations that can be detected as rhythms in the spontaneous firing of individual neurons. The period of single SCN neurons in a dissociated cell culture is dispersed in a wide range (from 20h to 28h in rats), but that of the locomotor rhythm is close to 24h, suggesting individual oscillators are coupled to generate an averaged circadian period in the nucleus. Electrical coupling via gap junctions, glial regulation, calcium spikes, ephaptic interactions, extracellular ion flux, and diffusible substances have been discussed as possible mechanisms that mediate the interneuronal rhythm synchrony. Recently, GABA (γ-aminobutyric acid), a major neurotransmitter in the SCN, was reported to regulate cellular communication and to synchronize rhythms through GABA_A receptors. At present, subsequent intracellular processes that are able to reset the genetic loop of oscillations are unknown. There may be diverse mechanisms for integrating the multiple circadian oscillators in the SCN. This article reviews the knowledge about the various circadian oscillations intrinsic to the SCN, with particular focus on the intercellular signaling of coupled oscillators. (Chronobiology International, 18(3), 371-387, 2001)     

Multiscale Model of Dynamic Neuromodulation Integrating Neuropeptide-Induced Signaling Pathway Activity with Membrane Electrophysiology

We developed a multiscale model to bridge neuropeptide receptor-activated signaling pathway activity with membrane electrophysiology. Typically, the neuromodulation of biochemical signaling and biophysics have been investigated separately in modeling studies. We studied the effects of Angiotensin II (AngII) on neuronal excitability changes mediated by signaling dynamics and downstream phosphorylation of ion channels. Experiments have shown that AngII binding to the AngII receptor type-1 elicits baseline-dependent regulation of cytosolic Ca²⁺ signaling. Our model simulations revealed a baseline Ca²⁺-dependent response to AngII receptor type-1 activation by AngII. Consistent with experimental observations, AngII evoked a rise in Ca²⁺ when starting at a low baseline Ca²⁺ level, and a decrease in Ca²⁺ when starting at a higher baseline. Our analysis predicted that the kinetics of Ca²⁺ transport into the endoplasmic reticulum play a critical role in shaping the Ca²⁺ response. The Ca²⁺ baseline also influenced the AngII-induced excitability changes such that lower Ca²⁺ levels were associated with a larger firing rate increase. We examined the relative contributions of signaling kinases protein kinase C and Ca²⁺/Calmodulin-dependent protein kinase II to AngII-mediated excitability changes by simulating activity blockade individually and in combination. We found that protein kinase C selectively controlled firing rate adaptation whereas Ca²⁺/Calmodulin-dependent protein kinase II induced a delayed effect on the firing rate increase. We tested whether signaling kinetics were necessary for the dynamic effects of AngII on excitability by simulating three scenarios of AngII-mediated K_DR channel phosphorylation: (1), an increased steady state; (2), a step-change increase; and (3), dynamic modulation. Our results revealed that the kinetics emerging from neuromodulatory activation of the signaling network were required to account for the dynamical changes in excitability. In summary, our integrated multiscale model provides, to our knowledge, a new approach for quantitative investigation of neuromodulatory effects on signaling and electrophysiology.     

Transient Activity of Excitatory Cl− Channels in Chara: Evidence for Quantal Release of a Gating Factor

In attached patches on the plasma membrane of nonexcited Chara corallina cells, randomly activating, transient Cl⁻ currents with variable amplitudes were recorded. The peak amplitudes of these currents could be grouped into distinct populations with approximately equidistant mean peak currents. Generally, the mean current of the smallest population measured about half of the distance between the means of subsequent populations. Currents of the smallest population occurred most frequently at all voltages; the frequency of observations decreased with increasing amplitudes of the currents. At all voltages transient currents from different populations were similar in duration with the exception of the smallest currents, which lasted only 0.6 times as long as larger currents. Furthermore, transient currents were most frequent at positive voltages, but once initiated at a positive conditioning pulse they were also observed during subsequent pulses to negative voltages. The results are consistent with the idea that Chara contains Ca²⁺ stores in the vicinity of the plasma membrane, which are indirectly filled from the external medium. Upon quantal Ca²⁺ discharge from adjacent stores, a process independent of membrane voltage, the concentration of Ca²⁺ in the cytoplasm increases transiently. Depending on the number of discharging stores, distinct numbers of Ca²⁺-stimulated Cl⁻ channels activate, giving rise to the macroscopic excitatory Cl⁻ current in these cells. Received: 27 October 1997/Revised: 26 February 1998     

A theoretical explanation for some effects of calcium on the facilitation of neurotransmitter release

A quantal model developed earlier by the authors is recast in terms of common macroscopic variables and applied to the well-documented T, P, N/L, AE neuron network of a leech ganglion. The electrical potential of a neuron (φ) and the ion potentials (φ_a) for Na⁺, K⁺, Cl^? and Ca²⁺ are featured, though it proves possible to reduce the resulting set of coupled non-linear diffusion equations to a single pair whose admissible solutions are defined by a simple algebraic dispersion relation. Less than 30 s is required to solve the system for a functional interval of 2·25 s on a CYBER 175 computer using a modified Runge-Kutta algorithm, the program for which is given. Irreversible effects are included but reversibility is stressed, since the neurons are seen to exchange energy with their environment only in the immediate neighborhood of firing peaks. Plasma oscillations, resulting from a disruption of the Debye layer, offer a sound physical mechanism whereby transient currents and ion exchanges of the observed magnitudes may be generated.The total energy H and information rate Γ_I transferred across the neural membrane are also calculated in terms of φ and φ_a. It is shown that while φ is determined primarily by the K⁺ potential, Γ_I depends mainly on the Ca²⁺ potential together with its time derivatives, and H depends on both the K⁺ and Ca²⁺ potentials. This also makes it possible, not only to compare φ (t) solutions for each of the neurons in the incrementally-loaded network to experimental measurements of φ(t) made for similar stimulus levels, but also to trace the correlated flows of energy and information through the system. Nearly all of the distinctive features of the experimental curves are reproduced, despite the presence of such complexities as wide variations in pulse frequencies and amplitudes, sudden suppression of firing in one neuron when another begins to fire, refractory phases of different durations, and facilitation building to plateau values only slightly less than peak amplitudes for the sensory neurons.While both the energy and information curves possess sharp maxima which coincide with the firing pulses of the potential curves, those for Γ_I (t) are bimodal with rounded maxima that must represent transmissions associated with ion motions instead of polarization effects. In the case of the L and AE neurons, these curves exhibit a series of discrete energy/information packets that could easily produce the proportional increases in muscle tension actually observed.     

Simulation of Spontaneous Ca Oscillations in Astrocytes Mediated by Voltage-Gated Calcium Channels

The purpose of this computational study was to investigate the possible role of voltage-gated Ca²⁺ channels in spontaneous Ca²⁺ oscillations of astrocytes. By incorporating different types of voltage-gated Ca²⁺ channels and a previous model, this study reproduced typical Ca²⁺ oscillations in silico. Our model could mimic the oscillatory phenomenon under a wide range of experimental conditions, including resting membrane potential (−75 to −60 mV), extracellular Ca²⁺ concentration (0.1 to 1500 μM), temperature (20 to 37°C), and blocking specific Ca²⁺ channels. By varying the experimental conditions, the amplitude and duration of Ca²⁺ oscillations changed slightly (both <25%), while the frequency changed significantly (∼400%). This indicates that spontaneous Ca²⁺ oscillations in astrocytes might be an all-or-none process, which might be frequency-encoded in signaling. Moreover, the properties of Ca²⁺ oscillations were found to be related to the dynamics of Ca²⁺ influx, and not only to a constant influx. Therefore, calcium channels dynamics should be used in studying Ca²⁺ oscillations. This work provides a platform to explore the still unclear mechanism of spontaneous Ca²⁺ oscillations in astrocytes.     

Bursting, chaos and birhythmicity originating from self-modulation of the inositol 1,4,5-trisphosphate signal in a model for intracellular Ca2+ oscillations

We investigate the various types of complex Ca²⁺ oscillations which can arise in a model based on the mechanism of Ca²⁺-induced Ca²⁺ release (CICR), that takes into account the Ca²⁺-stimulated degradation of inositol 1,4,5-trisphosphate (InsP₃) by a 3-kinase. This model was previously proposed in the course of an investigation of plausible mechanisms capable of generating complex Ca²⁺ oscillations (Borghans et al., 1997). Besides simple periodic behavior, this model for cytosolic Ca²⁺ oscillations in nonexcitable cells shows complex oscillatory phenomena like bursting or chaos. We show that the model also admits a coexistence between two stable regimes of sustained oscillations (birhythmicity). The occurrence of these various modes of oscillatory behavior is analysed by means of bifurcation diagrams. Complex oscillations are characterized by means of Poincaré sections, power spectra and Lyapounov exponents. The results point to the role of self-modulation of the InsP₃ signal by 3-kinase as a possible source for complex temporal patterns in Ca²⁺ signaling.