Diabetes-Induced Amplification of Nociceptive DRG Neuron Output by Upregulation of Somatic T-Type Ca2+ Channels.

The development of pain symptoms in peripheral diabetic neuropathy (PDN) is associated with the upregulation of T-type Ca2+ channels (T-channels) in the soma of nociceptive DRG neurons. Moreover, a block of these channels in DRG neurons effectively reversed mechanical and thermal hyperalgesia in animal diabetic models, indicating that T-channel functioning in these neurons is causally linked to PDN. However, no particular mechanisms relating the upregulation of T-channels in the soma of nociceptive DRG neurons to the pathological pain processing in PDN have been suggested. Here we have electrophysiologically identified voltage-gated currents expressed in nociceptive DRG neurons and developed a computation model of the neurons, including peripheral and central axons. Simulations showed substantially stronger sensitivity of neuronal excitability to diabetes-induced T-channel upregulation at the normal body temperature compared to the ambient one. We also found that upregulation of somatic T-channels, observed in these neurons under diabetic conditions, amplifies a single action potential invading the soma from the periphery into a burst of multiple action potentials further propagated to the end of the central axon. We have concluded that the somatic T-channel-dependent amplification of the peripheral nociceptive input to the spinal cord demonstrated in this work may underlie abnormal nociception at different stages of diabetes development.

Measurement of intracellular concentration of fluorescently-labeled targets in living cells.

Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells.

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 Gs 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.

Fast synchronous oscillations of firing rate in cultured rat suprachiasmatic nucleus neurons: possible role in circadian synchronization in the intact nucleus.

The coherent circadian rhythm of the brain's master circadian pacemaker, the suprachiasmatic nucleus (SCN), is a result of synchronization of electrical activity of many SCN neurons possessing their own circadian oscillators. However, how the activity of these neurons is synchronized is not precisely known. By plotting the electrical firing rates of dispersed rat SCN neurons in multi-electrode array dishes with 20-s averaging of action-potential activity, we have investigated a novel phenomenon: fast (relative to the circadian cycle) oscillations of firing rate (FOFR) with duration of bursts ∼10min and interburst interval varying in a range from 20 to 60min in different cells, remaining nevertheless rather regular in individual cells. In many cases, separate neurons in distant parts of the 1mm recording area of an array exhibited correlated FOFR. FOFR of individual cells were positively or negatively correlated with those of other cells in a functioning neural network. Intriguingly, in occasional neuron pairs, transformation of their irregular firing to circadian peaks was accompanied by appearance of FOFR and an increase in the magnitude of firing correlation. We hypothesize that this FOFR observed in cultured SCN neurons contribute to synchronization of the circadian rhythm in the intact SCN.

Modeling the spontaneous activity in suprachiasmatic nucleus neurons: role of cation single channels.

A population of interconnected neurons of the mammalian suprachiasmatic nuclei (SCN) controls circadian rhythms in physiological functions. In turn, a circadian rhythm of individual neurons is driven by intracellular processes, which via activation of specific membrane channels, produce circadian modulation of electrical firing rate. Yet the membrane target(s) of the cellular clock have remained enigmatic. Previously, subthreshold voltage-dependent cation (SVC) channels have been proposed as the membrane target of the cellular clock responsible for circadian modulation of the firing rate in SCN neurons. We tested this hypothesis with computational modeling based on experimental results from on-cell recording of SVC channel openings in acutely isolated SCN neurons and long-term continuous recording of activity from dispersed SCN neurons in a multielectrode array dish (MED). The model reproduced the circadian behavior if the number of SVC channels or their kinetics were modulated in accordance with protein concentration in a model of the intracellular clock (Scheper et al., 1999. J. Neurosci. 19, 40-47). Such modulation changed the average firing rate of the model neuron from zero ("subjective-night" silence) up to 18 Hz ("subjective-day" peak). Furthermore, the variability of interspike intervals (ISI) and the circadian pattern of firing rate (i.e. silence-to-activity ratio and shape of circadian peaks) are in reasonable agreement with experimental data obtained in dispersed SCN neurons in MED. These results suggest that the variability of ISI in intact SCN neurons is mostly due to stochastic single-channel openings, and that the circadian pattern of the firing rate is specified by threshold properties of dependence of the spontaneous firing rate on the number of single channels (R-N relationship). This plausible mathematical modeling supports the hypothesis that SVC channels could be a critical element in circadian modulation of firing rate in SCN neurons.

On the role of calcium and potassium currents in circadian modulation of firing rate in rat suprachiasmatic nucleus neurons: multielectrode dish analysis.

The master circadian clock of mammals in the suprachiasmatic nucleus (SCN) of the hypothalamus entrains to a 24-h daily light-dark cycle and regulates circadian rhythms. The SCN is composed of multiple neurons with cell autonomous clocks exhibiting robust firing rhythms with a high firing rate during the subjective day. The membrane target(s) of the cellular clock responsible for circadian modulation of the firing rate in SCN neurons still remain unclear. Previously, L-type Ca(2+) currents and fast delayed rectifier (FDR) K(+) currents have been suggested to contribute directly to circadian modulation of electrical activity. Using long-term continuous recording of activity from dispersed rat SCN neurons in multielectrode dish and ionic channel blockers, we tested these hypotheses. Neither an L-type Ca(2+) current blocker (20 microM of nifedipine for 2 days) nor an FDR current blocker (500 microM of 4-aminopyridine (4-AP) for 4 days) suppressed the circadian modulation of firing rate. A specific blocker of Na(+) persistent current (5 microM of riluzole for 1 day followed by 10 microM during the next day) reversibly suppressed firing activity in a dose-dependent manner. These data indicate that neither nifedipine-sensitive Ca(2+) current(s) nor 4-AP-sensitive K(+) current(s) are key membrane targets for circadian modulation of electrical firing rate in SCN neurons.

Circadian difference in firing rate of isolated rat suprachiasmatic nucleus neurons.

The suprachiasmatic nucleus (SCN) of the hypothalamus contains the primary circadian clock in mammals. Dissociated SCN neurons in long-term culture exhibit a circadian modulation of spontaneous electrical activity. To evaluate the presence of circadian differences in spontaneous activity of isolated SCN neurons without synaptic connections, dissociated rat SCN neurons were studied with on-cell recording 3-4 days after preparation, before the formation of dendrites, axons and synapses. A day-night difference in spontaneous electrical firing rate was found in acutely dissociated SCN neurons. During the first subjective day, the average firing rate (0.87+/-0.12 Hz) was significantly higher than during the first subjective night (0.24+/-0.06 Hz), while the firing rate on the next day (0.68+/-0.11 Hz) was significantly higher than during the preceding night. These data suggest that populations of isolated SCN neurons with no synaptic interactions contain a functioning circadian clock, and are particularly amenable to biophysical experiments.

Noise of the slowly inactivating Na current in suprachiasmatic nucleus neurons.

Slow depolarization induces a riluzole-sensitive persistent Na current (INa,P) in several types of neurons and a pharmacologically similar slowly inactivating Na current (INa,S) in rat suprachiasmatic nucleus neurons. In isolated suprachiasmatic nucleus neurons, INa,S fluctuations were analyzed to characterize the Na channel responsible for INa,S. The single-channel current near resting potential was -0.53+/-0.04 pA in an external solution containing 151 mM Na+ and 5 mM Na+ in the patch pipette. Tetrodotoxin completely blocked INa,S and single-channel current noise; 25 microM riluzole also suppressed INa,S and current noise by about 80% without a significant effect on mean single-channel current. The data suggest that single-channel noise is due to the opening of channels mediating INa,S with a conductance of about 3.4 pS.

Mechanism of irregular firing of suprachiasmatic nucleus neurons in rat hypothalamic slices.

The mechanisms of irregular firing of spontaneous action potentials in neurons from the rat suprachiasmatic nucleus (SCN) were studied in hypothalamic slices using cell-attached and whole cell recording. The firing pattern of spontaneous action potentials could be divided into regular and irregular, based on the interspike interval (ISI) histogram and the membrane potential trajectory between action potentials. Similar to previous studies, regular neurons had a firing rate about >3.5 Hz and irregular neurons typically fired about <3.5 Hz. The ISI of irregular-firing neurons was a linear function of the sum of inhibitory postsynaptic potentials (IPSPs) between action potentials. Bicuculline (10-30 microM) suppressed IPSPs and converted an irregular pattern to a more regular firing. Bicuculline also depolarized SCN neurons and induced bursting-like activity in some SCN neurons. Gabazine (20 microM), however, suppressed IPSPs without depolarization, and also converted irregular activity to regular firing. Thus GABAA receptor-mediated IPSPs appear responsible for irregular firing of SCN neurons in hypothalamic slices.

Riluzole-sensitive slowly inactivating sodium current in rat suprachiasmatic nucleus neurons.

The persistent (i.e., slowly inactivating) fraction of the Na current (I(Na,P)) regulates excitability of CNS neurons. In isolated rat suprachiasmatic nucleus (SCN) neurons with a ramp-type voltage-clamp protocol, we have studied the properties of a robust current that has the general properties of I(Na,P) but exhibits a slow inactivation (I(Na,S)). The time dependence of the development of the inactivation was also studied by clamping of the membrane potential at different levels: time constants ranging from approximately 50 to approximately 700 ms, depending on the voltage level, were revealed. The I(Na,S) (50-150 pA) was present in both spontaneously active and silent neurons. The neurons exhibited I(Na,S) without visible rundown during approximately 1-h recordings. I(Na,S) had a threshold between -65 and -60 mV and was maximal at about -45 mV. Tetrodotoxin (TTX; 1 microM) completely and reversibly blocked I(Na,S). Riluzole, an effective blocker of I(Na,P), inhibited reversibly I(Na,S) with an EC(50) of 1-2 microM. Microapplication of 10 microM riluzole during either extracellular or intracellular recording suppressed spontaneous activity in isolated SCN neurons. In the slice preparation, bath application of 20 microM riluzole resulted in decreased firing rate or complete suppression of spontaneous activity in some neurons (9/14) but had no effect on other neurons (5/14). In riluzole-resistant neurons in cell-attached experiments, low-amplitude current spikes were present in 1 microM TTX. We concluded that I(Na,S) is ubiquitously expressed by all SCN neurons and that this current is a necessary but not sufficient depolarizing component of the mechanism for spontaneous firing.