Cynthia A. Smeraski

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.

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.

Suprachiasmatic Nucleus Input to Autonomic Circuits Identified by Retrograde Transsynaptic Transport of Pseudorabies Virus from the Eye

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. J. Comp. Neurol. 471:298–313, 2004.

SUSCEPTIBILITY OF GREATER SAGE-GROUSE TO EXPERIMENTAL INFECTION WITH WEST NILE VIRUS

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 (103.2 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 106.4 PFUs/ml (±100.2 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% survival=9.5 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 102.3 PFUs/ml (±100.6 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.

Experimental infection of cliff swallows (Petrochelidon pyrrhonota) with varying doses of West Nile virus

Cliff swallows (Petrochelidon pyrrhonota) were inoculated with differing doses of West Nile virus (WNV) to evaluate their potential role as reservoir hosts in nature. Swallows often nest in large colonies in habitats and months associated with high mosquito abundance and early WNV transmission in North America. Additionally, cliff swallow diet consists of insects, including mosquitoes, leading to an additional potential route of WNV infection. The average peak viremia titer among infected cliff swallows was 10(6.3) plaque-forming units (PFU)/mL serum and the reservoir competence index was 0.34. There was no correlation between dose and probability of becoming infected or viremia peak and duration. Oral shedding was detected from 2 to 14 days post-inoculation with an average peak titer of 10(4.4) PFU/swab. These results suggest that cliff swallows are competent reservoir hosts of WNV and therefore, they may play a role in early seasonal amplification and maintenance of WNV.

West Nile virus infection in American Robins: new insights on dose response.

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.

Treatment of spatial memory impairment in hamsters infected with West Nile virus using a humanized monoclonal antibody MGAWN1

In addition to functional disorders of paresis, paralysis, and cardiopulmonary complications, subsets of West Nile virus (WNV) patients may also experience neurocognitive deficits and memory disturbances. A previous hamster study has also demonstrated spatial memory impairment using the Morris water maze (MWM) paradigm. The discovery of an efficacious therapeutic antibody MGAWN1 from pre-clinical rodent studies raises the possibility of preventing or treating WNV-induced memory deficits. In the current study, hamsters were treated intraperitoneally (i.p.) with 32 mg/kg of MGAWN1 at 4.5 days after subcutaneously (s.c.) challenging with WNV. As expected, MGAWN1 prevented mortality, weight loss, and improved food consumption of WNV-infected hamsters. The criteria for entry of surviving hamsters into the study were that they needed to have normal motor function (forelimb grip strength, beam walking) and normal spatial reference memory in the MWM probe task. Twenty-eight days after the acute phase of the disease had passed, MGAWN1- and saline-treated infected hamsters were again trained in the MWM. Spatial memory was evaluated 48 h after this training in which the hamsters searched for the location where a submerged escape platform had been positioned. Only 56% of infected hamsters treated with saline spent more time in the correct quadrant than the other three quadrants, as compared to 92% of MGAWN1-treated hamsters (P⩽0.05). Overall these studies support the possibility that WNV can cause spatial memory impairment and that therapeutic intervention may be considered.

Kainate‐activated cobalt uptake in the primary gustatory nucleus in goldfish: Visualization of the morphology and distribution of cells expressing AMPA/kainate receptors in the vagal lobe

Gustatory afferent fibers of the vagus nerve that innervate taste buds of the oropharynx of the goldfish, Carassius auratus, project to the vagal lobe, which is a laminated gustatory nucleus in the dorsal medulla. As in the mammalian gustatory system, responses by second‐order cells in the goldfish medulla are mediated by N‐methyl‐D‐aspartate (NMDA) and non‐NMDA ionotropic glutamate receptors. We utilized a cobalt uptake technique to label vagal lobe neurons that possess cobalt‐permeable ionotropic glutamate receptors. Vagal lobe slices were bathed in kainate (40 μM) or glutamate (0.5 or 1 mM) in the presence of CoCl2, which can pass into cells through the ligand‐gated cation channels of non‐NMDA receptors made up of certain subunit combinations. Cobalt‐filled cells and dendrites were observed in slices that were activated by kainate or glutamate, but not in control slices that were bathed in CoCl2 alone, nor in slices that were bathed with the non‐NMDA receptor antagonist 6,7‐dinitroquinoxaline‐2,3‐dione (10 μM) in addition to an agonist. Likewise, simple depolarization of the cells with KCl failed to induce cobalt loading. Cobalt‐filled round unipolar cells, elongate or globular bipolar cells, and multipolar cells with elongate or polygonal perikarya were distributed throughout the cell layers in the sensory zone of the vagal lobe. Numerous labeled neurons had dendrites spanning layers IV and VI, the two principal layers of primary afferent input. Apical and basal dendrites often extended radially through neighboring laminae, but many cells also extended dendrites tangential to the lamination of the sensory zone. In the motor layer, cell bodies and proximal dendrites of small, multipolar neurons, and large motoneurons were regularly loaded with cobalt. J. Comp. Neurol. 431:59–74, 2001.

West Nile virus infection of the nervous system

autonomic nervous system in these effects we experimentally manipulated stress levels in rhesus macaques infected with SIVmac251, and examined resulting changes SIV replication within axillary lymph nodes. Macaques were exposed to daily stable or unstable social interactions during the course of infection. Lymph nodes were biopsied during mid-stage infection and sites of viral replication were mapped by in situ hybridization against SIV env, gag, and nef RNA. Catecholaminergic neural fibers were mapped by SPG chemofluorescence. Spatial statistical analyses, showed that lymph nodes from experimentally stressed monkeys showed 50% increase in the density of SIV replication sites. This is the first demonstration that manipulation of stress can increase lentiviral gene expression in peripheral lymphoid tissues. We have previously shown that SIV replication is enhanced in the vicinity of ANS innervation. Increased SIV burden in macaques exposed to stressful social conditions may be a function of altered innervation in these animals. These studies suggest that adrenergic interventions aimed at blocking such interactions could potentially complement anti-retroviral therapies to delay disease progression and preserve the health of HIV-infected individuals.

#126 Experimental infection of birds with West Nile virus: Level of infection in the CNS

Clinical and epidemiological studies suggest that the host autonomic nervous system (ANS)might influenceHIVpathogenesis. To investigate the neuroanatomical basis for sympathetic nervous system (SNS) influences on lentiviral pathogenesis, we analyzed the spatial relationship between catecholaminergic neurons and SIV replication in lymph nodes from rhesus macaques experimentally infected with SIVmac251. Sites of viral replication were mapped by in situ hybridization for SIV env, gag, and nefRNA, and catecolaminergic neurons from the sympathetic nervous system were mapped by SPG chemofluorescence. Spatial statistical analyses showed that active SIV replication was 3.8-fold more likely to occur in the vicinity of catecholaminergic neurons (p < .0001). Density of both SNS innervation and SIV replication differed across cortical, paracortical, and medullary regions of the lymph node, but analyses of each region separately continued to show increased SIV replication in the vicinity of catecholaminergic neurons. Comparisons with lymph nodes fromuninfected rhesusmacaques revealed a 38% reduction in the density of SNSneurons in tissues from SIV-infected animals, suggesting that lentiviral pathogenesis might deplete lymph node neurons. Consistent with this hypothesis, the number of SIV replication sites was inversely correlated with the density of catecholaminergic neurons among SIV-infected animals. These data are consistent with human clinical studies and in vitro experimental studies in suggesting that catecholamine neurotransmitters from the SNS might enhance progression of lentiviral infection. Given the deleterious effects of SIV infection on neural density, these results also suggest that SNS influences may be most pronounced during the early phases of infection prior to virally induced depletion of lymph node neurons. Sources of support: National Institute of Mental Health (MH049033), the National Institutes of Allergy and Infectious Disease and Neurological Disorders and Stroke (AI/ NS52737), the University of California University wide AIDS Research Program (CC99-LA-02), the Norman Cousins Center for Psychoneuroimmunology, and the James B. Pendelton Charitable Trust.