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1.  O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination 
Cell metabolism  2013;17(2):303-310.
Circadian clocks are coupled to metabolic oscillations through nutrient-sensing pathways. Nutrient flux into the hexosamine biosynthesis pathway triggers covalent protein modification by O-linked β-D-N-acetylglucosamine (O-GlcNAc). Here we show that the hexosamine/O-GlcNAc pathway modulates peripheral clock oscillation. O-GlcNAc transferase (OGT) promotes expression of BMAL1/CLOCK target genes and affects circadian oscillation of clock genes in vitro and in vivo. Both BMAL1 and CLOCK are rhythmically O-GlcNAcylated and this protein modification stabilizes BMAL1 and CLOCK by inhibiting their ubiquitination. In vivo analysis of genetically modified mice with perturbed hepatic OGT expression shows aberrant circadian rhythms of glucose homeostasis. These results establish the counteraction between O-GlcNAcylation and ubiquitination as a key mechanism that regulates the circadian clock and suggest a crucial role for O-GlcNAc signaling in transducing nutritional signals to the core circadian timing machinery.
PMCID: PMC3647362  PMID: 23395176
2.  Peptide neuromodulation in invertebrate model systems 
Neuron  2012;76(1):82-97.
Neuropeptides modulate neural circuits controlling adaptive animal behaviors and physiological processes, such as feeding/metabolism, reproductive behaviors, circadian rhythms, central pattern generation, and sensorimotor integration. Invertebrate model systems have enabled detailed experimental analysis using combined genetic, behavioral, and physiological approaches. Here we review selected examples of neuropeptide modulation in crustaceans, mollusks, insects, and nematodes, with a particular emphasis on the genetic model organisms Drosophila melanogaster and Caenorhabditis elegans, where remarkable progress has been made. On the basis of this survey, we provide several integrating conceptual principles for understanding how neuropeptides modulate circuit function, and also propose that continued progress in this area requires increased emphasis on the development of richer, more sophisticated behavioral paradigms.
PMCID: PMC3466441  PMID: 23040808
3.  Expression Patterns of the Drosophila Neuropeptide CCHamide-2 and Its Receptor May Suggest Hormonal Signaling from the Gut to the Brain 
PLoS ONE  2013;8(10):e76131.
The insect neuropeptides CCHamide-1 and -2 are recently discovered peptides that probably occur in all arthropods. Here, we used immunocytochemistry, in situ hybridization, and quantitative PCR (qPCR), to localize the two peptides in the fruitfly Drosophila melanogaster. We found that CCHamide-1 and -2 were localized in endocrine cells of the midgut of larvae and adult flies. These endocrine cells had the appearance of sensory cells, projecting processes close to or into the gut lumen. In addition, CCHamide-2 was also localized in about forty neurons in the brain hemispheres and ventral nerve cord of larvae. Using qPCR we found high expression of the CCHamide-2 gene in the larval gut and very low expression of its receptor gene, while in the larval brain we found low expression of CCHamide-2 and very high expression of its receptor. These expression patterns suggest the following model: Endocrine CCHamide-2 cells in the gut sense the quality of food components in the gut lumen and transmit this information to the brain by releasing CCHamide-2 into the circulation; subsequently, after binding to its brain receptors, CCHamides-2 induces an altered feeding behavior in the animal and possibly other homeostatic adaptations.
PMCID: PMC3788761  PMID: 24098432
4.  Auditory Responses of Engrailed and Invected-Expressing Johnston’s Organ Neurons in Drosophila melanogaster 
PLoS ONE  2013;8(8):e71419.
The roles of the transcription factor Engrailed (En), and its paralogue Invected (Inv), in adult Drosophila Johnston’s Organ sensory neurons are unknown. We used en-GAL4 driven CD8-GFP and antibody staining to characterize these neurons in the pedicel (second antennal segment). The majority of En and Inv-expressing Johnston’s Organ neurons (En-JONs) are located in the ventral part of the posterior group of JONs, with only a few in the medial group. Anatomical classification of En-JON axon projections shows they are mainly type A and E, with a few type B. Extracellular recording of sound-evoked potentials (SEPs) from the antennal nerve was used along with Kir2.1 silencing to assess the contribution that En-JONs make to the auditory response to pure-tone sound stimuli. Silencing En-JONs reduces the SEP amplitude at the onset of the stimulus by about half at 100, 200 and 400 Hz, and also reduces the steady-state response to 200 Hz. En-JONs respond to 82 dB and 92 dB sounds but not 98 dB. Despite their asymmetrical distribution in the Johnston’s Organ they respond equally strongly to both directions of movement of the arista. This implies that individual neurons are excited in both directions, a conclusion supported by reanalysis of the morphology of the pedicel-funicular joint. Other methods of silencing the JONs were also used: RNAi against the voltage-gated Na+ channel encoded by the para gene, expression of attenuated diphtheria toxin, and expression of a modified influenza toxin M2(H37A). Only the latter was found to be more effective than Kir2.1. Three additional JON subsets were characterized using Flylight GAL4 lines. inv-GAL4 88B12 and Gycβ100B-GAL4 12G03 express in different subsets of A group neurons and CG12484-GAL4 91G04 is expressed in B neurons. All three contribute to the auditory response to 200 Hz tones.
PMCID: PMC3734059  PMID: 23940751
5.  Synchronized Bilateral Synaptic Inputs to Drosophila melanogaster Neuropeptidergic Rest/Arousal Neurons 
Neuropeptide Pigment-Dispersing Factor (PDF)-secreting large ventrolateral neurons (lLNvs) in the Drosophila brain regulate daily patterns of rest and arousal. These bilateral wake-promoting neurons are light-responsive and integrate information from the circadian system, sleep circuits, and light environment. In order to begin to dissect the synaptic circuitry of the circadian neural network, we performed simultaneous dual whole-cell patch clamp recordings of pairs of lLNvs. Both ipsilateral and contralateral pairs of lLNvs exhibit synchronous rhythmic membrane activity with a periodicity of about 5 to 10 seconds. This rhythmic lLNv activity is blocked by tetrodotoxin (TTX) voltage-gated sodium blocker, or α-bungarotoxin (α-BuTX) nicotinic acetylcholine receptor antagonist, indicating that action potential-dependent cholinergic synaptic connections are required for rhythmic lLNv activity. Since injecting current into one neuron of the pair had no effect on the membrane activity of the other neuron of the pair, this suggests that the synchrony is due to bilateral inputs and not coupling between the pairs of lLNvs. To further elucidate the nature of these synaptic inputs to lLNvs, we blocked or activated a variety of neurotransmitter receptors and measured effects on network activity and ionic conductances. These measurements indicate the lLNvs possess excitatory nicotinic ACh receptors, inhibitory ionotropic GABAA receptors, and inhibitory ionotropic glutamate-gated chloride (GluCl) receptors. We demonstrate that cholinergic input, but not GABAergic input, is required for synchronous membrane activity, while GABA can modulate firing patterns. We conclude that neuropeptidergic lLNvs that control rest and arousal receive synchronous synaptic inputs mediated by ACh.
PMCID: PMC3125135  PMID: 21632940
synaptic activity; clock neuron; nicotinic acetylcholine receptor; GABAA receptor; glutamate-gated Cl− channel; synchronous activity
6.  Tethering toxins and peptide ligands for modulation of neuronal function 
Tethering genetically encoded peptide toxins or ligands close to their point of activity at the cell plasma membrane provides a new approach to the study of cell networks and neuronal circuits, as it allows selective targeting of specific cell populations, enhances the working concentration of the ligand or blocker peptide, and permits the engineering of a large variety of t-peptides (e.g., including use of fluorescent markers, viral vectors and point mutation variants). This review describes the development of tethered toxins and peptides derived from the identification of the cell surface nAChR modulator lynx1, the existence of related endogenous cell surface modulators of nAChR and AMPA receptors, and the application of the t-toxin and t-neuropeptide technology to the dissection of neuronal circuits in metazoans.
PMCID: PMC3294089  PMID: 22119144
7.  Circadian control of membrane excitability in Drosophila melanogaster lateral ventral clock neurons 
Drosophila circadian rhythms are controlled by a neural circuit containing ∼150 clock neurons. While much is known about mechanisms of autonomous cellular oscillation, the connection between cellular oscillation and functional outputs that control physiological and behavioral rhythms is poorly understood. To address this issue, we performed whole-cell patch-clamp recordings on lateral ventral clock neurons (LNvs), including large and small LNvs (lLNvs, sLNvs), in situ in adult fly whole-brain explants. We found two distinct sizes of action potentials (APs) in more than 50% of lLNvs that fire APs spontaneously, and determined that large APs originate in the ipsilateral optic lobe and small APs in the contralateral. lLNv resting membrane potential (RMP), spontaneous AP firing rate, and membrane resistance are cyclically regulated as a function of time-of-day in 12hr:12hr light:dark conditions (LD). lLNv RMP becomes more hyperpolarized as time progresses from dawn to dusk with a concomitant decrease in spontaneous AP firing rate and membrane resistance. From dusk to dawn, lLNv RMP becomes more depolarized, with spontaneous AP firing rate and membrane resistance remaining stable. In contrast, circadian defective per0 null mutant lLNv membrane excitability is nearly constant in LD. Over 24hr in constant darkness (DD), wild-type lLNv membrane excitability is not cyclically regulated, although RMP gradually becomes slightly more depolarized. sLNv RMP is most depolarized around lights-on, with substantial variability centered around lights-off in LD. Our results indicate that LNv membrane excitability encodes time-of-day via a circadian clock-dependent mechanism, and likely plays a critical role in regulating Drosophila circadian behavior.
PMCID: PMC2680300  PMID: 18562620
circadian rhythms; Drosophila; membrane excitabiliy; time-of-day; clock neuron; cellular oscillation
8.  Autoreceptor Modulation of Peptide/Neurotransmitter Co-release from PDF Neurons Determines Allocation of Circadian Activity in Drosophila 
Cell reports  2012;2(2):332-344.
Drosophila melanogaster flies concentrate behavioral activity around dawn and dusk. This organization of daily activity is controlled by central circadian clock neurons, including the lateral ventral pacemaker neurons (LNvs) that secrete the neuropeptide PDF (Pigment Dispersing Factor). Previous studies have demonstrated the requirement for PDF signaling to PDF receptor (PDFR)-expressing dorsal clock neurons in organizing circadian activity. While LNvs also express functional PDFR, the role of these autoreceptors has remained enigmatic. Here we show that (1) PDFR activation in LNvs shifts the balance of circadian activity from evening to morning, similar to behavioral responses to summer-like environmental conditions and (2) this shift is mediated by stimulation of the Ga,s-cAMP pathway and a consequent change in PDF/neurotransmitter co-release from the LNvs. These results suggest a novel mechanism for environmental control of the allocation of circadian activity and provide new general insight into the role of neuropeptide autoreceptors in behavioral control circuits.
PMCID: PMC3432947  PMID: 22938867
9.  Circadian expression of clock genes in mouse macrophages, dendritic cells, and B cells 
Brain, behavior, and immunity  2011;26(3):407-413.
In mammals, circadian and daily rhythms influence nearly all aspects of physiology, ranging from behavior to gene expression. Functional molecular clocks have been described in the murine spleen and splenic NK cells. The aim of our study was to investigate the existence of molecular clock mechanisms in other immune cells. Therefore, we measured the circadian changes in gene expression of clock genes (Per1, Per2, Bmal1, and Clock) and clock-controlled transcription factors (Rev-erbα and Dbp) in splenic enriched macrophages, dendritic cells, and B cells in both mice entrained to a light-dark cycle and under constant environmental conditions. Our study reveals the existence of functional molecular clock mechanisms in splenic macrophages, dendritic cells, and B cells.
PMCID: PMC3336152  PMID: 22019350
Mouse splenic macrophages; dendritic cells; B cells possess functional circadian molecular clocks
10.  Altered Rest-Activity Patterns Evolve via Circadian Independent Mechanisms in Cave Adapted Balitorid Loaches 
PLoS ONE  2012;7(2):e30868.
Circadian rhythms and rest homeostasis are independent processes, each regulating important components of rest-activity patterns. Evolutionarily, the two are distinct from one another; total rest time is maintained unaffected even when circadian pacemaker cells are ablated. Throughout the animal kingdom, there exists a huge variation in rest-activity patterns, yet it is unclear how these behaviors have evolved. Here we show that four species of balitorid cavefish have greatly reduced rest times in comparison to rest times of their surface relatives. All four cave species retained biological rhythmicity, and in three of the four there is a pronounced 24-hour rhythm; in the fourth there is an altered rhythmicity of 38–40 hours. Thus, consistent changes in total rest have evolved in these species independent of circadian rhythmicity. Taken together, our data suggest that consistent reduction in total rest times were accomplished evolutionarily through alterations in rest homeostasis.
PMCID: PMC3278411  PMID: 22348026
11.  Dopamine Modulates Metabolic Rate and Temperature Sensitivity in Drosophila melanogaster 
PLoS ONE  2012;7(2):e31513.
Homeothermal animals, such as mammals, maintain their body temperature by heat generation and heat dissipation, while poikilothermal animals, such as insects, accomplish it by relocating to an environment of their favored temperature. Catecholamines are known to regulate thermogenesis and metabolic rate in mammals, but their roles in other animals are poorly understood. The fruit fly, Drosophila melanogaster, has been used as a model system for the genetic studies of temperature preference behavior. Here, we demonstrate that metabolic rate and temperature sensitivity of some temperature sensitive behaviors are regulated by dopamine in Drosophila. Temperature-sensitive molecules like dTrpA1 and shits induce temperature-dependent behavioral changes, and the temperature at which the changes are induced were lowered in the dopamine transporter-defective mutant, fumin. The mutant also displays a preference for lower temperatures. This thermophobic phenotype was rescued by the genetic recovery of the dopamine transporter in dopamine neurons. Flies fed with a dopamine biosynthesis inhibitor (3-iodo-L-tyrosine), which diminishes dopamine signaling, exhibited preference for a higher temperature. Furthermore, we found that the metabolic rate is up-regulated in the fumin mutant. Taken together, dopamine has functions in the temperature sensitivity of behavioral changes and metabolic rate regulation in Drosophila, as well as its previously reported functions in arousal/sleep regulation.
PMCID: PMC3274542  PMID: 22347491
12.  Large Scale Library Generation for High Throughput Sequencing 
PLoS ONE  2011;6(4):e19119.
Large efforts have recently been made to automate the sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols.
Methodology/Principal Findings
In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions.
The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.
PMCID: PMC3083417  PMID: 21589638
13.  The Batten Disease Palmitoyl Protein Thioesterase 1 Gene Regulates Neural Specification and Axon Connectivity during Drosophila Embryonic Development 
PLoS ONE  2010;5(12):e14402.
Palmitoyl Protein Thioesterase 1 (PPT1) is an essential lysosomal protein in the mammalian nervous system whereby defects result in a fatal pediatric disease called Infantile Neuronal Ceroids Lipofuscinosis (INCL). Flies bearing mutations in the Drosophila ortholog Ppt1 exhibit phenotypes similar to the human disease: accumulation of autofluorescence deposits and shortened adult lifespan. Since INCL patients die as young children, early developmental neural defects due to the loss of PPT1 are postulated but have yet to be elucidated. Here we show that Drosophila Ppt1 is required during embryonic neural development. Ppt1 embryos display numerous neural defects ranging from abnormal cell fate specification in a number of identified precursor lineages in the CNS, missing and disorganized neurons, faulty motoneuronal axon trajectory, and discontinuous, misaligned, and incorrect midline crossings of the longitudinal axon bundles of the ventral nerve cord. Defects in the PNS include a decreased number of sensory neurons, disorganized chordotonal neural clusters, and abnormally shaped neurons with aberrant dendritic projections. These results indicate that Ppt1 is essential for proper neuronal cell fates and organization; and to establish the local environment for proper axon guidance and fasciculation. Ppt1 function is well conserved from humans to flies; thus the INCL pathologies may be due, in part, to the accumulation of various embryonic neural defects similar to that of Drosophila. These findings may be relevant for understanding the developmental origin of neural deficiencies in INCL.
PMCID: PMC3008717  PMID: 21203506
14.  Heterogeneous Response to a Quorum-Sensing Signal in the Luminescence of Individual Vibrio fischeri 
PLoS ONE  2010;5(11):e15473.
The marine bacterium Vibrio fischeri regulates its bioluminescence through a quorum sensing mechanism: the bacterium releases diffusible small molecules (autoinducers) that accumulate in the environment as the population density increases. This accumulation of autoinducer (AI) eventually activates transcriptional regulators for bioluminescence as well as host colonization behaviors. Although V.fischeri quorum sensing has been extensively characterized in bulk populations, far less is known about how it performs at the level of the individual cell, where biochemical noise is likely to limit the precision of luminescence regulation. We have measured the time-dependence and AI-dependence of light production by individual V.fischeri cells that are immobilized in a perfusion chamber and supplied with a defined concentration of exogenous AI. We use low-light level microscopy to record and quantify the photon emission from the cells over periods of several hours as they respond to the introduction of AI. We observe an extremely heterogeneous response to the AI signal. Individual cells differ widely in the onset time for their luminescence and in their resulting brightness, even in the presence of high AI concentrations that saturate the light output from a bulk population. The observed heterogeneity shows that although a given concentration of quorum signal may determine the average light output from a population of cells, it provides far weaker control over the luminescence output of each individual cell.
PMCID: PMC2982848  PMID: 21103327
15.  Cellular dissection of circadian peptide signals using genetically encoded membrane-tethered ligands 
Current biology : CB  2009;19(14):1167-1175.
Neuropeptides regulate a broad range of physiological and behavioral processes. Elucidation of neuropeptide function requires identifying the cells that respond to neuropeptide signals and determining the molecular, cellular, physiological, and behavioral consequences of activation of their cognate GPCRs in those cells. As a novel tool for answering these questions, we have developed genetically encoded neuropeptides covalently tethered to a glycosylphosphatidyl inositol (GPI) glycolipid anchor on the extracellular leaflet of the plasma membrane (“t-peptides”).
We show that t-peptides cell-autonomously induce activation of their cognate GPCRs in cells that express both the t-peptide and its receptor. In the neural circuit controlling circadian rest-activity rhythms in Drosophila melanogaster, rhythmic secretion of the neuropeptide Pigment Dispersing Factor (PDF) and activation of its GPCR (PDFR) are important for intercellular communication of phase information and coordination of cellular oscillations of multiple circadian clock neurons. Broad expression of t-PDF in the circadian control circuit overcomes arrhythmicity induced by pdf01 null mutation, most likely due to activation of PDFR in PDFR-expressing clock neurons that do not themselves secrete PDF. More restricted cellular expression of t-PDF suggests that activation of PDFR accelerates cellular timekeeping in some clock neurons, while decelerating others.
The activation of PDFR in pdf01 null-mutant flies—and thus the absence of PDF-mediated intercellular transfer of phase information—induces strong rhythmicity in constant darkness, thus establishing a distinct role for PDF signaling in the circadian control circuit independent of the intercellular communication of temporal phase information. The t-peptide technology we have developed and validated should provide a useful tool for cellular dissection of bioactive peptide signaling in a variety of organisms and physiological contexts.
PMCID: PMC2719018  PMID: 19592252
16.  Do Decapod Crustaceans Have Nociceptors for Extreme pH? 
PLoS ONE  2010;5(4):e10244.
Nociception is the physiological detection of noxious stimuli. Because of its obvious importance, nociception is expected to be widespread across animal taxa and to trigger robust behaviours reliably. Nociception in invertebrates, such as crustaceans, is poorly studied.
Methodology/Principal Findings
Three decapod crustacean species were tested for nociceptive behaviour: Louisiana red swamp crayfish (Procambarus clarkii), white shrimp (Litopenaeus setiferus), and grass shrimp (Palaemonetes sp.). Applying sodium hydroxide, hydrochloric acid, or benzocaine to the antennae caused no change in behaviour in the three species compared to controls. Animals did not groom the stimulated antenna, and there was no difference in movement of treated individuals and controls. Extracellular recordings of antennal nerves in P. clarkii revealed continual spontaneous activity, but no neurons that were reliably excited by the application of concentrated sodium hydroxide or hydrochloric acid.
Previously reported responses to extreme pH are either not consistently evoked across species or were mischaracterized as nociception. There was no behavioural or physiological evidence that the antennae contained specialized nociceptors that responded to pH.
PMCID: PMC2857684  PMID: 20422026
17.  RNA-Interference Knockdown of Drosophila Pigment Dispersing Factor in Neuronal Subsets: The Anatomical Basis of a Neuropeptide's Circadian Functions 
PLoS ONE  2009;4(12):e8298.
In animals, neuropeptide signaling is an important component of circadian timekeeping. The neuropeptide pigment dispersing factor (PDF) is required for several aspects of circadian activity rhythms in Drosophila.
Methodology/Principal Findings
Here we investigate the anatomical basis for PDF's various circadian functions by targeted PDF RNA-interference in specific classes of Drosophila neuron. We demonstrate that PDF is required in the ventro-lateral neurons (vLNs) of the central brain and not in the abdominal ganglion for normal activity rhythms. Differential knockdown of PDF in the large or small vLNs indicates that PDF from the small vLNs is likely responsible for the maintenance of free-running activity rhythms and that PDF is not required in the large vLNs for normal behavior. PDF's role in setting the period of free-running activity rhythms and the proper timing of evening activity under light:dark cycles emanates from both subtypes of vLN, since PDF in either class of vLN was sufficient for these aspects of behavior.
These results reveal the neuroanatomical basis PDF's various circadian functions and refine our understanding of the clock neuron circuitry of Drosophila.
PMCID: PMC2788783  PMID: 20011537
18.  Studies of α-Helicity and Intersegmental Interactions in Voltage-Gated Na+ Channels: S2D4 
PLoS ONE  2009;4(11):e7674.
Much data, including crystallographic, support structural models of sodium and potassium channels consisting of S1–S4 transmembrane segments (the “voltage-sensing domain”) clustered around a central pore-forming region (S5–S6 segments and the intervening loop). Voltage gated sodium channels have four non-identical domains which differentiates them from the homotetrameric potassium channels that form the basis for current structural models. Since potassium and sodium channels also exhibit many different functional characteristics and the fourth domain (D4) of sodium channels differs in function from other domains (D1–D3), we have explored its structure in order to determine whether segments in D4 of sodium channels differ significantly from that determined for potassium channels. We have probed the secondary and tertiary structure and the role of the individual amino acid residues of the S2D4) of Nav1.4 by employing cysteine-scanning mutagenesis (with tryptophan and glutamine substituted for native cysteine). A Fourier transform power spectrum of perturbations in free energy of steady-state inactivation gating (using midpoint potentials and slopes of Boltzmann equation fits of channel availability, h∞-V plots) indicates a substantial amount of α-helical structure in S2D4 (peak at 106°, α-Periodicity Index (α-PI) of 3.10), This conclusion is supported by α-PI values of 3.28 and 2.84 for the perturbations in rate constants of entry into (β) and exit from (α) fast inactivation at 0 mV for mutant channels relative to WT channels assuming a simple two-state model for transition from the open to inactivated state. The results of cysteine substitution at the two most sensitive sites of the S2D4 α-helix (N1382 and E1392C) support the existence of electrostatic network interactions between S2 and other transmembrane segments within Nav1.4D4 similar to but not identical to those proposed for K+ channels.
PMCID: PMC2766034  PMID: 19881885
19.  Circadian Modulation of Gene Expression, but not Glutamate Uptake, in Mouse and Rat Cortical Astrocytes 
PLoS ONE  2009;4(10):e7476.
Circadian clocks control daily rhythms including sleep-wake, hormone secretion, and metabolism. These clocks are based on intracellular transcription-translation feedback loops that sustain daily oscillations of gene expression in many cell types. Mammalian astrocytes display circadian rhythms in the expression of the clock genes Period1 (Per1) and Period2 (Per2). However, a functional role for circadian oscillations in astrocytes is unknown. Because uptake of extrasynaptic glutamate depends on the presence of Per2 in astrocytes, we asked whether glutamate uptake by glia is circadian.
Methodology/Principal Findings
We measured glutamate uptake, transcript and protein levels of the astrocyte-specific glutamate transporter, Glast, and the expression of Per1 and Per2 from cultured cortical astrocytes and from explants of somatosensory cortex. We found that glutamate uptake and Glast mRNA and protein expression were significantly reduced in Clock/Clock, Per2- or NPAS2-deficient glia. Uptake was augmented when the medium was supplemented with dibutyryl-cAMP or B27. Critically, glutamate uptake was not circadian in cortical astrocytes cultured from rats or mice or in cortical slices from mice.
We conclude that glutamate uptake levels are modulated by CLOCK, PER2, NPAS2, and the composition of the culture medium, and that uptake does not show circadian variations.
PMCID: PMC2758999  PMID: 19829696
20.  Efferent Projections of Prokineticin 2 Expressing Neurons in the Mouse Suprachiasmatic Nucleus 
PLoS ONE  2009;4(9):e7151.
The suprachiasmatic nucleus (SCN) in the hypothalamus is the predominant circadian clock in mammals. To function as a pacemaker, the intrinsic timing signal from the SCN must be transmitted to different brain regions. Prokineticin 2 (PK2) is one of the candidate output molecules from the SCN. In this study, we investigated the efferent projections of PK2-expressing neurons in the SCN through a transgenic reporter approach. Using a bacterial artificial chromosome (BAC) transgenic mouse line, in which the enhanced green fluorescence protein (EGFP) reporter gene expression was driven by the PK2 promoter, we were able to obtain an efferent projections map from the EGFP-expressing neurons in the SCN. Our data revealed that EGFP-expressing neurons in the SCN, hence representing some of the PK2-expressing neurons, projected to many known SCN target areas, including the ventral lateral septum, medial preoptic area, subparaventricular zone, paraventricular nucleus, dorsomedial hypothalamic nucleus, lateral hypothalamic area and paraventricular thalamic nucleus. The efferent projections of PK2-expressing neurons supported the role of PK2 as an output molecule of the SCN.
PMCID: PMC2747004  PMID: 19784373
21.  Pregnenolone Sulfate Potentiates the Inwardly Rectifying K+ Channel Kir2.3 
PLoS ONE  2009;4(7):e6311.
Neurosteroids have various physiological and neuropsychopharmacological effects. In addition to the genomic effects of steroids, some neurosteroids modulate several neurotransmitter receptors and channels, such as N-methyl-D-aspartate receptors, γ-aminobutyric acid type A (GABAA) receptors, and σ1 receptors, and voltage-gated Ca2+ and K+ channels. However, the molecular mechanisms underlying the various effects of neurosteroids have not yet been sufficiently clarified. In the nervous system, inwardly rectifying K+ (Kir) channels also play important roles in the control of resting membrane potential, cellular excitability and K+ homeostasis. Among constitutively active Kir2 channels in a major Kir subfamily, Kir2.3 channels are expressed predominantly in the forebrain, a brain area related to cognition, memory, emotion, and neuropsychiatric disorders.
Methodology/Principal Findings
The present study examined the effects of various neurosteroids on Kir2.3 channels using the Xenopus oocyte expression assay. In oocytes injected with Kir2.3 mRNA, only pregnenolone sulfate (PREGS), among nine neurosteroids tested, reversibly potentiated Kir2.3 currents. The potentiation effect was concentration-dependent in the micromolar range, and the current-voltage relationship showed inward rectification. However, the potentiation effect of PREGS was not observed when PREGS was applied intracellularly and was not affected by extracellular pH conditions. Furthermore, although Kir1.1, Kir2.1, Kir2.2, and Kir3 channels were insensitive to PREGS, in oocytes injected with Kir2.1/Kir2.3 or Kir2.2/Kir2.3 mRNA, but not Kir2.1/Kir2.2 mRNA, PREGS potentiated Kir currents. These potentiation properties in the concentration-response relationships were less potent than for Kir2.3 channels, suggesting action of PREGS on Kir2.3-containing Kir2 heteromeric channels.
The present results suggest that PREGS acts as a positive modulator of Kir2.3 channels. Kir2.3 channel potentiation may provide novel insights into the various effects of PREGS.
PMCID: PMC2710005  PMID: 19621089
22.  PINP: A New Method of Tagging Neuronal Populations for Identification during In Vivo Electrophysiological Recording 
PLoS ONE  2009;4(7):e6099.
Neural circuits are exquisitely organized, consisting of many different neuronal subpopulations. However, it is difficult to assess the functional roles of these subpopulations using conventional extracellular recording techniques because these techniques do not easily distinguish spikes from different neuronal populations. To overcome this limitation, we have developed PINP (Photostimulation-assisted Identification of Neuronal Populations), a method of tagging neuronal populations for identification during in vivo electrophysiological recording. The method is based on expressing the light-activated channel channelrhodopsin-2 (ChR2) to restricted neuronal subpopulations. ChR2-tagged neurons can be detected electrophysiologically in vivo since illumination of these neurons with a brief flash of blue light triggers a short latency reliable action potential. We demonstrate the feasibility of this technique by expressing ChR2 in distinct populations of cortical neurons using two different strategies. First, we labeled a subpopulation of cortical neurons—mainly fast-spiking interneurons—by using adeno-associated virus (AAV) to deliver ChR2 in a transgenic mouse line in which the expression of Cre recombinase was driven by the parvalbumin promoter. Second, we labeled subpopulations of excitatory neurons in the rat auditory cortex with ChR2 based on projection target by using herpes simplex virus 1 (HSV1), which is efficiently taken up by axons and transported retrogradely; we find that this latter population responds to acoustic stimulation differently from unlabeled neurons. Tagging neurons is a novel application of ChR2, used in this case to monitor activity instead of manipulating it. PINP can be readily extended to other populations of genetically identifiable neurons, and will provide a useful method for probing the functional role of different neuronal populations in vivo.
PMCID: PMC2702752  PMID: 19584920
23.  Perturbing Dynamin Reveals Potent Effects on the Drosophila Circadian Clock 
PLoS ONE  2009;4(4):e5235.
Transcriptional feedback loops are central to circadian clock function. However, the role of neural activity and membrane events in molecular rhythms in the fruit fly Drosophila is unclear. To address this question, we expressed a temperature-sensitive, dominant negative allele of the fly homolog of dynamin called shibirets1 (shits1), an active component in membrane vesicle scission.
Principal Findings
Broad expression in clock cells resulted in unexpectedly long, robust periods (>28 hours) comparable to perturbation of core clock components, suggesting an unappreciated role of membrane dynamics in setting period. Expression in the pacemaker lateral ventral neurons (LNv) was necessary and sufficient for this effect. Manipulation of other endocytic components exacerbated shits1's behavioral effects, suggesting its mechanism is specific to endocytic regulation. PKA overexpression rescued period effects suggesting shits1 may downregulate PKA pathways. Levels of the clock component PERIOD were reduced in the shits1-expressing pacemaker small LNv of flies held at a fully restrictive temperature (29°C). Less restrictive conditions (25°C) delayed cycling proportional to observed behavioral changes. Levels of the neuropeptide PIGMENT-DISPERSING FACTOR (PDF), the only known LNv neurotransmitter, were also reduced, but PERIOD cycling was still delayed in flies lacking PDF, implicating a PDF-independent process. Further, shits1 expression in the eye also results in reduced PER protein and per and vri transcript levels, suggesting that shibire-dependent signaling extends to peripheral clocks. The level of nuclear CLK, transcriptional activator of many core clock genes, is also reduced in shits1 flies, and Clk overexpression suppresses the period-altering effects of shits1.
We propose that membrane protein turnover through endocytic regulation of PKA pathways modulates the core clock by altering CLK levels and/or activity. These results suggest an important role for membrane scission in setting circadian period.
PMCID: PMC2668759  PMID: 19384421
24.  BK Channels Regulate Spontaneous Action Potential Rhythmicity in the Suprachiasmatic Nucleus 
PLoS ONE  2008;3(12):e3884.
Circadian (∼24 hr) rhythms are generated by the central pacemaker localized to the suprachiasmatic nucleus (SCN) of the hypothalamus. Although the basis for intrinsic rhythmicity is generally understood to rely on transcription factors encoded by “clock genes”, less is known about the daily regulation of SCN neuronal activity patterns that communicate a circadian time signal to downstream behaviors and physiological systems. Action potentials in the SCN are necessary for the circadian timing of behavior, and individual SCN neurons modulate their spontaneous firing rate (SFR) over the daily cycle, suggesting that the circadian patterning of neuronal activity is necessary for normal behavioral rhythm expression. The BK K+ channel plays an important role in suppressing spontaneous firing at night in SCN neurons. Deletion of the Kcnma1 gene, encoding the BK channel, causes degradation of circadian behavioral and physiological rhythms.
Methodology/Principal Findings
To test the hypothesis that loss of robust behavioral rhythmicity in Kcnma1−/− mice is due to the disruption of SFR rhythms in the SCN, we used multi-electrode arrays to record extracellular action potentials from acute wild-type (WT) and Kcnma1−/− slices. Patterns of activity in the SCN were tracked simultaneously for up to 3 days, and the phase, period, and synchronization of SFR rhythms were examined. Loss of BK channels increased arrhythmicity but also altered the amplitude and period of rhythmic activity. Unexpectedly, Kcnma1−/− SCNs showed increased variability in the timing of the daily SFR peak.
These results suggest that BK channels regulate multiple aspects of the circadian patterning of neuronal activity in the SCN. In addition, these data illustrate the characteristics of a disrupted SCN rhythm downstream of clock gene-mediated timekeeping and its relationship to behavioral rhythms.
PMCID: PMC2586654  PMID: 19060951
25.  Electrical Hyperexcitation of Lateral Ventral Pacemaker Neurons Desynchronizes Downstream Circadian Oscillators in the Fly Circadian Circuit and Induces Multiple Behavioral Periods 
Coupling of autonomous cellular oscillators is an essential aspect of circadian clock function but little is known about its circuit requirements. Functional ablation of the pigment-dispersing factor-expressing lateral ventral subset (LNV ) of Drosophila clock neurons abolishes circadian rhythms of locomotor activity. The hypothesis that LNVs synchronize oscillations in downstream clock neurons was tested by rendering the LNVs hyperexcitable via transgenic expression of a low activation threshold voltage-gated sodium channel. When the LNVs are made hyperexcitable, free-running behavioral rhythms decompose into multiple independent superimposed oscillations and the clock protein oscillations in the dorsal neuron 1 and 2 subgroups of clock neurons are phase-shifted. Thus, regulated electrical activity of the LNVs synchronize multiple oscillators in the fly circadian pacemaker circuit.
PMCID: PMC2597197  PMID: 16407545
arrhythmia; behavior; circadian rhythms; desynchronization; Drosophila; sodium channel

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