Hypoxia enhances high-voltage-activated calcium currents in rat primary cortical neurons via calcineurin

Hypoxia regulates neuronal ion channels, sometimes resulting in seizures. We evaluated the effects of brief sustained hypoxia (1% O(2), 4h) on voltage-gated calcium channels (VGCCs) in cultured rat primary cortical neurons. High-voltage activated (HVA) Ca(2+) currents were acquired immediately after hypoxic exposure or after 48h recovery in 95% air/5% CO(2). Maximal Ca(2+) current density increased 1.5-fold immediately after hypoxia, but reverted to baseline after 48h normoxia. This enhancement was primarily due to an increase in L-type VGCC activity, since nimodipine-insensitive residual Ca(2+) currents were unchanged. The half-maximal potentials of activation and steady-state inactivation were unchanged. The calcineurin inhibitors FK-506 (in the recording pipette) or cyclosporine A (during hypoxia) prevented the post-hypoxic increase in HVA Ca(2+) currents, while rapamycin and okadaic acid did not. L-type VGCCs were the source of Ca(2+) for calcineurin activation, as nimodipine during hypoxia prevented post-hypoxic enhancement. Hypoxia transiently potentiated L-type VGCC currents via calcineurin, suggesting a positive feedback loop to amplify neuronal calcium signaling that may contribute to seizure generation.     

Omega-conotoxin CVIB differentially inhibits native and recombinant N- and P/Q-type calcium channels

Omega-conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist omega-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. Omega-conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 +/- 0.05 and 7.64 +/- 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (approximately 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (> or = 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (approximately 22% of total) was attributable to P/Q-type VGCCs. Omega-conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Ca(V)2.2) and P/Q- (Ca(V)2.1), but not R- (Ca(V)2.3) type VGCCs expressed in Xenopus oocytes. The alpha2delta1 auxiliary subunit co-expressed with Ca(V)2.2 and Ca(V)2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. Omega-conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.     

Neuropeptide Y applied in vitro can block the phase shifts induced by light in vivo

The mammalian suprachiasmatic nuclei (SCN) can be synchronized by light, with direct glutamatergic input from the retina. Input to the SCN from the intergeniculate leaflet contains neuropeptide Y (NPY) and can modulate photic responses. NPY can reduce the phase-resetting effect of light or glutamate. We investigated the effect of NPY applied in vitro on light-induced phase shifts of the SCN neural activity rhythm. Light pulses delivered in vivo induced phase shifts in brain slice preparations similar to those as measured by behavioral activity rhythms. NPY applied after the light pulse blocked the phase shifts during both the early and late subjective night. NPY applied 30 min after the light pulse could block the phase delay induced by light. Our results show that NPY can inhibit photic resetting of the clock during the subjective night. The time course of this inhibitory effect suggests a mechanism downstream of the glutamate receptor.     

Nitric oxide modulates calcium entry through P/Q-type calcium channels and N-methyl-d-aspartate receptors in rat cortical neurons

Voltage-gated calcium channels (VGCC) and N-methyl-d-aspartate receptors (NMDAR) account for most of the depolarization-induced neuronal calcium entry. The susceptibility of individual routes of calcium entry for nitric oxide (NO) is largely unknown. We loaded cultured rat cortical neurons with fluo-4 acetoxymethylester to study the effect of the NO synthase inhibitor Nomega-nitro-l-arginine and the NO donor S-nitroso-N-acetylpenicillamine on the intracellular calcium concentration ([Ca2+]i). The potassium-induced [Ca2+]i increase was amplified by Nomega-nitro-l-arginine and attenuated by S-nitroso-N-acetylpenicillamine. This modulation was abolished by either the P/Q-type VGCC antagonist omega-agatoxin IVA or by the NMDAR antagonist MK-801, but not by N-type (omega-conotoxin GVIA) or L-type (nimodipine) VGCC blockers. These results suggest that NO can modulate neuronal calcium entry during depolarization by interacting with P/Q-type VGCC and NMDAR.     

Triptans attenuate circadian responses to light

Daily exposure to light synchronizes the circadian clock, located in the suprachiasmatic nucleus (SCN), to external day/night cycles. These responses to light can be modified by serotonergic drugs, such as serotonin 5HT_1B receptor agonists. Triptans are specific 5HT_1B agonists prescribed to treat migraines. Here, we examined the effects of two triptans (zolmitriptan and sumatriptan) on photic phase resetting in Syrian hamsters. Pre‐treatment with intra‐SCN sumatriptan significantly attenuates, and at higher doses completely blocks, phase advances to light during the late night. Pre‐treatment with systemic zolmitriptan significantly attenuates both light‐induced phase advances and phase delays. Neither of these drugs, nor their vehicles, causes phase shifts on their own. Pre‐treatment with zolmitriptan also significantly reduces the expression of light‐induced c‐fos in the SCN. Neither zolmitriptan nor vehicle alone induces significant c‐fos expression in the SCN. Finally, pre‐treatment with zolmitriptan does not attenuate phase shifts to intra‐SCN N‐methyl‐d ‐aspartate injections, indicating that the mechanism of action for zolmitriptan is likely to be through activation of presynaptic 5HT_1B receptors on retinal terminals, thereby decreasing light‐induced neurotransmitter release. As triptans are commercially available medications, there is potential for their use in blocking unwanted photic phase shifting during shift‐work or jet‐lag. Additionally, triptans may also affect the circadian clock in patients receiving them regularly for migraines. Finally, our results may hint at the mechanism by which triptans can alleviate the photophobia that frequently accompanies migraines, namely by activating 5HT_1B receptors on retinal terminals elsewhere in the brain, and thereby diminishing visually‐evoked neurotransmitter signalling in those areas.     

Hamster circadian rhythms are phase-shifted by electrical stimulation of the geniculo-hypothalamic tract

The suprachiasmatic nuclei (SCN) contain the major pacemaker for mammalian circadian rhythms. The SCN receive photic input both directly, via the retinohypothalamic tract (RHT), and indirectly, via the geniculohypothalamic tract (GHT), which originates in cells in the intergeniculate leaflet (IGL) and anterior portions of the ventral lateral geniculate nucleus (vLGN). We tested whether electrical stimulation of the GHT would induce phase shifts in wheel-running activity rhythms of Syrian hamsters housed in continuous darkness or continuous illumination. In both lighting conditions, electrical stimulation of the GHT induced mainly phase advances when given during the late subjective day and small phase delays when given during the late subjective night and early subjective day. Stimulation in the thalamus outside the GHT failed to produce similar phase shifts. Repeated daily stimulation had only a weak entraining effect on the activity rhythm. Activation of GHT neurons appears to influence the pacemaker for activity rhythms in a phase-dependent manner.     

GABA(B) receptor activation inhibits N- and P/Q-type calcium channels in cultured lamprey sensory neurons

In lamprey, sensory transmission from mechanosensory receptors (dorsal cells) to central neurons is presynaptically inhibited by GABA(B) receptor activation. The mechanisms underlying this effect were investigated using isolated dorsal cells, where voltage-dependent calcium currents were recorded in the whole-cell configuration. Activation of GABA(B) receptors by baclofen decreased the peak amplitude of high voltage-activated (HVA) calcium currents and slowed the activation phase. The role of G-proteins in mediating the effects of baclofen was examined. Intracellular dialysis of GTPgammaS occluded the effects of baclofen. Intracellular dialysis of GDPbetaS and preincubation in pertussis toxin both attenuated the effect of baclofen. Specific calcium channel blockers were used to study the types of HVA calcium channels involved in the GABA(B)-mediated modulation. The baclofen-induced inhibition was not affected by the L-type calcium channel antagonist nimodipine, but was partially blocked by the N-type blocker omega-conotoxin GVIA, and completely occluded by omega-conotoxin MVIIC, a blocker of both N- and P/Q-type channels. The pharmacology of dorsal cell GABA(B) receptors was studied using two agonists, baclofen and CGP 27492, and four antagonists, CGP 35348, CGP 55845, phaclofen and saclofen. The inhibition induced by either of the two agonists was blocked by CGP 55845, phaclofen and saclofen. The antagonist CGP 35348 completely blocked the inhibition of HVA calcium current induced by the agonist CGP 27492, but had no effect on baclofen-induced GABA(B) receptor activation. This study thus demonstrates that GABA(B) receptor activation in lamprey mechanosensory neurons inhibits N- and P/Q-type calcium channels in a voltage- and G-protein-dependent manner.     

Calcium-dependent subthreshold oscillations determine bursting activity induced by N-methyl-D-aspartate in rat subthalamic neurons in vitro

We used whole-cell patch recordings in current clamp to investigate the ionic dependence of burst firing induced by N-methyl-d-aspartate (NMDA) in neurons of the subthalamic nucleus (STN) in slices of rat brain. NMDA (20 microm) converted single-spike firing to burst firing in 87% of STN neurons tested. NMDA-induced bursting was blocked by AP5 (50 microm), and was not mimicked by the non-NMDA receptor agonist AMPA (0.6 microm). Tetrodotoxin (1 microm) converted bursts to oscillations of membrane potential, which were most robust when oscillations ranged between -50 and -70 mV. The NMDA bursts were blocked by an elevated extracellular concentration of Mg(2+), but superfusate containing no added Mg(2+) either reduced or increased burst firing, depending upon the amount of intracellular current injection. Block of K(+) conductances by apamin and tetraethylammonium prolonged burst duration, but iberiotoxin had no effect. NMDA-induced burst firing and membrane oscillations were completely blocked by superfusate containing no added Ca(2+), and they were significantly reduced when patch pipettes contained BAPTA. Selective antagonists for T-type (mibefradil, 10 microm), L-type (nifedipine, 3 microm), and N-type (omega-conotoxin GVIA, 1 micro m) Ca(2+) channels had no effect on NMDA burst firing. Superfusate containing a low concentration of Na(+) (20 mm) completely abolished NMDA-induced burst firing. Flufenamic acid (10 microm), which blocks current mediated by Ca(2+)-activated nonselective cation channels (I(CAN)), reversibly abolished NMDA-depended bursting. These results are consistent with the hypothesis that NMDA-induced burst firing in STN neurons requires activation of either an I(CAN) or a Na(+)-Ca(2+) exchanger.     

Sustained activation of GABAA receptors in the suprachiasmatic nucleus mediates light‐induced phase delays of the circadian clock: a novel function of ionotropic receptors

The suprachiasmatic nucleus (SCN) contains a circadian clock that generates endogenous rhythmicity and entrains that rhythmicity with the day–night cycle. The neurochemical events that transduce photic input within the SCN and mediate entrainment by resetting the molecular clock have yet to be defined. Because GABA is contained in nearly all SCN neurons we tested the hypothesis that GABA serves as this signal in studies employing Syrian hamsters (Mesocricetus auratus). Activation of GABA_A receptors was found to be necessary and sufficient for light to induce phase delays of the clock. Remarkably, the sustained activation of GABA_A receptors for more than three consecutive hours was necessary to phase‐delay the clock. The duration of GABA_A receptor activation required to induce phase delays would not have been predicted by either the prevalent theory of circadian entrainment or by expectations regarding the duration of ionotropic receptor activation necessary to produce functional responses. Taken together, these data identify a novel neurochemical mechanism essential for phase‐delaying the ‘master’ circadian clock within the SCN as well as identifying an unprecedented action of an amino acid neurotransmitter involving the sustained activation of ionotropic receptors.     

Circadian control during the day and night: Role of neuropeptide Y Y5 receptors in the suprachiasmatic nucleus

Circadian rhythms are reset by light during the night or by nonphotic stimuli during the day. Neuropeptide Y (NPY), which appears to mediate at least some nonphotic phase shifts by its actions in the suprachiasmatic nucleus (SCN), induces phase advances during the day and inhibits light-induced phase advances during the night. In this study, we used a highly selective Y5-like agonist to test whether activation of NPY Y5 receptors is sufficient to mimic NPY during the day and late night in Syrian hamsters. We also tested whether NPY in the early night reduces light-induced phase delays in a dose-dependent manner. Microinjection of a selective Y5 receptor agonist, (Ala(31), Aib(32))-NPY, into the SCN significantly inhibited light-induced phase advances during the late night, but did not induce phase advances during the day. In addition, concentrations of NPY ranging from 0.23 to 23 mM did not attenuate light-induced phase delays in the early night. These results suggest that activation of Y5-like receptors is sufficient to inhibit light-induced phase advances during the late night but is not sufficient to induce phase advances during the day. Furthermore, this study provided no evidence that NPY can inhibit light-induced phase shifts early in the night.     

Regulation of voltage-gated Ca2 + currents by Ca2 +/calmodulin-dependent protein kinase II in resting sensory neurons

Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca^2 + channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca^2 + currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0 μM) reduced depolarization-induced I_Ca by 16–30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total I_Ca. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent.     

Modulation of photic resetting in rats by lesions of projections to the suprachiasmatic nuclei expressing p75 neurotrophin receptor

The suprachiasmatic nuclei of the hypothalamus (SCN) are the site of the master circadian clock in mammals. The SCN clock is mainly entrained by the light–dark cycle. Light information is conveyed from the retina to the SCN through direct, retinohypothalamic fibres. The SCN also receive other projections, like cholinergic fibres from basal forebrain. To test whether cholinergic afferents are involved in photic resetting, lesions of cholinergic projections were performed in rats with intracerebroventricular (i.c.v.) injections or intra‐SCN microinjections of 192 IgG‐saporin. When injected in the SCN, this immunotoxin destroys the cholinergic projections and retinohypothalamic afferents that express p75 low‐affinity nerve growth factor (p75^NGF) receptors. The extent of lesions in the basal forebrain and SCN was assessed by acetylcholinesterase histochemistry, p75^NGF receptor, choline acetyl‐transferase, calbindin‐D28K and VIP immunocytochemistry. The intra‐SCN treatment reduced light‐induced phase advances by 30%, and induced a complete loss of forebrain and retinal afferents expressing p75^NGF receptors within the SCN and a decrease of forebrain cholinergic neurons, most likely those projecting to the SCN. The i.c.v. treatment reduced light‐induced phase advances by 40%, increased phase delays and led to extensive damage of forebrain p75^NGF‐expressing neurons, while sparing half of the fibres expressing p75^NGF receptors (retinal afferents?) in the SCN. Because the integrity of forebrain p75^NGF‐expressing neurons appears to be critical in mediating the effects on light‐induced phase advances, we therefore suggest that anterior cholinergic projections expressing p75^NGF receptors modulate the sensitivity of the SCN clock to the phase advancing effects of light.     

Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain

The effect of membrane electrical activity on spiral ganglion neuron (SGN) neurite growth remains unknown despite its relevance to cochlear implant technology. We demonstrate that membrane depolarization delays the initial formation and inhibits the subsequent extension of cultured SGN neurites. This inhibition depends directly on the level of depolarization with higher levels of depolarization causing retraction of existing neurites. Cultured SGNs express subunits for L-type, N-type, and P/Q type voltage-gated calcium channels (VGCCs) and removal of extracellular Ca(2+) or treatment with a combination of L-type, N-type, and P/Q-type VGCC antagonists rescues SGN neurite growth under depolarizing conditions. By measuring the fluorescence intensity of SGNs loaded with the fluorogenic calpain substrate t-butoxy carbonyl-Leu-Met-chloromethylaminocoumarin (20 microM), we demonstrate that depolarization activates calpains. Calpeptin (15 microM), a calpain inhibitor, prevents calpain activation by depolarization and rescues neurite growth in depolarized SGNs suggesting that calpain activation contributes to the inhibition of neurite growth by depolarization.     

Neuropeptide-mediated calcium signaling in the suprachiasmatic nucleus network

Neuroactive peptides and the intracellular calcium concentration ([Ca(2+) ](i) ) play important roles in light-induced modulation of gene expression in the suprachiasmatic nucleus (SCN) neurons that ultimately control behavioral rhythms. Vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) are expressed rhythmically within populations of SCN neurons. Pituitary adenylate cyclase-activating peptide (PACAP) is released from retinohypothalamic tract (RHT) terminals synapsing on SCN neurons. Nociceptin/orphanin FQ (OFQ) receptors are functionally expressed in the SCN. We examined the role of several neuropeptides on Ca(2+) signaling, simultaneously imaging multiple neurons within the SCN neural network. VIP reduced the [Ca(2+) ](i) in populations of SCN neurons during the day, but had little effect at night. Stimulation of the RHT at frequencies that simulate light input signaling evoked transient [Ca(2+) ](i) elevations that were not altered by VIP. AVP elevated the [Ca(2+) ](i) during both the day and night, PACAP produced variable responses, and OFQ induced a reduction in the [Ca(2+) ](i) similar to VIP. During the day, VIP lowered the [Ca(2+) ](i) to near nighttime levels, while AVP elevated [Ca(2+) ](i) during both the day and night, suggesting that the VIP effects on [Ca(2+) ](i) were dependent, and the AVP effects independent of the action potential firing activity state of the neuron. We hypothesize that VIP and AVP regulate, at least in part, Ca(2+) homeostasis in SCN neurons and may be a major point of regulation for SCN neuronal synchronization.     

Attenuation of Zn2+ neurotoxicity by aspirin: role of N-type Ca2+ channel and the carboxyl acid group

Synaptically released Zn2+ ions enter into neurons primarily through voltage-gated Ca2+ channels (VGCC) or N-methyl-d-aspartate (NMDA) receptors, which can mediate pathological neuronal death. We studied the possibility (and underlying mechanisms) that aspirin, known to prevent NMDA neurotoxicity, would also attenuate Zn2+ neurotoxicity. Administration of 3 to 10 mM aspirin, in cortical cell cultures, attenuated the evolution of neuronal death following exposure to 300 microM Zn2+ for 30 min. This neuroprotective effect of aspirin was attributable to the prevention of Zn2+ ion entry. Aspirin interfered with inward currents and an increase in [Ca2+]i through VGCC and selective binding of omega-conotoxin, sensitive to N-type Ca2+ channel. The omega-conotoxins GVIA or MVIIC, the selective inhibitors of N-type Ca2+ channels, attenuated Zn2+ neurotoxicity. Aspirin derivatives lacking the carboxyl acid group did not reduce Zn2+ neurotoxicity. The present findings suggest that aspirin prevents Zn2+-mediated neuronal death by interfering with VGCC, and its action specifically requires the carboxyl acid group.     

Distribution of high-voltage-activated calcium channels in cultured gamma-aminobutyric acidergic neurons from mouse cerebral cortex.

The localization of voltage-gated calcium channel (VGCC) alpha(1) subunits in cultured GABAergic mouse cortical neurons was examined by immunocytochemical methods. Ca(v)1.2 and Ca(v)1.3 subunits of L-type VGCCs were found in cell bodies and dendrites of GABA-immunopositive neurons. Likewise, the Ca(v)2.3 subunit of R-type VGCCs was expressed in a somatodendritic pattern. Ca(v)2.2 subunits of N-type channels were found exclusively in small varicosities that were identified as presynaptic nerve terminals based on their expression of synaptic marker proteins. Two splice variants of the Ca(v)2.1 subunit of P/Q-type VGCCs showed widely differing expression patterns. The rbA isoform displayed a purely somatodendritic staining pattern, whereas the BI isoform was confined to axon-like fibers and nerve terminals. The nerve terminals of these cultured GABAergic neurons express Ca(v)2.2 either alone or in combination with Ca(v)2.1 (BI isoform) but never express Ca(v)2.1 alone. The functional association between VGCCs and the neurotransmitter release machinery was probed using the FM1-43 dye-labeling technique. N-type VGCCs were found to be tightly coupled to exocytosis in these cultured cortical neurons, and P-type VGCCs were also important in a fraction of the cells. The predominant role of N-type VGCCs in neurotransmitter release and the specific localization of the BI isoform of Ca(v)2.1 in the nerve terminals of these neurons distinguish them from previously studied central neurons. The complementary localization patterns observed for two different isoforms of the Ca(v)2.1 subunits provide direct evidence for alternative splicing as a means of generating functional diversity among neuronal calcium channels.     

Involvement of T-type Ca2+ channels in the potentiation of synaptic and visual responses during the critical period in rat visual cortex

Neocortical neuronal circuits are refined by experience during the critical period of early postnatal life. The shift of ocular dominance in the visual cortex following monocular deprivation has been intensively studied to unravel the mechanisms underlying the experience-dependent modification. Synaptic plasticity is considered to be involved in this process. We previously showed in layer 2/3 pyramidal neurons of rat visual cortex that low-frequency stimulation-induced long-term potentiation (LTP) at excitatory synapses, which requires the activation of Ni²⁺-sensitive (R-type or T-type) voltage-gated Ca²⁺ channels (VGCCs) for induction, shared a similar age and experience dependence with ocular dominance plasticity. In this study, we examined whether this LTP is involved in ocular dominance plasticity. In visual cortical slices, LTP was blocked by mibefradil, kurtoxin and R -(−)-efonidipine, T-type VGCC blockers, but not by SNX-482, an R-type VGCC blocker, indicating that LTP induction requires T-type VGCC activation. Mibefradil did not affect synaptic transmission even at a dose about 30 times higher than that required for LTP blockade. Therefore, this drug was used to test the effect of T-type VGCC blockade on ocular dominance shift produced by 6 days of monocular deprivation during the critical period using visual evoked potentials (VEPs). Although this monocular deprivation commonly produced both depression of deprived eye responses and potentiation of nondeprived eye responses, only the former change occurred when mibefradil was infused into the visual cortex during monocular deprivation. Mibefradil infusion produced no acute effects on VEPs. These results suggest that T-type VGCC-dependent LTP contributes to the experience-dependent enhancement of visual responses.     

Nimodipine potentiates light-induced phase shifts of circadian activity rhythms but not c-fos expression in the suprachiasmatic nucleus of mice

The present study assessed whether treatment with the L-type calcium channel antagonist nimodipine affects the responsiveness of the circadian pacemaker to light in C3H/HeN mice. Nimodipine (10 mg/kg, sc) increased the magnitude of light-induced phase delays (P<0.01) and c-fos mRNA expression in the paraventricular nuclei (P<0.01), but not in the suprachiasmatic nuclei (SCN). These results suggest that nimodipine may affect phase shifts of circadian activity rhythms through a mechanism independent of c-fos expression in the SCN.     

Investigation Into the Regulation of the Circadian System by Dopamine and Melatonin in the Adult Siberian Hamster (Phodopus sungorus)

Dopamine and melatonin have both been implicated in mediating maternal influences on the developing circadian system of altricial rodents. The aim of these studies was to investigate their role in the entrainment of the circadian system of the adult Siberian hamster (Phodopus sungorus). In-situ hybridization revealed that D₁-dopamine receptor (D₁-R) mRNA was expressed in the adult suprachiasmatic nucleus (SCN) at levels comparable to neonates. As dopamine has been postulated to mimic photic stimulation during early development, experiment 1 compared the effects of a D₁-R agonist and a light pulse on free-running wheel running rhythms in hamsters maintained in constant dim red light. A phase response curve to light was generated, revealing clear phase delays early in the subjective night, and large phase advances in the late subjective night. However, the D₁-R agonist (SKF 81297, 2 mg/kg, s.c.) did not produce consistent phase shifts at any circadian phase. Experiment 2 tested the ability of this dopaminergic agonist to modulate photic responses of the circadian system. Free-running animals were pre-treated with SKF 81297 (2 mg/kg, s.c.) 30 min before a 15 min light pulse given early or late in the subjective night. This agonist had no effect on the magnitude of phase shifts at either circadian time. In experiment 3, light pulses at CT13–15 induced expression of the immediate early gene c-fos in the SCN, as assessed by immunocytochemistry for the protein product. In contrast, SKF 81297 (2 mg/kg, s.c.) at the same phase did not induce c-fos in the SCN, despite marked c-fos induction in the caudate-putamen, nor did it affect photic induction of c-fos in the SCN. To investigate whether dopamine might be involved in nonphotic regulation of the circadian system in adult hamsters, experiment 4 compared the response of free-running hamsters to a series of injections of SKF 81297 (2 mg/kg, s.c.) or melatonin (1 mg/kg, s.c.), since melatonin receptor expression in the SCN also persists into adulthood. Animals were treated every 23.5 h for 6 days. The serial injections of melatonin produced cumulative phase advances of up to 3 h when delivered in late subjective day, but not when presented in late subjective night. Hamsters did not respond to SKF 81297 or vehicle treatment at either circadian phase. Moreover, pre-treatment with the dopaminergic agonist did not affect the phase-advancing effects of melatonin when both were given in the serial injection protocol. These results demonstrate clear phase-dependent effects of light pulses and melatonin on circadian rhythms in Siberian hamsters, but suggest that D₁-Rs in the SCN no longer modulate photic or melatonin-dependent entrainment pathways in the adult.     

Differential effects of glutamatergic blockade on circadian and photic regulation of gene expression in the hamster suprachiasmatic nucleus

Nocturnal light exposure induces immediate-early gene (IEG) expression in the hypothalamic suprachiasmatic nucleus (SCN) and causes phase shifts of activity rhythms in mammals. Some IEGs also show a circadian rhythm of expression in the SCN. While excitatory amino acids (EAAs) are known to be involved in mediating photic regulation of entrainment and gene expression, their involvement in spontaneous rhythms of gene expression has not been studied. We assessed the role of NMDA receptors in the expression of NGFI-A, junB and fosB mRNAs induced by light pulses of different intensities late in the night (Zeitgeber Time [ZT] 18). We also examined the spontaneous expression of junB mRNA near subjective dawn (ZT 0). Both dim (5 lx) and bright (100 lx) light pulses induced similar levels of expression of NGFI-A and junB in the SCN late in the night. fosB mRNA was strongly induced by bright light but was less sensitive to dim light. At ZT 18, dizocilpine (MK-801) (3 mg/kg, i.p. ), a non-competitive NMDA receptor antagonist, almost completely blocked light-evoked expression of IEG mRNAs in the ventral SCN but not in the dorsolateral region at a mid-caudal level using either light intensity. At ZT 0, MK-801 strongly reduced light-evoked expression of junB mRNA in both SCN subdivisions, but inhibited spontaneous expression significantly only in the dorsal region. NMDA receptors appear to play an important role in mediating photic input regulating IEG expression only in the ventral SCN at night. At dawn, however, NMDA receptors are involved in mediating photic effects in both parts of the SCN, as well as being involved in spontaneous activation of junB expression selectively in the dorsal SCN. These findings support the idea that the effects in the dorsolateral SCN of nocturnal light exposure are mediated by different mechanisms than those in other portions of the nucleus.