Gary Pickard - US grants

neural information processing; circadian rhythms; suprachiasmatic nucleus;

9319634 Pickard Living organism possess internal biological clocks that control the timing of many different biological processes. Jet lag, ability to concentrate, the way you respond to anesthesia are all affected by daily or seasonal biological cycles. Organisms isolated without exposure to day:night cycles or other environmental time cues continue to express rhythmic processes but with a period only approximating 24 hours. One striking day/night rhythmicity is the synthesis and secretion of the hormone melatonin by the pineal gland. While it is clear that the pineal gland plays a major role in the control of circadian activity rhythms, a major question is whether single isolated cells also exhibit these 24 hour circadian oscillations. Dr. Pickard is establishing the technology to accurately detect rhythmic fluctuations in hormone secretion from single cells. Using an in vitro preparation of dissociated pineal cells in short-term culture, he is developing a reverse hemolytic plaque assay to measure melatonin secretion in single isolated cells. This technology will enable Dr. Pickard to detect rhythmic hormone secretion in the attamol range (10-18mol). With this level of sensitivity, he will establish the procedures to sequentially assay melatonin secretion from the same isolated cells, thus providing an answer to one of the major fundamental questions regarding the organization of biological clocks. ***

DESCRIPTION: The goal of this research is to furnish an understanding of the complex neural substrates underlying the regulation of the circadian oscillator located in the hypothalamic suprachiasmatic nucleus (SCN), which functions as our biological clock. Photic information essential for daily phase resetting of the SCN circadian clock is conveyed directly to the SCN from retinal ganglion cells via the retinohypothalamic tract (RHT). The SCN also receives a dense serotonergic innervation arising from the midbrain raphe. RHT and serotonergic afferents are coextensive in the SCN and serotonergic agonists can modify the pacemakers response to light. In this proposal the applicants will test the hypothesis that the 5HT1B receptor subtype is located presynaptically on retinal axon terminals in the SCN and that it plays a major role in regulating photic input to the SCN. Using behavioral analysis of wheel running activity the applicants will test whether 5HT1B receptors mediate serotonergic inhibition of light-induced phase shifts in hamsters and in transgenic knockout mice lacking the 5HT1B receptor. Using morphological techniques the applicants will test whether the 5HT1B receptor can regulate the light-induce expression of c-Fos protein in SCN neurons, and more critically examine the expression of 5HT1B receptors in RHT terminals. Finally, using patch clamp electrophysiology the applicants will test the effects of 5HT on RHT-SCN synaptic transmission in slices from both hamsters and knockout mice.

The goal of this research is to furnish an understanding of the complex neural substrates underlying the regulation of the circadian oscillator located in the hypothalamic suprachiasmatic nucleus (SCN). Increased knowledge of the functional organization of the hypothalamus and the circadian timing system is relevant to major public health concerns such as sleep disorders, sleep disruption (as in jet lag or shift work) and serious mood disorders such as seasonal affective disorder (SAD). Photic information essential for the daily resetting of the SCN circadian clock is conveyed directly to the SCN from the retina via the retinohypothalamic tract. The SCN also receives a dense serotonergic innervation arising from the midbrain raphe. We have provided evidence that the 5HT1B receptor subtype is located in RHT terminals in the SCN and that stimulation of these receptors attenuates photic input to the SCN. We now show that animals lacking functional 5HT1B receptors synchronize to winter-like photoperiods (i.e., short days) similar to people suffering from winter-depression or SAD. Behavioral analyses of 5HT1B knockout (KO) mice will be used to describe further the behavioral phenotype of these animals. A detailed phase response curve to acute light pulses will be generated. The mechanism(s) underlying the altered entrainment to short days will be examined. We hypothesize that loss of functional 5HT1B receptors on GABA terminals in the SCN results in disinhibition of GABA release. This produces GABA "spillover" and stimulation of GABAB receptors on RHT terminals. This will be investigated using electron microscopy for GABA receptors on RHT terminals, in vitro SCN electrophysiology, and behavioral pharmacology using GABAB antagonists. Altered PER2 expression in the brain in 5HT1B KO mice in short days will also be examined as a potential correlate of seasonal depression

[unreadable] DESCRIPTION (provided by applicant): Light entrains the suprachiasmatic nucleus (SCN), a circadian oscillator and a primary component of the mammalian circadian system. A subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) express melanopsin, a novel vertebrate opsin, and pituitary adenylate cyclase-activating peptide (PACAP). These cells convey signals to the SCN providing information about ambient lighting conditions. However, the phototransduction system(s) used by these ipRGCs and the retinal neurons that are presynaptic to them are unknown. Moreover, the potential role of ipRGCs in regulating intra-retinal physiology is unexplored. The long term goals of this application are to provide an understanding of the phototransduction cascade used by ipRGCs, to describe the intra-retinal circuitry of this system of neurons to better understand their function, and to explore the role of melanopsin retinal ganglion cells in the regulation of intra-retinal dopamine. To study the phototransduction process, a highly enriched population of ipRGCs is generated using an immunopanning procedure. Isolated cells are examined using in vitro calcium imaging to observe light- stimulated calcium influx. The ipRGCs use melanopsin as a chromophore and neurons afferent to melanopsin-expressing cells will be identified using pseudorabies virus (PRV) as a transneuronal tracer. Mice in which Cre-recombinase is under the control of the melanopsin promoter will be infected with a conditional PRV that replicates only in neurons that express Cre-recombinase and in neurons in synaptic contact with the originally infected cells. Electron microscopy and calcium imaging will be used to explore the relationship between ipRGCs and the dopaminergic system of the retina. Finally, the role PACAP in modulating the effects of light on ipRGCs and the circadian system is examined using calcium imaging and in behavioral studies with PACAP null mice. Disturbances in the entrainment or phasing of our biological clock are responsible for abnormal phasing of sleep rhythms and may underlie serious affective disorders. Understanding the retinal neurons afferent to the SCN will aid in our ability to understand and treat these disturbances of phase. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The transsynaptic retrograde transport of the Bartha strain of pseudorabies virus (PRV Bartha) has become an important neuroanatomical tract-tracing technique for the characterization of neuronal circuits in the central nervous system. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful technique for defining interactions between parallel neural circuits in the brain. However, the current use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods, limits the potential of these recombinant PRV Bartha strains as dual transsynaptic tracers. We have developed two isogenic recombinant strains of PRV Bartha (i.e., PRV152 and PRV614) differing only in the fluorescent reporter protein they express. PRV152 expresses the enhanced green fluorescent protein (EGFP), is driven by the human cytomeglovirus (CMV) promoter, and is inserted in the middle of the gG gene in the middle of the viral genome. PRV152 is in wide use and is well characterized. PRV614 expresses a novel monomeric red fluorescent protein (mRFP1) driven by the CMV promoter also inserted in the middle of the gG gene. It has only recently been constructed and is uncharacterized. The availability of two viral transneuronal tracers expressing different fluorescent reporters that can be visualized concurrently without additional tissue processing has enormous utility. In this application, we propose to characterize the newly developed PRV614 as a transsynaptic retrograde viral tracer that can be used in combination with PRV152 to further define neuronal circuits in the brain. The kinetics of PRV614 infection and retrograde transport will be determined in vivo using the retrograde transport of PRV through autonomic circuits innervating the eye. The ability of two isogenic strains of PRV to infect the same neuron when one PRV arrives later than the other will be determined both in vivo and in vitro. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; PRV 614 will eliminate many of the pitfalls associated with the currently used dual PRV recombinants. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The transsynaptic retrograde transport of the Bartha strain of pseudorabies virus (PRV Bartha) has become an important neuroanatomical tract-tracing technique for the characterization of neuronal circuits in the central nervous system. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful technique for defining interactions between parallel neural circuits in the brain. However, the current use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods, limits the potential of these recombinant PRV Bartha strains as dual transsynaptic tracers. We have developed two isogenic recombinant strains of PRV Bartha (i.e., PRV152 and PRV614) differing only in the fluorescent reporter protein they express. PRV152 expresses the enhanced green fluorescent protein (EGFP), is driven by the human cytomeglovirus (CMV) promoter, and is inserted in the middle of the gG gene in the middle of the viral genome. PRV152 is in wide use and is well characterized. PRV614 expresses a novel monomeric red fluorescent protein (mRFP1) driven by the CMV promoter also inserted in the middle of the gG gene. It has only recently been constructed and is uncharacterized. The availability of two viral transneuronal tracers expressing different fluorescent reporters that can be visualized concurrently without additional tissue processing has enormous utility. In this application, we propose to characterize the newly developed PRV614 as a transsynaptic retrograde viral tracer that can be used in combination with PRV152 to further define neuronal circuits in the brain. The kinetics of PRV614 infection and retrograde transport will be determined in vivo using the retrograde transport of PRV through autonomic circuits innervating the eye. The ability of two isogenic strains of PRV to infect the same neuron when one PRV arrives later than the other will be determined both in vivo and in vitro. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; PRV 614 will eliminate many of the pitfalls associated with the currently used dual PRV recombinants. [unreadable] [unreadable]

Project Summary The suprachiasmatic nucleus (SCN) is the primary circadian oscillator in the central nervous system, entrained to the day/night cycle via the retinohypothalamic tract. The circadian-timing system has a complex architecture. In addition to the SCN, subsidiary clocks are located in most, if not all, tissues, organs, and cells of the body including brain regions distinct from the SCN. Peripheral clocks directly regulate local rhythms in cellular metabolism and hormone secretion and require daily entraining cues from the SCN for coordinated timing of behavioral, physiologic and metabolic circadian rhythms, a primary requisite for a healthy body and mind. The SCN maintains global circadian synchrony via its connections with autonomic circuits innervating peripheral organs and by its regulation of rhythmic hormone secretion such as adrenal glucocorticoids. Rhythmic corticosterone (CORT) signals induce the rhythmic expression of a diverse array of genes including clock genes. Temporal homeostasis is a complex interplay between central and autonomic neural circuits and hormonal feedback from the adrenal. Changes in circadian function and the accompanying changes in phase have been associated with several human disorders. A reduction in the amplitude of the CORT diurnal rhythm may exert a wide range of effects on metabolism and central nervous system function. Preliminary data demonstrate that alterations in entrainment of the SCN to the day/night cycle produce changes in the diurnal CORT rhythm; as entrainment phase angle is progressively more delayed relative to light offset the amplitude of the diurnal corticosterone rhythm is progressively reduced, up to as much as 50%. Specific Aim 1 uses transcriptional profiles of clock genes to extend preliminary findings and examines potential mechanisms by which altered entrainment to the day/night cycle reduces the amplitude of the diurnal CORT rhythm. Specific Aim 2 describes the neural circuits (that may circumvent the SCN) that send signals to the adrenal. Retinal input to pre-autonomic neurons is identified by anterograde tracing of retinal efferents to the hypothalamus in conjunction with labeling of pre-autonomic neurons in the hypothalamus via transneuronal retrograde tracing using pseudorabies virus injected into the adrenal. Functional experiments target identified pre-autonomic hypothalamic neurons for neurotoxic lesioning to determine effects on adrenal function. Specific Aim 3 utilizes transplantation of adrenals from mice with arrhythmic adrenal oscillators (Per2/Cry1 dKO mice) into adrenalectomized wild type mice with altered entrainment to dissect the functional roles of the SCN and adrenal oscillators, and the L:D cycle on the regulation of the diurnal rhythm of CORT secretion. Understanding how retinal circuits and the central clock regulate peripheral oscillators via autonomic circuits will aid in our ability to better understand and treat altered circadian rhythms.