Gary Pickard

Functional neuroanatomy of the circadian timing system. Before the identification of retinal ganglion cell (RGC) afferents terminating in the hypothalamic suprachiasmatic nucleus (SCN) in the early 1970s, virtually nothing was known regarding the neural circuitry underlying the generation and regulation of mammalian circadian behavior. I therefore directed my research efforts in the early part of my career to defining the neuroanatomical substrates which comprise the circadian system. The rationale being that the neuroanatomical ‘wiring diagram’ developed by this approach would provide a framework upon which we could build a more complete understanding of the mechanisms by which time-keeping functions are integrated in the central nervous system. Using intracerebral injections of tracer molecules, I described the afferent connections of the SCN which included the first morphological description of SCN-projecting RGCs and inputs from the intergeniculate leaflet (IGL), a retinorecipient nucleus in the thalamus. I subsequently showed that the SCN and IGL were innervated by the same retinal ganglion cells via axon collaterals thereby establishing the IGL as an integral component of the circadian timing system. The neuroanatomical data provided the basis for lesion studies that began to probe the function of identified circuits. IGL lesions altered the response of the circadian system to light and showed that the IGL contributed to the endogenous period of the circadian behavioral rhythm. A particularly important observation made from these studies was that unilateral SCN ablation in animals with two circadian rhythms (i.e., split rhythms) eliminated only one of the two circadian rhythms, providing evidence that each SCN could function as an independent circadian oscillator. This work contributed significantly to an emerging principle: the SCN was a component of a distributed circadian timing system. We now know that the SCN is the primary circadian clock entrained to the external day/night cycle via retinal ganglion cell afferents and that the SCN in turn entrains molecular oscillations in virtually all tissues and organs of the body via descending autonomic neural circuits.

• Pickard GE (1982) The afferent connections of the suprachiasmatic nucleus of the golden hamster with emphasis on the retinohypothalamic projection. J Comp Neurol 211:65-83. [PMID: 7174884]
• Pickard GE, Turek FW (1982) Splitting of the circadian rhythm of activity is abolished by unilateral lesions of the suprachiasmatic nuclei. Science 215:1119-1121. [PMID: 7063843]
• Pickard GE (1985) Bifurcating axons of retinal ganglion cells terminate in the hypothalamic suprachiasmatic nucleus and the intergeniculate leaflet of the thalamus. Neurosci Lett 55:211-217. [PMID: 4000547]
• Pickard GE, Ralph M, Menaker M (1987) The intergeniculate leaflet partially mediates the effects of light on circadian rhythms. J Biol Rhythms 2:35-56. [PMID: 2979650]

(collectively: 1235 citations Google Scholar)

Serotonergic innervation of the SCN. Based on tracing studies and serotonin immunocytochemical analyses, we and others had documented that the SCN received a dense serotonergic input from the midbrain raphe. However, the function of this major afferent system innervating the SCN circadian clock was unknown. Using a multidisciplinary approach to examine the role of serotonin (5-HT) in the SCN (i.e., behavioral pharmacology, ligand binding assays, light-evoked gene induction, whole-cell patch-clamp electrophysiology in an SCN slice preparation, 5-HT1B receptor knockout mice, and electron microscopic immunocytochemistry) we specifically explored the role of 5-HT activation of two 5-HT receptor subtypes in the SCN (5-HT1B and 5-HT7). We demonstrated that 5-HT1B receptors were located presynaptically on retinal ganglion cell terminals in the SCN and that activation of these receptors inhibited the release of glutamate; 5-HT7 receptors were postsynaptic receptors on SCN dendrites. Using 5-HT1B receptor knockout animals we described a reduction in the sensitivity of the SCN circadian system to light and the effect was more pronounced in the late subjective night when light evokes phase advances in the clock. These studies showed that 5-HT released in the SCN modulated the response of the circadian oscillator to light in a phase dependent manner and firmly established a functional role for the serotonergic innervation of the SCN. The data suggested that pathophysiology of serotonergic neurotransmission could lead to alterations in entrainment and it is known that alterations in entrainment are associated with affective disorders.

• Pickard GE, Weber ET, Scott PA, Riberdy AF, Rea MA (1996) 5-HT1B receptor agonists inhibit lightinduced phase shifts of behavioral circadian rhythms and expression of the immediate-early gene c-fos in the suprachiasmatic nucleus. J Neurosci 16:8208-8220. [PMID: 8987845]
• Pickard GE, Smith BN, Belenky M, Rea MA, Dudek FE, Sollars PJ (1999) 5-HT1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus. J Neurosci 19:4034-4045. [PMID: 10234032]
• Smith BN, Sollars PJ, Dudek FE, Pickard GE (2001) Serotonergic modulation of retinal input to the mouse suprachiasmatic nucleus mediated by 5-HT1B and 5-HT7 receptors. J Biol Rhythms 16:25-38. [PMID: 11220775]
• Belenky M, Pickard GE (2001) Subcellular distribution of 5-HT1B and 5-HT7 receptors in the mouse suprachiasmatic nucleus. J Comp Neurol 432:371-388. [PMID: 11246214]

(collectively: 568 citations Google Scholar)

Pseudorabies virus as a neurobiological tool. Pseudorabies virus (PRV) began to be used as a selfamplifying transneuronal neuroanatomical circuit tracer in the 1980s. At that time immunocytochemistry was used to identify infected neurons. This was slow and laborious and often only selected brain regions were examined. In the late 1990s Lynn Enquist at Princeton University, generated a recombinant of the Bartha strain of PRV expressing EGFP (PRV152). We began collaborating with Lynn Enquist at this time using PRV152 to study retinal circuits. Because PRV152 expressed EGFP as a cytoplasmic reporter, labeled neurons were easily detected and immunocytochemistry was not necessary, allowing examination of serial sections of the entire brain after peripheral infection. This allowed us to discover that PRV Bartha, carrying a deletion in the Us region, was incapable of anterograde transport in axons. Moreover, we also demonstrated that neurons infected with PRV152 in vivo could be targeted for electrophysiological recording in vitro, providing a powerful tool for further probing the functional properties of defined neural circuits. We later utilized PRV152 to identify different types of RGCs afferent to the SCN. In collaboration with Bruce Banfield (Univ. Colorado), we made and characterized the first PRV strain expressing a red fluorescent reporter (PRV614) isogenic with PRV152. These recombinant PRV strains have been shared with many dozens of investigators around the world.

• Smith BN, Banfield BW, Smeraski CA, Wilcox CL, Dudek FE, Enquist LW, Pickard GE (2000) Pseudorabies virus expressing enhanced green fluorescent protein: a tool for in vitro electrophysiological analysis of transsynaptically labeled neurons in identified CNS circuits. Proc Natl Acad Sci USA 97:9264-9269. [PMID: 10922076}
• Pickard GE, Smeraski CA, Tomlinson CC, Banfield BW, Kaufman J, Wilcox CL, Enquist LW, Sollars PJ (2002) Intravitreal injection of the attenuated pseudorabies virus PRV-Bartha results in infection of the hamster suprachiasmatic nucleus only by retrograde transsynaptic transport via autonomic circuits. J Neurosci 22:27012710. [PMID: 11923435]
• Banfield BW, Kaufman JD, Randall J, Pickard GE (2003) Development of pseudorabies virus strains expressing red fluorescent proteins: New tools for multisynaptic labeling applications. J Virol 77:10106-10112. [PMID: 12941921]
• Sollars PJ, Smeraski CA, Kaufman JD, Ogilvie MD, Provencio I, Pickard GE (2003) Melanopsin and nonmelanopsin expressing retinal ganglion cells innervate the hypothalamic suprachiasmatic nucleus. Vis Neurosci 20:601-610. [PMID: 15088713]

(collectively: 641 citations Google Scholar)

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells. In 2002 it was discovered that the RGCs innervating the SCN expressed an opsin, termed melanopsin that rendered these neurons photosensitive (intrinsically photosensitive retinal ganglion cells, ipRGCs). This discovery of a new photoreceptor type in the mammalian retina with invertebrate-like characteristics opened a new field in retinal biology and raised many questions. We reported that ipRGCs received synaptic input from rod and cone photoreceptors via bipolar cells and that amacrine cells also were in synaptic contact with ipRGCs. Since the response of ipRGCs to light in situ was modulated by this retinal circuitry, we developed an immuno-panning assay to study single, isolated ipRGCs in short-term culture. Using calcium imaging and electrophysiological recording we showed that these cells responded to light in isolation without the need of pharmacological blockade of neural transmission required to study these cells in the intact retina. We also identified a transient receptor potential (TRP) channel (i.e., TRPC6,7) as the putative channel responsible for the initial light-evoked depolarization of these neurons and showed that light-evoked calcium influx was mediated via L-type voltagegated calcium channels. We then described that there were distinct morphological types of ipRGC that differentially innervated retinorecipient targets in the brain; five types of ipRGC have now been described. In collaboration with Doug McMahon at Vanderbilt University and Dave Berson at Brown University, we reported that the M1 type of ipRGC sent signals back into the retina (via intraretinal collaterals) to drive action potentials in dopaminergic amacrine cells in the retinal inner nuclear layer. These results established that information flow in the retina is truly bi-directional and that ipRGCs act as both interneurons for intraretinal visual signaling as well as projection neurons transmitting visual signals to central visual nuclei.

• Belenky MA, Smeraski CA, Provencio I, Sollars PJ, Pickard GE (2003) Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses. J Comp Neurol 460:380-393. [PMID: 12692856]
• Hartwick ATE, Bramley JR, Yu J, Stevens KT, Allen CN, Baldridge WH, Sollars PJ, Pickard GE (2007) Light-evoked calcium responses of isolated melanopsin-expressing retinal ganglion cells. J Neurosci 27:1346813480. PMID: 18057205] 
• Baver SB, Pickard GE, Sollars PJ, Pickard GE (2008) Two types of melanopsin retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic nucleus and the olivary pretectal nucleus. Eur J Neurosci 27:1763-1770. [PMID: 18371076]
• Zhang D-Q, Wong KY, Sollars PJ, Berson DM, Pickard GE, McMahon DG (2008) Intra-retinal signaling by ganglion cell photoreceptors to dopaminergic amacrine neurons. Proc Natl Acad Sci USA 105:14181-14186. [PMID: 18779590]

(collectively: 1048 citations Google Scholar)

Alpha-herpesvirus neuroinvasion and transport mechanisms. In collaboration with Dr. Greg Smith at Northwestern University, we are identifying the mechanisms by which alpha-herpesviruses gain access to the nervous system. In vivo experiments conducted in our lab have complemented in vitro work done by Dr. Smith and have contributed to the discovery of virus invasive mechanisms and host intrinsic defenses that could not have been unveiled in cultured neurons alone and without our expertise in neuroanatomy and viral tracing. This has been a very exciting and rewarding collaboration that has resulted in identification of viral factors that engage microtubule motors and in the dynamic regulation of ubiquitination of viral proteins that promotes neuroinvasion from epithelial cells to axon terminal and retrograde transport into the nervous system. Ongoing work described in this application has also identified mutations that render viruses incapable of neuroinvasion, indicating we are near to cracking the herpesvirus neuroinvasive code and offering the promise for developing live-attenuated vaccines.

• Lee JI, Sollars PJ, Baver SB, Pickard GE, Leelawong M, Smith GA (2009) A herpesvirus encoded deubiquitinase is a novel neuroinvasive determinant. PLoS Path 5:e1000387. [PMID: 19381253]
• Zaichick SV, Bohannon KP, Hughes A, Sollars PJ, Pickard GE, Smith GA (2013) The herpesvirus VP1/2 protein is an effector of dynein-mediated capsid transport and neuroinvasion. Cell Host & Microbe 13:193-203. [PMID: 23414759] 
• Huffmaster NJ, Sollars PJ, Richards AL, Pickard GE, Smith GA (2015) Dynamic ubiquitination drives herpesvirus neuroinvasion. Proc Natl Acad Sci USA 112:12818-12823. [PMID: 26407585]
• Richards AL, Sollars PJ, Pitts JD, Stults AM, Heldwein EE, Pickard GE, Smith GA (2017) The pUL37 tegument protein guides alpha-herpesvirus retrograde axonal transport to promote neuroinvasion. PLoS Pathogens 13:e1006741. [PMID: 29216315]

(collectively: 258 citations Google Scholar)

Complete List of Published Work in MyBibliography.