The circadian visual system, 2005.

The primary mammalian circadian clock resides in the suprachiasmatic nucleus (SCN), a recipient of dense retinohypothalamic innervation. In its most basic form, the circadian rhythm system is part of the greater visual system. A secondary component of the circadian visual system is the retinorecipient intergeniculate leaflet (IGL) which has connections to many parts of the brain, including efferents converging on targets of the SCN. The IGL also provides a major input to the SCN, with a third major SCN afferent projection arriving from the median raphe nucleus. The last decade has seen a blossoming of research into the anatomy and function of the visual, geniculohypothalamic and midbrain serotonergic systems modulating circadian rhythmicity in a variety of species. There has also been a substantial and simultaneous elaboration of knowledge about the intrinsic structure of the SCN. Many of the developments have been driven by molecular biological investigation of the circadian clock and the molecular tools are enabling novel understanding of regional function within the SCN. The present discussion is an extension of the material covered by the 1994 review, "The Circadian Visual System."

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN).

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.

Potential pathways for intercellular communication within the calbindin subnucleus of the hamster suprachiasmatic nucleus.

In mammals, the suprachiasmatic nucleus (SCN) is the master circadian pacemaker. Within the caudal hamster SCN, a cluster of neurons containing the calcium binding protein, calbindin-D28K (CB), has been implicated in circadian locomotion. However, calbindin-immunoreactive (CB+) neurons in the calbindin subnucleus (CBsn) do not display a circadian rhythm in spontaneous firing [Eur J Neurosci 16 (2002) 2469]. Previously, we proposed that intercellular communication might be essential in integrating outputs from rhythmic (CB-) neurons and nonrhythmic (CB+) neurons to produce a circadian output in the intact animal. The primary aim of this study is to provide a neuroanatomical framework to better understand intercellular communication within the CBsn. Using reconstructions of previously recorded neurons, we demonstrate that CB+ neurons have significantly more dendrites than CB- neurons. In addition, CBsn neurons have dorsally oriented dendritic arbors. Using double-label confocal microscopy, we show that GABA colocalizes with CB+ neurons and GABA(A) receptor subunits make intimate contacts with neurons in the CBsn. Transforming growth factor alpha (TGFalpha), a substance shown to inhibit locomotion [Science 294 (2001) 2511], is present within the CBsn. In addition, neurons in this region express the epidermal growth factor receptor, the only receptor for TGFalpha. Lastly, we show that CB+ neurons are coupled to CB+ and CB- neurons by gap junctions. The current study provides a structural framework for synaptic communication, electrical coupling, and signaling via a growth factor within the CBsn of the hamster SCN. Our results reveal connections that have the potential for integrating cellular communication within a subregion of the SCN that is critically involved in circadian locomotion.

Characterization of an apamin-sensitive potassium current in suprachiasmatic nucleus neurons.

In neurons of the suprachiasmatic nucleus, spike frequency adaptation and membrane afterhyperpolarization occur during a train of action potentials. Extracellular Ca2+ may regulate neuronal excitability by several mechanisms, including activation of small conductance and large conductance Ca(2+)-activated K+ channels. The overall goal of this study was to examine the role of Ca(2+)-activated K+ currents in individual suprachiasmatic nucleus neurons. To this end, we used the nystatin-perforated patch technique to record currents from suprachiasmatic nucleus neurons. Iberiotoxin and tetraethylammonium, antagonists of large conductance Ca(2+)-activated K+ channels, had no effect on the membrane afterhyperpolarization. However, antagonists of small conductance Ca(2+)-activated K+ channels, apamin and d-tubocurarine, reduced the amplitude of the membrane afterhyperpolarization and inhibited the spike frequency adaptation that occurred during a train of action potentials. Although there was no significant difference in membrane AHP between different portions of the circadian day, apamin and d-tubocurarine increased the spontaneous firing frequency of suprachiasmatic nucleus neurons during the daytime. In voltage-clamp mode, membrane depolarization-activated currents were followed by an outward tail current reversing near the K+ equilibrium potential. The tail current decayed with a time constant of 220 ms at +20 mV and 149 ms at -40 mV. Apamin irreversibly and d-tubocurarine reversibly inhibited the tail current. The tail current amplitude was also reduced by the GABAA receptor antagonist, bicuculline methiodide, while picrotoxin (another GABAA receptor antagonist) was without effect. Removal of extracellular Ca2+ or the addition of Cd2+ reversibly inhibited the tail current. These results indicate that apamin- and d-tubocurarine-sensitive small conductance Ca(2+)-activated K+ channels have a modulatory function on the action potential firing frequency as well as the membrane afterhyperpolarization that follows a train of action potentials in suprachiasmatic nucleus neurons. Importantly, our data also indicate that a portion of the effects of bicuculline methiodide on suprachiasmatic nucleus neurons may be mediated by inhibition of small conductance Ca(2+)-activated K+ channels.

The sulphydryl reagent, N-ethylmaleimide, disrupts sleep and blocks A1 adenosine receptor-mediated inhibition of intracellular calcium signaling in the in vitro ventromedial preoptic nucleus.

To explore the neuronal signaling mechanisms underlying sleep regulation in the rat, the present study examined continuous intra-third ventricle infusion of N-ethylmaleimide (NEM), a sulphydryl reagent that inhibits G(i/o) protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01-10 micromol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 micromol/10 h) dramatically inhibited day-time sleep (-57% for non-rapid eye movement sleep and -89% for rapid eye movement sleep) with a compensatory increase of sleep during the subsequent night-time (+33% for non-rapid eye movement sleep and +259% for rapid eye movement sleep). The day-time brain temperature was also increased by NEM, demonstrating effects of NEM on both sleep and body temperature levels. Immunostaining of the rat hypothalamus with a monoclonal antibody against the A1 adenosine receptor (A1R) was used to explore the distribution of a sleep-related G(i/o) protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca(2+) imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N(6)-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca(2+) oscillations and high potassium (80 mM)-induced Ca(2+) flux in the ventromedial preoptic nucleus, while NEM (100-300 microM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca(2+) flux. From these results we suggest that NEM-sensitive G protein-coupled receptor(s) may play an important role in the regulation of sleep and body temperature in the rat and one possible mechanism is an A1R-mediated regulation of intracellular Ca(2+) concentrations in the ventromedial preoptic nucleus.

Rudimentary TCR signaling triggers default IL-10 secretion by human Th1 cells.

Understanding the process of inducing T cell activation has been hampered by the complex interactions between APC and inflammatory Th1 cells. To dissociate Ag-specific signaling through the TCR from costimulatory signaling, rTCR ligands (RTL) containing the alpha1 and beta1 domains of HLA-DR2b (DRA*0101:DRB1*1501) covalently linked with either the myelin basic protein peptide 85-99 (RTL303) or CABL-b3a2 (RTL311) peptides were constructed to provide a minimal ligand for peptide-specific TCRs. When incubated with peptide-specific Th1 cell clones in the absence of APC or costimulatory molecules, only the cognate RTL induced partial activation through the TCR. This partial activation included rapid TCR zeta-chain phosphorylation, calcium mobilization, and reduced extracellular signal-related kinase activity, as well as IL-10 production, but not proliferation or other obvious phenotypic changes. On restimulation with APC/peptide, the RTL-pretreated Th1 clones had reduced proliferation and secreted less IFN-gamma; IL-10 production persisted. These findings reveal for the first time the rudimentary signaling pattern delivered by initial engagement of the external TCR interface, which is further supplemented by coactivation molecules. Activation with RTLs provides a novel strategy for generating autoantigen-specific bystander suppression useful for treatment of complex autoimmune diseases.

Regulation of melatonin 1a receptor signaling and trafficking by asparagine-124.

Melatonin is a pineal hormone that regulates seasonal reproduction and has been used to treat circadian rhythm disorders. The melatonin 1a receptor is a seven- transmembrane domain receptor that signals predominately via pertussis toxin-sensitive G-proteins. Point mutations were created at residue N124 in cytoplasmic domain II of the receptor and the mutant receptors were expressed in a neurohormonal cell line. The acidic N124D- and E-substituted receptors had high-affinity (125)I-melatonin binding and a subcellular localization similar to the neutral N124N wild-type receptor. Melatonin efficacy for the inhibition of cAMP by N124D and E mutations was significantly decreased. N124D and E mutations strongly compromised melatonin efficacy and potency for inhibition of K(+)-induced intracellular Ca(++) fluxes and eliminated control of spontaneous calcium fluxes. However, these substitutions did not appear to affect activation of Kir3 potassium channels. The hydrophobic N124L and N124A or basic N124K mutations failed to bind (125)I-melatonin and appeared to aggregate or traffic improperly. N124A and N124K receptors were retained in the Golgi. Therefore, mutants at N124 separated into two sets: the first bound (125)I-melatonin with high affinity and trafficked normally, but with reduced inhibitory coupling to adenylyl cyclase and Ca(++) channels. The second set lacked melatonin binding and exhibited severe trafficking defects. In summary, asparagine-124 controls melatonin receptor function as evidenced by changes in melatonin binding, control of cAMP levels, and regulation of ion channel activity. Asparagine-124 also has a unique structural effect controlling receptor distribution within the cell.

Cyclic AMP regulates the calcium transients released from IP(3)-sensitive stores by activation of rat kappa-opioid receptors expressed in CHO cells.

We analyzed intracellular Ca(2+)and cAMP levels in Chinese hamster ovary cells expressing a cloned rat kappa opioid receptor (CHO-kappa cells). Although expression of kappa(kappa)-opioid receptors was confirmed with a fluorescent dynorphin analog in almost all CHO-kappa cells, the kappa-specific agonists, U50488H or U69593, induced a Ca(2+) transient only in 35% of the cells. The Ca(2+) response occurred in all-or-none fashion and the half-maximal dosage of U50488H (812.1nM) was higher than that (3.2nM) to inhibit forskolin-stimulated cAMP. The kappa-receptors coupled to G(i/o)proteins since pertussis toxin significantly reduced the U50488H actions on intracellular Ca(2+) and cAMP. The Ca(2+) transient originates from IP(3)-sensitive internal stores since the Ca(2+) response was blocked by a PLC inhibitor (U73122) or by thapsigargin depletion of internal stores while removal of extracellular Ca(2+) had no effect. Interestingly, application of dibutyryl cAMP (+ 56.2%) or 8-bromo-cAMP (+ 174.7%) significantly increased the occurrence of U50488H-induced Ca(2+) mobilization while protein kinase A (PKA) inhibitors, Rp-cAMP (-32.3%) or myr-psi PKA (-73.9%) significantly reduced the response. Therefore, it was concluded that cAMP and PKA activity can regulate the Ca(2+) mobilization. These results suggest that the kappa receptor-linked cAMP cascade regulates the occurrence of kappa-opioid-mediated Ca(2+) mobilization.

PLC beta 4-independent Ca2+ rise via muscarinic receptors in the mouse suprachiasmatic nucleus.

Cholinergic regulation of the suprachiasmatic nucleus (SCN) has been extensively studied although the intracellular signaling mechanisms are not well understood. We examined immunostaining for phospholipase C-beta (PLC-beta) families that couple to muscarinic acetylcholine receptors (mAChR) and demonstrated the expression of PLC-beta 1 and beta 4 in the mouse SCN. Ca2+ imaging analysis indicated that the MI-mAChR antagonist, pirenzepine blocked carbachol-induced Ca2+ elevation in the SCN and the response was equivalent between the wild type and the PLC-beta 4-knockout mice. In addition, the knockout mice displayed locomotor and temperature rhythms coupling to 24 h light/dark cycles. Therefore, it was proposed that PLC-beta 1 but not PLC-beta 4 was involved in the mAChR-mediated Ca2+ signaling in the SCN.

Pre- and postsynaptic actions of serotonin on rat suprachiasmatic nucleus neurons.

Serotoninergic transmission is implicated in the photic and non-photic regulation of circadian rhythms. 5-HT (1-100 microM), carboxamidotryptamine (5-CT 0.1-10 microM) and (+)-8-hydroxy-dipropylaminotetraline (8-OH-DPAT, 1-30 microM) dose-dependently activated an outward current (5-100 pA) in 30% of neurons voltage-clamped at -60 mV in the suprachiasmatic nucleus (SCN) in vitro slice. EC(50) values were 7.0 microM for 5-HT and 0.2 microM for 5-CT. Serotonin-induced outward current was associated with an increase in input conductance, and the current was blocked by Ba(2+) (1 mM). The amplitude of the current was enhanced by depolarization, reduced by hyperpolarization, and reversed its polarity during a hyperpolarization beyond the potassium equilibrium potential. Mean amplitudes of the 5-HT outward current changed with time of the subjective circadian day. The value near CT2 (23.8 pA) was about 4 times greater than that around CT14 (6.7 pA). Cells that responded with an outward current showed four types of morphology: monopolar, simple bipolar, curly bipolar and radial shaped; they were localized in all parts of the SCN. The EPSC evoked by retino-hypothalamic-tract (RHT) stimulation was inhibited 26% but the inward current induced by exogenously applied glutamate or NMDA was not affected by serotonin agonists. Focal stimulation-induced and spontaneous IPSC but not the exogenous GABA-induced outward current were inhibited by 5-HT agonists in a subpopulation of cells. In conclusion, 5-HT regulates SCN neurons by both pre- and post-synaptic inhibitory mechanisms; the latter may play a key role in modulating SCN circadian rhythm by activation of 5-HT receptors and opening of a potassium channel.

Neurotoxic potential of three structural analogs of beta-N-oxalyl-alpha,beta-diaminopropanoic acid (beta-ODAP).

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of beta-ODAP were synthesized. Carboxymethyl-alpha,beta-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 microM), but not by CNQX (10 microM). N-acetyl-alpha,beta-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three beta-ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2-3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [3H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for beta-ODAP. These data indicate that analogs of beta-ODAP may interact with glutamate receptors without producing neurotoxicity.

Melatonin receptor potentiation of cyclic AMP and the cystic fibrosis transmembrane conductance regulator ion channel.

We have used the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as a model system to study the cAMP signal transduction pathways coupled to the Xenopus melatonin receptor. During forskolin (Fsk) stimulation, melatonin reduced the amplitude of the CFTR currents in oocytes injected with in vitro transcribed cRNAs for the Xenopus melatonin receptor and CFTR. Pertussis toxin (Ptx) treatment eliminated melatonin inhibition of Fsk stimulated CFTR currents. In oocytes injected with cRNA for melatonin receptors, serotonin receptors (5-HT7), and CFTR Cl- channels, application of melatonin together with serotonin (5-HT) activated an additional inward current showing potentiation of adenylyl cyclases by melatonin receptors. Subthreshold activation of 5-HT7 receptors was sufficient and necessary to permit activation of CFTR channels by melatonin. Preexposure to melatonin desensitized the melatonin receptor mediated response. Therefore, based on this model system, the effects of melatonin in vivo could be either positive or negative modulation of other neuronal inputs, depending on the mode of adenylyl cyclase stimulation.

Orphanin-FQ/nociceptin (OFQ/N) modulates the activity of suprachiasmatic nucleus neurons.

Neurons in the suprachiasmatic nucleus (SCN) constitute the principal circadian pacemaker of mammals. In situ hybridization studies revealed expression of orphanin-FQ/nociceptin (OFQ/N) receptor (NOR) mRNA in the SCN, whereas no expression of mRNA for preproOFQ/N (ppOFQ/N) was detected. The presence of OFQ/N peptide in the SCN was demonstrated by radioimmunoassay. SCN neurons (88%) responded dose-dependently to OFQ/N with an outward current (EC50 = 22.3 nM) that was reduced in amplitude by membrane hyperpolarization and reversed polarity near the theoretical potassium equilibrium potential. [Phe1psi(Ch2-NH)Gly2]OFQ/N(1-13)NH2 (3 microM), a putative NOR antagonist, activated a small outward current and significantly reduced the amplitude of the OFQ/N-stimulated current. OFQ/N reduced the NMDA receptor-mediated increase in intracellular Ca2+. When injected unilaterally into the SCN of Syrian hamsters housed in constant darkness, OFQ/N (1-50 pmol) failed to alter the timing of the hamsters' wheel-running activity. However, injection of OFQ/N (0.1-50 pmol) before a brief exposure to light during the midsubjective night significantly attenuated the light-induced phase advances of the activity rhythm. These data are consistent with the interpretation that OFQ/N acting at specific receptors modulates the activity of SCN neurons and, thereby, the response of the circadian clock to light.

Lead inhibition of N-methyl-D-aspartate receptors containing NR2A, NR2C and NR2D subunits.

The potency of Pb2+ inhibition of glutamate-activated currents mediated by N-methyl-D-aspartate (NMDA) receptors was dependent on the subunits composing the receptors when functionally expressed in Xenopus laevis oocytes. Pb2+ reduced the amplitudes of glutamate-activated currents and shifted the agonist EC50 values of NMDA receptors consisting of different subunit compositions. The IC50 values for Pb2+ ranged from 1.52 to 8.19 microM, with a rank order of potency of NR1b-2A > NR1b-2C > NR1b-2D > NR1b-2AC. For NR1b-2AC NMDA receptors, the IC50 value was dependent on the agonist concentration; at saturating agonist concentrations (300 microM), the IC50 value was 8.19 microM, whereas at 3 microM glutamate, the IC50 value was 3.39 microM. Pb2+ was a noncompetitive inhibitor of NR1b-2A, NR1b-2C and NR1b-2D NMDA receptors. At low concentrations (<1 microM) Pb2+ potentiated NR1b-2AC NMDA receptors. These data provide further evidence to support the hypothesis that the actions of Pb2+ on NMDA receptors are determined by the receptor subunit composition.

Cloning and characterization of Kir3.1 (GIRK1) C-terminal alternative splice variants.

Southern blot analysis of RT-PCR products from brain and heart revealed multiple products for a C-terminal region of Kir3.1. Sequencing yielded clones for wild-type Kir3.1 and three Kir3.1 C-terminal alternative splice variants, including a unique alternative exon. Two of these variants encoded truncated Kir3.1 molecules. Tissue distribution and electrophysiological characterization of a single truncated variant, Kir3.1(00) were then examined. Kir3.1 channels are gated by G-protein beta gamma-subunits binding to the C-terminal domain, thus, the truncation of Kir3.1(00) removes a major functional domain. When incorporated into heteromeric channels with other family members (Kir3.1, 3.2 or 3.4) several functional changes were observed: (1) Kir3.1(00) changes G-protein activation of Kir3 channels; (2) Kir3.1(00) is restricted in its ability to assemble with other channel subunits as heteromers; and (3) incorporation of Kir3.1(00) into heteromeric channel complexes alters the kinetics of channel re-activation.

Tracer and electrical coupling of rat suprachiasmatic nucleus neurons.

Whole-cell recording from single neurons of the suprachiasmatic nucleus with an electrode containing the tracer neurobiotin resulted in the staining of multiple neurons in 30% of the cases. Typically, one neuron was darkly stained with dendritic processes and an axon clearly visible while other neurons were lightly stained. The darkly-stained cells were identified as the recorded neuron and tracer-coupled to one to five lightly stained neurons. The resting membrane potential, input membrane conductance, membrane capacitance, the decay time constant and the maximum H-current amplitude of the recorded neurons with tracer-coupled cells were not significantly different from those of neurons not showing tracer coupling. Stimulation of the preoptic area activated an antidromic action potential or an all-or-none small slow inward current in some neurons when the synaptic transmission was blocked by a calcium-free/Mn2+ solution. The small slow inward current did not "collide" with an orthodromically activated action spike suggesting that the current represents the signal from an electrotonically-coupled neuron. In addition, the frequency of biphasic field currents from a neighbouring cell firing were increased by depolarization and decreased by hyperpolarization of the recorded cell. These data demonstrate a chemical and electrical low-resistance coupling of suprachiasmatic nucleus neurons, which could be important in synthesizing the suprachiasmatic nucleus circadian rhythm.

Membrane properties and synaptic inputs of suprachiasmatic nucleus neurons in rat brain slices.

1. Whole-cell recordings were made from 390 neurons of the suprachiasmatic nucleus (SCN) in horizontal brain slices during different portions of the circadian day. The locomotor activity of the rats was measured prior to the preparation of brain slices to insure that each rat was entrained to a 12 h-12 h light-dark cycle. 2. The mean input conductance was 42% higher (1.58 nS) in neurons recorded near the subjective dawn than those (1.11 nS) recorded near the subjective dusk. The current required to hold the neurons at -60 mV also showed a circadian variation with a peak in the middle of the subjective day and a nadir in the middle of the subjective night. Analysis of the variations in the input conductance and the holding current at -60 mV suggested that at least two ion conductances are involved in the pacemaking of the circadian rhythms. 3. Voltage-clamped SCN neurons often had both outward and inward spontaneous postsynaptic currents. The outward currents were blocked by bicuculline but not by strychnine, and were identified as IPSCs mediated by GABAA receptors. The inward currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and were identified as EPSCs mediated by glutamate. Most spontaneous synaptic currents were miniature currents but action potential-dependent large events were seen more often in IPSCs than in EPSCs. 4. Stimulation of the optic nerve or chiasm usually evoked a monosynaptic EPSC which was mediated by both NMDA and non-NMDA receptors. In 13% of cells, optic nerve stimulation evoked an outward current or an inward current followed by an outward current; all the evoked currents were blocked by 4-aminophosphonovaleric acid (APV) and CNQX whereas the outward current only was blocked by bicuculline, suggesting involvement of an inhibitory interneuron. 5. SCN neurons sum the excitatory inputs from both optic nerves; on average each SCN cell receives innervation from at least 4.8 retinohypothalamic tract (RHT) axons. 6. Focal stimulation in the vicinity of the recorded neuron revealed that nearly all SCN neurons receive local or extranuclear GABAergic inputs operating via GABAA receptors. The EPSCs activated by such stimulation were not significantly different in amplitude and pharmacological properties from those induced by RHT stimulation. 7. One hundred and one neurons were labelled with neurobiotin during whole-cell recording. Based on the dendritic structures, four types of SCN neurons (monopolar, radial, simple bipolar and curly bipolar) were identified. The curly bipolar cells had a higher membrane conductance, holding current and hyperpolarization-activated current (Ih) amplitude than the other neuronal types. Radial neurons did not respond to optic nerve stimulation, which activated EPSCs in the other cell types.

The sensitivity of N-methyl-D-aspartate receptors to lead inhibition is dependent on the receptor subunit composition.

Pb+2 is a potent inhibitor of N-methyl-D-aspartate (NMDA) receptors and its action is dependent on neuronal maturation. Developmentally regulated expression of NMDA receptor subunits may underlie the changing sensitivity to Pb+2. In oocytes expressing in vitro transcribed cRNAs for zeta 1 epsilon 1 or zeta 1 epsilon 2 NMDA receptor subunits, Pb+2 inhibited glutamate-activated currents with IC50 values of 0.87 +/- 0.25 and 1.21 +/- 0.22 microM, respectively, and NMDA-activated currents with IC50 values of 1.37 +/- 0.47 and 1.11 +/- 0.33 microM, respectively. In oocytes expressing zeta 1 epsilon 1 epsilon 2 subunits, the IC50 values for Pb+2 blockade of NMDA- or glutamate-activated currents were significantly larger when compared to zeta 1 epsilon 1 or zeta 1 epsilon 2 combinations. Pb+2 concentrations greater than 1 microM inhibited glutamate-activated currents with an IC50 of 6.1 +/- 1.22 microM and NMDA-activated currents with an IC50 of 6.64 +/- 3.34 microM. Pb+2 reduced the maximal current amplitude consistent with a noncompetitive block. zeta 1 epsilon 1 epsilon 2 NMDA receptors were potentiated by low concentrations of Pb+2 ( < 1.0 microM). These data suggest that brain regions with zeta 1 epsilon 1 or zeta 1 epsilon 2 NMDA receptors subunits would be more vulnerable to Pb+2 toxicity than those with zeta 1 epsilon 1 epsilon 2 NMDA-receptors, which are expressed later in development. These data provide a mechanism for the reported changes in the efficacy of block of NMDA receptors by Pb+2 during development.

Melatonin receptors activate heteromeric G-protein coupled Kir3 channels.

The effects of melatonin on circadian pacemaker activity in the central nervous system may be the result of melatonin receptor activation of G-protein coupled potassium channels which inhibit the action potential firing of neurons. Xenopus laevis and human1a melatonin receptors stimulated heteromeric G-protein activated inwardly rectifying potassium channels (Kir3.1/Kir3.2) when expressed in vitro in oocytes. Pertussis toxin reduced iodo-melatonin (87.1% reduction) and melatonin (90.3% reduction) stimulated currents in a time-dependent manner for cells expressing X. laevis receptors. A similar pertussis toxin inhibition was observed for human melatonin receptors (melatonin, 78.9% reduction). This suggests a potential role for heteromeric Kir3 channels in the receptor-mediated actions of melatonin in vivo.

Melatonin activates an outward current and inhibits Ih in rat suprachiasmatic nucleus neurons.

Whole-cell voltage-clamp recordings were made from suprachiasmatic nucleus (SCN) neurons maintained in horizontal brain slices. The majority of neurons exhibited spontaneous and evoked excitatory and inhibitory synaptic currents (EPSC and IPSC), mediated by glutamate and GABA respectively. Melatonin had no effect on either the spontaneous or evoked EPSC or IPSC. Application of melatonin (0.1-30 microM) during circadian time (CT) 9-12 activated an outward current at -60 mV and increased the membrane conductance in a concentration-dependent manner. The current was augmented by depolarization, reduced by hyperpolarization and, in some cells, reversed its polarity near the potassium equilibrium potential. Some neurons also responded to melatonin during other times of the circadian day (CT 3-9 or CT 12-15). Hyperpolarizing steps, in a portion of cells, activated an inward cation current which resembled the Ih described in other neurons. Melatonin (10 microM) inhibited activation of the Ih. These data indicate that melatonin may inhibit SCN neurons by activating a potassium current and inhibiting the Ih.