Gary E. Pickard

Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses

Melanopsin is a novel opsin synthesized in a small subset of retinal ganglion cells. Ganglion cells expressing melanopsin are capable of depolarizing in response to light in the absence of rod or cone input and are thus intrinsically light sensitive. Melanopsin ganglion cells convey information regarding general levels of environmental illumination to the suprachiasmatic nucleus, the intergeniculate leaflet, and the pretectum. Typically, retinal ganglion cells communicate information to central visual structures by receiving input from retinal photoreceptors via bipolar and amacrine cells. Because melanopsin ganglion cells do not require synaptic input to generate light‐induced signals, these cells need not receive synapses from other neurons in the retina. In this study, we examined the ultrastructure of melanopsin ganglion cells in the mouse retina to determine the type (if any) of synaptic input these cells receive. Melanopsin immunoreaction product was associated primarily with the plasma membrane of (1) perikarya in the ganglion cell layer, (2) dendritic processes in the inner plexiform layer (IPL), and (3) axons in the optic fiber layer. Melanopsin‐immunoreactive dendrites in the inner (ON) region of the IPL were postsynaptic to bipolar and amacrine terminals, whereas melanopsin dendrites stratifying in the outer (OFF) region of the IPL received only amacrine terminals. These observations suggested that rod and/or cone signals may be capable of modifying the intrinsic light response in melanopsin‐expressing retinal ganglion cells. J. Comp. Neurol. 460:380–393, 2003.

Two types of melanopsin retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic nucleus and the olivary pretectal nucleus

Melanopsin‐expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) innervate the hypothalamic suprachiasmatic nucleus (SCN) and the olivary pretectal nucleus (OPN), providing irradiance information for entrainment of circadian rhythms and for stimulating the pupillary light reflex. In this study, mice were used in which the melanopsin gene was replaced with the tau‐lacZ gene. Heterozygous (tau‐lacZ+/–) mice express both melanopsin and β‐galactosidase. In tau‐lacZ+/– mice, only ∼50% of melanopsin ipRGCs contain β‐galactosidase, and these cells are specifically labeled with a C‐terminus melanopsin antibody. Retrograde tracer injection into the SCN labels β‐galactosidase‐expressing ipRGCs (termed M1) that comprise ∼80% of the SCN‐projecting ipRGCs. M1 ipRGCs and an additional set of ipRGCs (termed M2) are labeled with a melanopsin antiserum targeted against the N‐terminus of the melanopsin protein; M2 ipRGCs do not contain detectable β‐galactosidase, and these cells make up the remainder of the SCN‐projecting RGCs. Tracer injection into the OPN labeled non‐melanopsin RGCs and both types of melanopsin ipRGC: 45% M1 and 55% M2. Infection of the iris with pseudorabies virus (PRV) results in retrograde transneuronal label of OPN projection neurons that innervate preganglionic parasympathetic neurons of the Edinger‐Westphal nucleus; PRV‐labeled cells were located almost exclusively within the terminal field of M1 ipRGCs in the periphery (shell) of the OPN. The OPN core receives retinal input, and we hypothesize that the OPN core receives input from the M2 ipRGCs. Two subtypes of melanopsin ipRGCs project differentially to the SCN and OPN; the functional significance of ipRGCs subtypes is currently unknown.

Intraretinal signaling by ganglion cell photoreceptors to dopaminergic amacrine neurons

Retinal dopaminergic amacrine neurons (DA neurons) play a central role in reconfiguring retinal function according to prevailing illumination conditions, yet the mechanisms by which light regulates their activity are poorly understood. We investigated the means by which sustained light responses are evoked in DA neurons. Sustained light responses were driven by cationic currents and persisted in vitro and in vivo in the presence of L-AP4, a blocker of retinal ON-bipolar cells. Several characteristics of these L-AP4-resistant light responses suggested that they were driven by melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), including long latencies, marked poststimulus persistence, and a peak spectral sensitivity of 478 nm. Furthermore, sustained DA neuron light responses, but not transient DA neuron responses, persisted in rod/cone degenerate retinas, in which ipRGCs account for virtually all remaining retinal phototransduction. Thus, ganglion-cell photoreceptors provide excitatory drive to DA neurons, most likely by way of the coramification of their dendrites and the processes of DA neurons in the inner plexiform layer. This unprecedented centrifugal outflow of ganglion-cell signals within the retina provides a novel basis for the restructuring of retinal circuits by light.

Development of Pseudorabies Virus Strains Expressing Red Fluorescent Proteins: New Tools for Multisynaptic Labeling Applications

ABSTRACT The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. 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 method for defining connections of single neurons to multiple neural circuits in the brain. However, the present 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 virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.

Melanopsin and non-melanopsin expressing retinal ganglion cells innervate the hypothalamic suprachiasmatic nucleus

Retinal input to the hypothalamic suprachiasmatic nucleus (SCN) synchronizes the SCN circadian oscillator to the external day/night cycle. Retinal ganglion cells that innervate the SCN via the retinohypothalamic tract are intrinsically light sensitive and express melanopsin. In this study, we provide data indicating that not all SCN-projecting retinal ganglion cells express melanopsin. To determine the proportion of ganglion cells afferent to the SCN that express melanopsin, ganglion cells were labeled following transsynaptic retrograde transport of a recombinant of the Bartha strain of pseudorabies virus (PRV152) constructed to express the enhanced green fluorescent protein (EGFP). PRV152 injected into the anterior chamber of the eye retrogradely infects four retinorecipient nuclei in the brain via autonomic circuits to the eye, resulting in transneuronally labeled ganglion cells in the contralateral retina 96 h after intraocular infection. In animals with large bilateral lesions of the lateral geniculate body/optic tract, ganglion cells labeled with PRV152 are retrogradely infected from only the SCN. In these animals, most PRV152-infected ganglion cells were immunoreactive for melanopsin. However, a significant percentage (10-20%) of EGFP-labeled ganglion cells did not express melanopsin. These data suggest that in addition to the intrinsically light-sensitive melanopsin-expressing ganglion cells, conventional ganglion cells also innervate the SCN. Thus, it appears that the rod/cone system of photoreceptors may provide signals to the SCN circadian system independent of intrinsically light-sensitive melanopsin ganglion cells.

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.

Subcellular distribution of 5-HT1b and 5-HT7 receptors in the mouse suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN), a circadian oscillator, receives glutamatergic afferents from the retina and serotonergic (5‐HT) afferents from the median raphe. 5‐HT1B and 5‐HT7 receptor agonists inhibit the effects of light on SCN circadian activity. Electron microscopic (EM) immunocytochemical procedures were used to determine the subcellular localization of 5‐HT1B and 5‐HT7 receptors in the SCN. 5‐HT1B receptor immunostaining was associated with the plasma membrane of thin unmyelinated axons, preterminal axons, and terminals of optic and nonoptic origin. 5‐HT1B receptor immunostaining in terminals was almost never observed at the synaptic active zone. To a much lesser extent, 5‐HT1B immunoreaction product was noted in dendrites and somata of SCN neurons. 5‐HT7 receptor immunoreactivity in γ‐aminobutyric acid (GABA), vasoactive intestinal polypeptide (VIP), and vasopressin (VP) neuronal elements in the SCN was examined by using double‐label procedures. 5‐HT7 receptor immunoreaction product was often observed in GABA‐, VIP‐, and VP‐immunoreactive dendrites as postsynaptic receptors and in axonal terminals as presynaptic receptors. 5‐HT7 receptor immunoreactivity in terminals and dendrites was often associated with the plasma membrane but very seldom at the active zone. In GABA‐, VIP‐, and VP‐immunoreactive perikarya, 5‐HT7 receptor immunoreaction product was distributed throughout the cytoplasm often in association with the endoplasmic reticulum and the Golgi complex. The distribution of 5‐HT1B receptors in presynaptic afferent terminals and postsynaptic SCN processes, as well as the distribution of 5‐HT7 receptors in both pre‐ and postsynaptic GABA, VIP, and VP SCN processes, suggests that serotonin plays a significant role in the regulation of circadian rhythms by modulating SCN synaptic activity. J. Comp. Neurol. 432:371–388, 2001.

Serotonergic innervation of the hypothalamic suprachiasmatic nucleus and photic regulation of circadian rhythms

Converging lines of evidence have firmly established that the hypothalamic suprachiasmatic nucleus (SCN) is a light‐entrainable circadian oscillator in mammals, critically important for the expression of behavioral and physiological circadian rhythms. Photic information essential for the daily phase resetting of the SCN circadian clock is conveyed directly to the SCN from retinal ganglion cells via the retinohypothalamic tract. The SCN also receives a dense serotonergic innervation arising from the mesencephalic raphe. The terminal fields of retinal and serotonergic afferents within the SCN are co‐extensive, and serotonergic agonists can modify the response of the SCN circadian oscillator to light. However, the functional organization and subcellular localization of 5HT receptor subtypes in the SCN are just beginning to be clarified. This information is necessary to understand the role 5HT afferents play in modulating photic input to the SCN. In this paper, we review evidence suggesting that the serotonergic modulation of retinohypothalamic neurotransmission may be achieved via at least two different cellular mechanisms: 1) a postsynaptic mechanism mediated via 5HT1A or 5ht7 receptors located on SCN neurons; and 2) a presynaptic mechanism mediated via 5HT1B receptors located on retinal axon terminals in the SCN. Activation of either of these 5HT receptor mechanisms in the SCN by specific 5HT agonists inhibits the effects of light on circadian function. We hypothesize that 5HT modulation of photic input to the SCN may serve to set the gain of the SCN circadian system to light.

Heterogeneous expression of γ‐aminobutyric acid and γ‐aminobutyric acid‐associated receptors and transporters in the rat suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN) is the primary mammalian circadian clock that regulates rhythmic physiology and behavior. The SCN is composed of a diverse set of neurons arranged in a tight intrinsic network. In the rat, vasoactive intestinal peptide (VIP)‐ and gastrin‐releasing peptide (GRP)‐containing neurons are the dominant cell phenotypes of the ventral SCN, and these cells receive photic information from the retina and the intergeniculate leaflet. Neurons expressing vasopressin (VP) are concentrated in the dorsal and medial aspects of the SCN. Although the VIP/GRP and VP cell groups are concentrated in different regions of the SCN, the separation of these cell groups is not absolute. The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is expressed in most SCN neurons irrespective of their location or peptidergic phenotype. In the present study, immunoperoxidase labeling, immunofluorescence confocal microscopy, and ultrastructural immunocytochemistry were used to examine the spatial distribution of several markers associated with SCN GABAergic neurons. Glutamate decarboxylase, a marker of GABA synthesis, and vesicular GABA transporter were more prominently observed in the ventral SCN. KCC2, a K+/Cl– cotransporter, was highly expressed in the ventral SCN in association with VIP‐ and GRP‐producing neurons, whereas VP neurons in the dorsal SCN were devoid of KCC2. On the other hand, GABAB receptors were observed predominantly in VPergic neurons dorsally, whereas, in the ventral SCN, GABAB receptors were associated almost exclusively with retinal afferent fibers and terminals. The differential expression of GABAergic markers within the SCN suggests that GABA may play dissimilar roles in different SCN neuronal phenotypes. J. Comp. Neurol. 506:708–732, 2008.

Response of the mouse circadian system to serotonin 1A/2/7 agonists in vivo: Surprisingly little

Serotonin (5-HT) is thought to play a role in regulating nonphotic phase shifts and modulating photic phase shifts of the mammalian circadian system, but results with different species (rats vs. hamsters) and techniques (in vivo vs. in vitro; systemic vs. intracerebral drug delivery) have been discordant. Here we examined the effects of the 5-HT1A/7 agonist 8-OH-DPAT and the 5-HT1/2 agonist quipazine on the circadian system in mice, with some parallel experiments conducted with hamsters for comparative purposes. In mice, neither drug, delivered systemically at a range of circadian phases and doses, induced phase shifts significantly different from vehicle injections. In hamsters, quipazine intraperitoneally (i.p.) did not induce phase shifts, whereas 8-OH-DPAT induced phase shifts after i.p. but not intra-SCN injections. In mice, quipazine modestly increased c-Fos expression in the SCN (site of the circadian pacemaker) during the subjective day, whereas 8-OH-DPAT did not affect SCN c-Fos. In hamsters, both drugs suppressed SCN c-Fos in the subjective day. In both species, both drugs strongly induced c-Fos in the paraventricular nucleus (within-subject positive control). 8-OH-DPAT did not significantly attenuate light-induced phase shifts in mice but did in hamsters (between-species positive control). These results indicate that in the intact mouse in vivo, acute activation of 5-HT1A/2/7 receptors in the circadian system is not sufficient to reset the SCN pacemaker or to oppose phase-shifting effects of light. There appear to be significant species differences in the susceptibility of the circadian system to modulation by systemically delivered serotonergics.