Charles N. Allen

Circadian Dynamics of Cytosolic and Nuclear Ca2+ in Single Suprachiasmatic Nucleus Neurons

Intracellular free Ca(2+) regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca(2+)-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca(2+) concentration were observed in SCN neurons. The cytosolic Ca(2+) rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca(2+) rhythms, while ryanodine damped both Ca(2+) and MUA rhythms. These results demonstrate cytosolic Ca(2+) rhythms regulated by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons.

Intrinsic light responses of retinal ganglion cells projecting to the circadian system

In mammals, light entrainment of the circadian clock, located in the suprachiasmatic nuclei (SCN), requires retinal input. Traditional rod and cone photoreceptors, however, are not required. Instead, the SCN‐projecting retinal ganglion cells (RGCs) function as autonomous photoreceptors and exhibit light responses independent of rod‐ and cone‐driven input. Using whole‐cell patch‐clamp recording techniques, we have investigated the morphological and electrophysiological properties of this unique class of RGCs. Although SCN‐projecting RGCs resemble Type III cells in form, they display strikingly different physiological properties from these neurons. First, in response to the injection of a sustained depolarizing current, SCN‐projecting cells fired in a transient fashion, in contrast to most RGCs which fired robust trains of action potentials. Second, in response to light, SCN‐projecting RGCs exhibited an intensity‐dependent transient depolarization in the absence of rod and cone input. This depolarization reached a peak within 5 s and generated increased spiking activity before decaying to a plateau. Voltage‐clamp recordings were used to characterize the light‐activated conductance which generated this depolarization. In response to varying light intensities, SCN‐projecting RGCs exhibited a graded transient inward current which peaked within 5 s and decayed to a plateau. The voltage dependence of the light‐activated current was obtained by subtracting currents elicited by a voltage ramp before and during illumination. The light‐activated current displayed both inward and outward rectification and was largely unaffected by substitution of extracellular Na+ with choline. In both respects, the intrinsic light‐activated current observed in SCN‐projecting RGCs resembles currents carried by ion channels of the transient receptor potential (trp) family, which are known to mediate the light response of invertebrate photoreceptors.

The light-activated signaling pathway in SCN-projecting rat retinal ganglion cells

In mammals, the master circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus. The period and phase of the circadian pacemaker are calibrated by direct photic input from retinal ganglion cells (RGCs). SCN‐projecting RGCs respond to light in the absence of rod‐ and cone‐driven synaptic input, a property for which they are termed intrinsically photosensitive. In SCN‐projecting RGCs, light activates a nonselective cationic current that displays inward and outward rectification. The goal of the present study was to investigate the identity of the light‐activated ion channel and the intracellular signaling pathway leading to its activation. We considered two candidate channels, cyclic nucleotide‐gated (CNG) channels and transient receptor potential (TRP) channels, which mediate vertebrate and invertebrate phototransduction, respectively. We report that the intrinsic light response relies upon a G‐protein‐dependent process. Although our data indicate that cyclic nucleotides modulate the signaling pathway, CNG channels do not appear to conduct the light‐activated current because (i) cyclic nucleotides in the pipette solution do not activate a conductance or completely block the light response, (ii) CNG channel blockers fail to inhibit the light response, (iii) the effects of internal and external divalent cations are inconsistent with their effects on CNG channels, and (iv) immunohistochemistry reveals no CNG channels in SCN‐projecting RGCs. Finally, we show that the pharmacology of the light‐activated channel resembles that of some TRPC channel family members; the response is blocked by lanthanides and ruthenium red and SK&F 96365, and is enhanced by flufenamic acid and 1‐oleoyl‐2‐acetyl‐sn‐glycerol. Furthermore, immunohistochemical experiments reveal that TRPC6 is expressed in many RGCs, including those that express melanopsin.

Mutations of Presenilin Genes in Dilated Cardiomyopathy and Heart Failure

Two common disorders of the elderly are heart failure and Alzheimer disease (AD). Heart failure usually results from dilated cardiomyopathy (DCM). DCM of unknown cause in families has recently been shown to result from genetic disease, highlighting newly discovered disease mechanisms. AD is the most frequent neurodegenerative disease of older Americans. Familial AD is caused most commonly by presenilin 1 (PSEN1) or presenilin 2 (PSEN2) mutations, a discovery that has greatly advanced the field. The presenilins are also expressed in the heart and are critical to cardiac development. We hypothesized that mutations in presenilins may also be associated with DCM and that their discovery could provide new insight into the pathogenesis of DCM and heart failure. A total of 315 index patients with DCM were evaluated for sequence variation in PSEN1 and PSEN2. Families positive for mutations underwent additional clinical, genetic, and functional studies. A novel PSEN1 missense mutation (Asp333Gly) was identified in one family, and a single PSEN2 missense mutation (Ser130Leu) was found in two other families. Both mutations segregated with DCM and heart failure. The PSEN1 mutation was associated with complete penetrance and progressive disease that resulted in the necessity of cardiac transplantation or in death. The PSEN2 mutation showed partial penetrance, milder disease, and a more favorable prognosis. Calcium signaling was altered in cultured skin fibroblasts from PSEN1 and PSEN2 mutation carriers. These data indicate that PSEN1 and PSEN2 mutations are associated with DCM and heart failure and implicate novel mechanisms of myocardial disease.

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.

Light-Evoked Calcium Responses of Isolated Melanopsin-Expressing Retinal Ganglion Cells

A small number (<2%) of mammalian retinal ganglion cells express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). Light depolarizes ipRGCs and increases intracellular calcium levels ([Ca2+]i) but the signaling cascades underlying these responses have yet to be elucidated. To facilitate physiological studies on these rare photoreceptors, highly enriched ipRGC cultures from neonatal rats were generated using anti-melanopsin-mediated plate adhesion (immunopanning). This novel approach enabled experiments on isolated ipRGCs, eliminating the potential confounding influence of rod/cone-driven input. Light induced a rise in [Ca2+]i (monitored using fura-2 imaging) in the immunopanned ipRGCs and the source of this Ca2+ signal was investigated. The Ca2+ responses were inhibited by 2-aminoethoxydiphenyl borate, SKF-96365 (1–2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole), flufenamic acid, lanthanum, and gadolinium, consistent with the involvement of canonical transient receptor potential (TRP) channels in ipRGC phototransduction. However, the contribution of direct Ca2+ flux through a putative TRP channel to ipRGC [Ca2+]i was relatively small, as most (∼90%) of the light-induced Ca2+ responses could be blocked by preventing action potential firing with tetrodotoxin. The L-type voltage-gated Ca2+ channel (VGCC) blockers verapamil and (+)-cis-diltiazem significantly reduced the light-evoked Ca2+ responses, while the internal Ca2+ stores depleting agent thapsigargin had negligible effect. These results indicate that Ca2+ influx through VGCCs, activated after action potential firing, was the primary source for light-evoked elevations in ipRGC [Ca2+]i. Furthermore, concurrent Ca2+ imaging and cell-attached electrophysiological recordings demonstrated that the Ca2+ responses were highly correlated to spike frequency, thereby establishing a direct link between action potential firing and somatic [Ca2+]i in light-stimulated ipRGCs.

Inhibition of energy metabolism by 3-nitropropionic acid activates ATP-sensitive potassium channels

3-Nitropropionic acid (1 mM), which inhibits succinate dehydrogenase activity and reduces cellular energy, produces in the pyramidal cell layer of the hippocampal region CA1 a hyperpolarization for variable lengths of time before evoking an irreversible depolarization. Hyperpolarization is caused by an increased potassium conductance that is attenuated by glibenclamide (1-10 microM), a selective antagonist of ATP-sensitive potassium channels; in contrast, diazoxide (0.5 mM), an agonist at this channel, induces a hyperpolarization in CA1 neurons of rat hippocampal slices. The transient hyperpolarization after prolonged (ca. 1 h) application of 3-NPA is followed by a depolarization that is incompletely reversed by brief application of the glutamate antagonists (D-2-amino-5-phosphonopentanoic acid (APV), 6,7-dichloroquinoxaline-2,3-dione (CNQX), 3-(+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), 7-chloro-kynurenic acid (7Cl-KYN)). Early application of glibenclamide (within the initial 5 min) blocked or reduced hyperpolarization and accelerated the depolarization. These data suggest that metabolic inhibition by 3-NPA initially activates ATP-sensitive potassium channels. Events other than activation of glutamate receptors participate in the final depolarization resulting from uncoupling of oxidative phosphorylation.

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.

Calbindin neurons in the hamster suprachiasmatic nucleus do not exhibit a circadian variation in spontaneous firing rate.

The role of the mammalian suprachiasmatic nuclei (SCN) in generating circadian rhythms in behaviours and other physiological processes is well established. A prominent feature of SCN neurons is the circadian oscillation in action potential firing frequency, with a peak near midday. A subset of calbindin‐immunoreactive (CB+) neurons form a compact subnucleus (CBsn) in the hamster SCN. Restoration of rhythmicity using fetal SCN grafts in SCN‐lesioned hamsters is critically dependent upon the presence of CB+ neurons within the transplanted grafts [LeSauter & Silver (1999) J. Neurosci., 5574–5585]. The aim of the current study was to determine whether CB+ neurons within the CBsn of the hamster SCN fire action potentials in a circadian pattern as part of their output signal. Using patch‐clamp recording, we demonstrated that CB+ neurons in the CBsn do not express a circadian rhythm in spontaneous firing frequency under diurnal conditions in vitro. Furthermore, the percentage of silent CB– cells varies with zeitgeber time, whereas the percentage of silent CB+ cells does not. Immunohistochemical analysis revealed that the CBsn is a nonhomogeneous nucleus, containing many more CB– than CB+ cells. Our results reveal that CB+ neurons within the CBsn represent a functionally distinct neuronal subpopulation in which rhythmic action potential output may not be necessary for the restoration of behavioural circadian rhythmicity.

Synaptic inputs to retinal ganglion cells that set the circadian clock.

Melanopsin‐containing retinal ganglion cells (RGCs) project to the suprachiasmatic nuclei (SCN) and mediate photoentrainment of the circadian system. Melanopsin is a novel retinal‐based photopigment that renders these cells intrinsically photosensitive (ip). Although genetic ablation of melanopsin abolishes the intrinsic light response, it has a surprisingly minor effect on circadian photoentrainment. This and other non‐visual responses to light are lost only when the melanopsin deficiency is coupled with mutations that disable classical rod and cone photoreceptors, suggesting that melanopsin‐containing RGCs also receive rod‐ and cone‐driven synaptic inputs. Using whole‐cell patch‐clamp recording, we demonstrate that light triggers synaptic currents in ipRGCs via activation of ionotropic glutamate and γ‐aminobutyric acid (GABA) receptors. Miniature postsynaptic currents (mPSCs) were clearly observed in ipRGCs, although they were less robust and were seen less frequently than those seen in non‐ip cells. Pharmacological treatments revealed that the majority of ipRGCs receive excitatory glutamatergic inputs that were blocked by DNQX and/or kynurenic acid, as well as inhibitory GABAergic inputs that were blocked by bicuculline. Other ipRGCs received either glutamatergic or GABAergic inputs nearly exclusively. Although strychnine (Strych)‐sensitive mPSCs were evident on many non‐ipRGCs, indicating the presence of glycinergic inputs, we saw no evidence of Strych‐sensitive events in ipRGCs. Based on these results, it is clear that SCN‐projecting RGCs can respond to light both via an intrinsic melanopsin‐based signaling cascade and via a synaptic pathway driven by classical rod and/or cone photoreceptors. It remains to be determined how the ipRGCs integrate these temporally distinct inputs to generate the signals that mediate circadian photoentrainment and other non‐visual responses to light.