Jason N. Itri

Fast delayed rectifier potassium current is required for circadian neural activity

In mammals, the precise circadian timing of many biological processes depends on the generation of oscillations in neural activity of pacemaker cells in the suprachiasmatic nucleus (SCN). The ionic mechanisms that underlie these rhythms are largely unknown. Using the mouse brain slice preparation, we show that the magnitude of fast delayed rectifier (FDR) potassium currents has a diurnal rhythm that peaks during the day. Notably, this rhythm continues in constant darkness, providing the first demonstration of the circadian regulation of an intrinsic voltage-gated current in mammalian cells. Blocking this current prevented the daily rhythm in firing rate in SCN neurons. Kv3.1b and Kv3.2 potassium channels were widely distributed within the SCN, with higher expression during the day. We conclude that the FDR is necessary for the circadian modulation of electrical activity in SCN neurons and represents an important part of the ionic basis for the generation of rhythmic output.

Circadian Regulation of A-Type Potassium Currents in the Suprachiasmatic Nucleus

In mammals, the precise circadian timing of many biological processes depends on the generation of oscillations in neural activity of pacemaker cells in the suprachiasmatic nucleus (SCN) of the hypothalamus. Understanding the ionic mechanisms underlying these rhythms is an important goal of research in chronobiology. Previous work has shown that SCN neurons express A-type potassium currents (IAs), but little is known about the properties of this current in the SCN. We sought to characterize some of these properties, including the identities of IA channel subunits found in the SCN and the circadian regulation of IA itself. In this study, we were able to detect significant hybridization for Shal-related family members 1 and 2 (Kv4.1 and 4.2) within the SCN. In addition, we used Western blot to show that the Kv4.1 and 4.2 proteins are expressed in SCN tissue. We further show that the magnitude of the IA current exhibits a diurnal rhythm that peaks during the day in the dorsal region of the mouse SCN. This rhythm seems to be driven by a subset of SCN neurons with a larger peak current and a longer decay constant. Importantly, this rhythm in neurons in the dorsal SCN continues in constant darkness, providing an important demonstration of the circadian regulation of an intrinsic voltage-gated current in mammalian cells. We conclude that the anatomical expression, biophysical properties, and pharmacological profiles measured are all consistent with the SCN IA current being generated by Kv4 channels. Additionally, these data suggest a role for IA in the regulation of spontaneous action potential firing during the transitions between day/night and in the integration of synaptic inputs to SCN neurons throughout the daily cycle.

Regulation of glutamatergic signalling by PACAP in the mammalian suprachiasmatic nucleus

BackgroundPrevious studies indicate that light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells that contain both glutamate and pituitary adenylyl cyclase activating peptide (PACAP). While the role of glutamate in this pathway has been well studied, the involvement of PACAP and its receptors are only beginning to be understood. Speculating that PACAP may function to modulate how neurons in the suprachiasmatic nucleus respond to glutamate, we used electrophysiological and calcium imaging tools to examine possible cellular interactions between these co-transmitters.ResultsExogenous application of PACAP increased both the amplitude and frequency of spontaneous excitatory postsynaptic currents recorded from SCN neurons in a mouse brain slice preparation. PACAP also increased the magnitude of AMPA-evoked currents through a mechanism mediated by PAC1 receptors and the adenylyl cyclase-signalling cascade. This enhancement of excitatory currents was not limited to those evoked by AMPA as the magnitude of NMDA currents were also enhanced by application of PACAP. Furthermore, PACAP enhanced AMPA and NMDA evoked calcium transients while PACAP alone produced very little change in resting calcium in most mouse SCN neurons. Finally, in rat SCN neurons, exogenous PACAP enhanced AMPA evoked currents and calcium transients as well evoked robust calcium transients on its own.ConclusionThe results reported here show that PACAP is a potent modulator of glutamatergic signalling within the SCN in the early night.

Dopaminergic modulation of early signs of excitotoxicity in visualized rat neostriatal neurons

Cell swelling induced by activation of excitatory amino acid receptors is presumably the first step in a toxic cascade that may ultimately lead to cell death. Previously we showed that bath application of N‐methyl‐d‐aspartate (NMDA) or kainate (KA) produces swelling of neostriatal cells. The present experiments examined modulation of NMDA and KA‐induced cell swelling by dopamine (DA) and its receptor agonists. Nomarski optics and infra‐red videomicroscopy were utilized to visualize neostriatal medium‐sized neurons in thick slices from rat pups (12–18 postnatal days). Increase in somatic cross‐sectional area served as the indicator of swelling induced by bath application of glutamate receptor agonists. NMDA induced cell swelling in a dose‐dependent manner. Activation of DA receptors in the absence of NMDA did not produce swelling. DA and the D1 receptor agonist SKF 38393, increased the magnitude of swelling produced by NMDA. This effect was reduced in the presence of the D1 receptor antagonist, SCH 23390. In contrast, activation of D2 receptors by quinpirole decreased the magnitude of NMDA‐induced cell swelling. DA slightly attenuated cell swelling induced by activation of KA receptors. Quinpirole produced a significant concentration‐dependent reduction in KA‐induced swelling while SKF38393 increased KA‐induced swelling, but only at a low concentration of KA. Together, these results provide additional support for the hypothesis that the direction of DA modulation depends on the glutamate receptor subtype, as well as the DA receptor subtype activated. One possible consequence of these observations is that endogenous DA may be an important contributing factor in the mechanisms of cell death in Huntingtons disease.

Differential sensitivity of medium- and large-sized striatal neurons to NMDA but not kainate receptor activation in the rat.

Infrared videomicroscopy and differential interference contrast optics were used to identify medium‐ and large‐sized neurons in striatal slices from young rats. Whole‐cell patch‐clamp recordings were obtained to compare membrane currents evoked by application of N‐methyl‐d‐aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were significantly smaller in large‐ than in medium‐sized striatal neurons. The negative slope conductance for NMDA currents was greater in medium‐ than in large‐sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast, currents induced by kainate were significantly greater in large‐sized neurons whilst current densities were approximately equal in both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium‐sized neurons but were relatively infrequent in large‐sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large‐ than in medium‐sized neurons. A final set of experiments assessed a functional consequence of the differential sensitivity of medium‐ and large‐sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after prolonged application of NMDA or kainate. NMDA produced a time‐dependent increase in somatic area in medium‐sized neurons whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced significant swelling in both medium‐ and large‐sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor subunit composition and/or the relative density of receptors in the two cell types. These findings help define the conditions that put neurons at risk for excitotoxic damage in neurological disorders.

Ionotropic glutamate receptor expression and dopaminergic modulation in the developing subthalamic nucleus of the rat: an immunohistochemical and electrophysiological analysis.

Using standard immunohistochemical procedures, we investigated the changes in the expression of ionotropic glutamate receptor (GluR) subunits, GluRl, GluR5/6/7, and NMDAR1, in the subthalamic nucleus of developing rats. The general sequence of development for each subunit was the same. At early postnatal ages, there was dense neuropil staining and cellular clustering which progressed to decreased neuropil staining and an even distribution of conspicuous cells in the later postnatal ages and in the adult. GluR5/6/7 displayed the earliest maturation, while GluR1 exhibited the slowest maturation. These morphological changes suggest a different time course for the functionality of GluR subtypes in the developing subthalamic nucleus. Correlative electrophysiological studies demonstrated functional GluRs as early as 16 days of age. All neurons tested displayed robust responses to kainate and N-methyl-D-aspartate, and these responses were modulated by dopamine.