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.

PRV-Bartha ͉ PRV 152 ͉ suprachiasmatic nucleus ͉ retinal ganglion cell T he use of neurotropic alphaherpesviruses has greatly advanced our ability to visualize ensembles of neurons that contribute to multisynaptic circuits in the central nervous system (CNS) (1). In particular, the attenuated vaccine strain of pseudorabies virus (PRV-Bartha) has been used successfully as a self-amplifying neural tracer after peripheral application or direct injection into brain parenchyma (2-4). The usefulness of PRV as a neural tracer relies on its ability to infect chains of hierarchically connected neurons via specific transsynaptic passage of progeny virus rather than infection by lytic release into the extracellular space (4, 5). Typically, PRV infects the CNS by invading neurons in the periphery and then replicating and spreading to the CNS via synaptically linked neurons. However, PRV can also invade neurons through their somata if the viral concentration is sufficient (6), as evidenced by primary infection of retinal ganglion cells (RGCs) after intravitreal injection of PRV (7-9). Infection of RGCs with PRV-Bartha, followed by viral replication, results in the anterograde transsynaptic infection of a restricted set of retinorecipient neurons [i.e., suprachiasmatic nucleus (SCN), intergeniculate leaflet (IGL), pretectum (PT), and lateral terminal nucleus]. Intravitreal injection of the wild-type virus, PRV-Becker, produces transneuronal infection of neurons in all retinorecipient subcortical regions (7). The factors that determine the specificity of PRV-Bartha infection of selective retinorecipient targets are not completely understood, although deletion of specific genes in PRV-Becker results in a restricted neurotropism identical to that demonstrated with PRV-Bartha (10).Although viral transsynaptic tracing represents an important methodological advance for the analysis of CNS circuits, functional analysis of virus-infected neurons has been limited to sensory or sympathetic ganglia in culture (11, 12) because of the inability to identify virus-infected neurons in situ. Analyses of electrophysiological properties of neurons, in the context of known functional connections of the recorded neuron, would represent a further important methodological advance for the analysis of CNS circuits.The development of retrogradely transported fluorescent tracers has allowed investigators to examine the physiology of neurons with known projections (13-15). However, such studies usually require direct and accurate injection of a target region followed by retrograde transport to identify first-order neurons projecting to the target. Other ''prelabeling'' studies have used constructs of green fluorescent protein to label neurons that possess a particular genetic phenotype, such as expression of gonadotropin-releasing hormone (16). Both of these techniques have allowed examination of the physiological properties of neurons in vitro that possess presumed anatomical or functional correlates in the intact animal.We now report a neuron-labeling...

Intravitreal injection of the attenuated strain of pseudorabies virus (PRV Bartha) results in transneuronal spread of virus to a restricted set of central nuclei in the rat and mouse. We examined the pattern of central infection in the golden hamster after intravitreal inoculation with a recombinant strain of PRV Bartha constructed to express enhanced green fluorescent protein (PRV 152). Neurons in a subset of retinorecipient nuclei [i.e., suprachiasmatic nucleus (SCN), intergeniculate leaflet, olivary pretectal nucleus (OPN), and lateral terminal nucleus] and autonomic nuclei [i.e., paraventricular hypothalamic nucleus and Edinger-Westphal nucleus (EW)] are labeled by late stages of infection. Infection of the EW precedes infection in retinorecipient structures, raising the possibility that the SCN becomes infected by retrograde transsynaptic infection via autonomic (i.e., EW) circuits. We tested this hypothesis in two ways: (1) by removing the infected eye 24 hr after PRV 152 inoculation, well before viral infection first appears in the SCN; and (2) by examining central infection after intravitreal PRV 152 injection in animals with ablation of the EW. The pattern and time course of central infection were unchanged after enucleation, whereas EW ablation before intravitreal inoculation eliminated viral infection in the SCN. The results of EW lesions along with known connections between EW, OPN, and SCN indicate that intravitreal injection of PRV Bartha produces a retrograde infection of the autonomic innervation of the eye, which subsequently labels a restricted set of retinorecipient nuclei via retrograde trans-synaptic infection. These results, taken together with other genetic data, indicate that the mutations in PRV Bartha render the virus incapable of anterograde transport. PRV Bartha is thus a retrograde transsynaptic marker in the CNS.

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.

West Nile virus (WNV) is a vector-borne pathogen that was first detected in the United States in 1999. The natural transmission cycle of WNV involves mosquito vectors and avian hosts, which vary in their competency to transmit the virus. American robins are an abundant backyard species in the United States and appear to have an important role in the amplification and dissemination of WNV. In this study we examine the response of American robins to infection with various WNV doses within the range of those administered by some natural mosquito vectors. Thirty American robins were assigned a WNV dosage treatment and needle inoculated with 100.95 PFU, 101.26 PFU, 102.15 PFU, or 103.15 PFU. Serum samples were tested for the presence of infectious WNV and/or antibodies, while oral swabs were tested for the presence of WNV RNA. Five of the 30 (17%) robins had neutralizing antibodies to WNV prior to the experiment and none developed viremia or shed WNV RNA. The proportion of WNV-seronegative birds that became viremic after WNV inoculation increased in a dose dependent manner. At the lowest dose, only 40% (2/5) of the inoculated birds developed productive infections while at the highest dose, 100% (7/7) of the birds became viremic. Oral shedding of WNV RNA followed a similar trend where robins inoculated with the lower two doses were less likely to shed viral RNA (25%) than robins inoculated with one of the higher doses (92%). Viremia titers and morbidity did not increase in a dose dependent manner; only two birds succumbed to infection and, interestingly, both were inoculated with the lowest dose of WNV. It is clear that the disease ecology of WNV is a complex interplay of hosts, vectors, and viral dose delivered.

ABSTRACT:Populations of greater sage-grouse (Centrocercus urophasianus) have declined 45-80% in North America since 1950. Although much of this decline has been attributed to habitat loss, recent field studies have indicated that West Nile virus (WNV) has had a significant negative impact on local populations of grouse. We confirm the susceptibility of greater sage-grouse to WNV infection in laboratory experimental studies. Grouse were challenged by subcutaneous injection of WNV (10 3.2 plaque-forming units [PFUs]). All grouse died within 6 days of infection. The Kaplan-Meier estimate for 50% survival was 4.5 days. Mean peak viremia for nonvaccinated birds was 10 6.4 PFUs/ml (610 0.2 PFUs/ml, standard error of the mean [SEM]). Virus was shed cloacally and orally. Four of the five vaccinated grouse died, but survival time was increased (50% survival59.5 days), with 1 grouse surviving to the end-point of the experiment (14 days) with no signs of illness. Mean peak viremia for the vaccinated birds was 10 2.3 PFUs/ml (610 0.6 PFUs/ml, SEM). Two birds cleared the virus from their blood before death or euthanasia. These data emphasize the high susceptibility of greater sage-grouse to infection with WNV.

Intraocular injection of the Bartha strain of pseudorabies virus (PRV Bartha) results in transsynaptic infection of the hypothalamic suprachiasmatic nucleus (SCN), a retinorecipient circadian oscillator. PRV Bartha infection of a limited number of retinorecipient structures, including the SCN, was initially interpreted as the differential infection of a subpopulation of rat retinal ganglion cells, followed by replication and anterograde transport via the optic nerve. A recent report that used a recombinant strain of PRV Bartha (PRV152) expressing enhanced green fluorescent protein demonstrated that SCN infection actually results from retrograde transneuronal transport of the virus via the autonomic innervation of the eye in the golden hamster. In the present study using the rat, the pattern of infection after intravitreal inoculation with PRV152 was examined to determine if infection of the rat SCN is also restricted to retrograde transsynaptic transport. It was observed that infection in preganglionic autonomic nuclei (i.e., Edinger-Westphal nucleus, superior salivatory nucleus, and intermediolateral nucleus) precedes infection in the SCN. Sympathetic superior cervical ganglionectomy did not abolish label in the SCN after intraocular infection, nor did lesions of parasympathetic preganglionic neurons in the Edinger-Westphal nucleus. However, combined Edinger-Westphal nucleus ablation and superior cervical ganglionectomy eliminated infection of the SCN. This observation allowed a detailed examination of the SCN contribution to descending autonomic circuits afferent to the eye. The results indicate that in the rat, as in the hamster, SCN infection after intraocular PRV152 inoculation is by retrograde transsynaptic transport via autonomic pathways to the eye.

Primary gustatory afferents from the oropharynx of the goldfish, Carassius auratus, terminate in the vagal lobe, a laminated structure in the dorsal medulla comparable to the gustatory portion of the nucleus of the solitary tract in mammals. We utilized an in vitro brain slice preparation to test the role of different ionotropic glutamate receptor subtypes in synaptic transmission of gustatory information by recording changes in field potentials after application of various glutamate receptor antagonists. Electrical stimulation of the vagus nerve (NX) evokes two short-latency postsynaptic field potentials from sensory layers of the vagal lobe. 6,7-Dinitroquinoxaline-2,3-dione and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, two non-N-methyl-D-aspartate (NMDA) ionotropic receptor antagonists, blocked these short-latency potentials. Slower potentials that were revealed under Mg2+ -free conditions, were abolished by the NMDA receptor antagonist, D(-)-2-amino-5-phosphonovaleric acid (APV). Repetitive stimulation produced short-term facilitation, which was attenuated by application of APV. These results indicate that the synaptic responses in the vagal lobe produced by stimulation of the gustatory roots of the NX involve both NMDA and non-NMDA receptors. An NMDA receptor-mediated facilitation may serve to amplify incoming bursts of primary afferent activity.