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Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established1. Recurrent infections arise sporadically from the peripheral nervous system throughout the life of the host, and invasion of the central nervous system may occur, with severe outcomes2. These viruses directly recruit cellular motors for transport along microtubules in nerve axons, but how the motors are manipulated to deliver the virus to neuronal nuclei is not understood. Here, using herpes simplex virus type I and pseudorabies virus as model alphaherpesviruses, we show that a cellular kinesin motor is captured by virions in epithelial cells, carried between cells, and subsequently used in neurons to traffic to nuclei. Viruses assembled in the absence of kinesin are not neuroinvasive. The findings explain a critical component of the alphaherpesvirus neuroinvasive mechanism and demonstrate that these viruses assimilate a cellular protein as an essential proviral structural component. This principle of viral assimilation may prove relevant to other virus families and offers new strategies to combat infection. Herpes simplex virus type I and pseudorabies virus assimilate kinesin from host epithelial cells and repurpose the motor to traffic to the nuclei of neurons in the peripheral nervous system.

Following exposure and replication at mucosal surfaces, most alphaherpesviruses invade the peripheral nervous system by retrograde axonal transport and establish lifelong latent infections in the peripheral ganglia. Reactivation of ganglionic infections is followed by anterograde axonal transport of virions back to body surfaces where viral replication results in disease that can range from moderate to severe in presentation. In the case of bovine herpesvirus 1 (BoHV-1), replication in the epithelial mucosa presents as infectious bovine rhinotracheitis (IBR), a respiratory disease of significant economic impact. In this study, we provide a live-cell analysis of BoHV-1 retrograde axonal transport relative to the model alphaherpesvirus pathogen pseudorabies virus (PRV) and demonstrate that this critical neuroinvasive step is conserved between the two viruses. In addition, we report that the BoHV-1 pUL37 tegument protein supports processive retrograde motion in infected axons and invasion of the calf peripheral nervous system. IMPORTANCE A molecular and cellular understanding of the retrograde axonal transport process that underlies the neuroinvasive properties of the alphaherpesviruses is established from studies of herpes simplex virus and pseudorabies virus. The degree to which this phenotype is conserved in other related viruses has largely not been examined. We provide a time-lapse analysis of the retrograde axonal transport kinetics of bovine herpesvirus 1 and demonstrate that mutation of the pUL37 region 2 effector affords a strategy to produce live-attenuated vaccines for enhanced protection of cattle.

Herpes simplex virus (HSV) infections are common and can cause severe illness but no vaccine is currently available. The recent failure of subunit HSV vaccines has highlighted the need for vaccines that present a diverse array of antigens, including the development of next-generation live-attenuated vaccines. However, most attenuated HSV strains propagate poorly, limiting their ability to elicit protective immune responses. A live-attenuated vaccine that replicates in non-neural tissue but is ablated for transmission into the nervous system may elicit protective immune responses without evoking neurologic complications or establishing life-long infections. Initial studies of R2, a live-attenuated vaccine that is engineered to be unable to invade the nervous system, used the guinea pig genital HSV model to evaluate the ability of R2 to replicate at the site of inoculation, cause disease and infect neural tissues. R2 was then evaluated as a vaccine using three routes of inoculation: intramuscular (IM), intradermal (ID) and intravaginal (IVag) and compared to IM administered gD2+MPL/Alum vaccine in the same model. R2 replicated in the genital tract but did not produce acute or recurrent disease and did not infect the neural tissue. The R2 vaccine-induced neutralizing antibody and decreased the severity of acute and recurrent HSV-2 disease as well as recurrent shedding. The ID route was the most effective. ID administered R2 was more effective than gD2+MPL/Alum at inducing neutralizing antibody, suppressing acute disease, and acute vaginal virus replication. R2 was especially more effective at reducing recurrent virus shedding, the most common source of HSV transmission. The live-attenuated prophylactic HSV vaccine, R2, was effective in the guinea pig model of genital HSV-2 especially when administered by the ID route. The use of live-attenuated HSV vaccines that robustly replicate in mucosal tissues but are ablated for neuroinvasion offers a promising approach for HSV vaccines.

Neurotropic alpha-herpesviruses that infect mammals establish life-long latent infections in the peripheral nervous system after initial infection of exposed mucosal tissues. The neuroinvasive properties can lead to severe complications both with clinical and veterinary alpha-herpesviruses, and vaccines are often unavailable or provide limited protection. Here we assess the properties and efficacy of an R2 vaccine derived from the alpha-herpesvirus, pseudorabies virus (PRV), in pigs. We demonstrate that the PRV R2 vaccine does not invade the porcine peripheral nervous system within the limits of detection. Furthermore, after a single intranasal vaccination, R2 conferred protection to pigs subsequently challenged with a virulent PRV field strain (NIA-3). These findings support that the R2 vaccine design is non-neuroinvasive and is an effective vaccine in the context of a natural host.

Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established

Intrinsically photosensitive retinal ganglion cells (ipRGCs) innervate the hypothalamic suprachiasmatic nucleus (SCN), a circadian oscillator that functions as a biological clock. ipRGCs use vesicular glutamate transporter 2 (vGlut2) to package glutamate into synaptic vesicles and light-evoked resetting of the SCN circadian clock is widely attributed to ipRGC glutamatergic neurotransmission. Pituitary adenylate cyclase-activating polypeptide (PACAP) is also packaged into vesicles in ipRGCs and PACAP may be coreleased with glutamate in the SCN. vGlut2 has been conditionally deleted in ipRGCs in mice [conditional knock-outs (cKOs)] and their aberrant photoentrainment and residual attenuated light responses have been ascribed to ipRGC PACAP release. However, there is no direct evidence that all ipRGC glutamatergic neurotransmission is eliminated in vGlut2 cKOs. Here, we examined two lines of ipRGC vGlut2 cKO mice for SCN-mediated behavioral responses under several lighting conditions and for ipRGC glutamatergic neurotransmission in the SCN. Circadian behavioral responses varied from a very limited response to light to near normal photoentrainment. After collecting behavioral data, hypothalamic slices were prepared and evoked EPSCs (eEPSCs) were recorded from SCN neurons by stimulating the optic chiasm. In cKOs, glutamatergic eEPSCs were recorded and all eEPSC parameters examined (stimulus threshold, amplitude, rise time or time-to-peak and stimulus strength to evoke a maximal response) were similar to controls. We conclude that a variable number but functionally significant percentage of ipRGCs in two vGlut2 cKO mouse lines continue to release glutamate. Thus, the residual SCN-mediated light responses in these cKO mouse lines cannot be attributed solely to ipRGC PACAP release.

There is a growing recognition that the coordinated timing of behavioral, physiologic, and metabolic circadian rhythms is a requirement for a healthy body and mind. In mammals, the primary circadian oscillator is the hypothalamic suprachiasmatic nucleus (SCN), which is responsible for circadian coordination throughout the organism. Temporal homeostasis is recognized as a complex interplay between rhythmic clock gene expression in brain regions outside the SCN and in peripheral organs. Abnormalities in this intricate circadian orchestration may alter sleep patterns and contribute to the pathophysiology of affective disorders.

Retinal ganglion Y (alpha) cells are found in retinas ranging from frogs to mice to primates. The highly conserved nature of the large, fast conducting retinal Y cell is a testament to its fundamental task, although precisely what this task is remained ill-defined. The recent discovery that Y-alpha retinal ganglion cells send axon collaterals to the serotonergic dorsal raphe nucleus (DRN) in addition to the lateral geniculate nucleus (LGN), medial interlaminar nucleus (MIN), pretectum and the superior colliculus (SC) has offered new insights into the important survival tasks performed by these cells with highly branched axons. We propose that in addition to its role in visual perception, the Y-alpha retinal ganglion cell provides concurrent signals via axon collaterals to the DRN, the major source of serotonergic afferents to the forebrain, to dramatically inhibit 5-HT activity during orientation or alerting/escape responses, which dis-facilitates ongoing tonic motor activity while dis-inhibiting sensory information processing throughout the visual system. The new data provide a fresh view of these evolutionarily old retinal ganglion cells.

Reactivation from latency results in transmission of neurotropic herpesviruses from the nervous system to body surfaces, referred to as anterograde axonal trafficking. The virus-encoded protein pUS9 promotes axonal dissemination by sorting virus particles into axons, but whether it is also an effector of fast axonal transport within axons is unknown. To determine the role of pUS9 in anterograde trafficking, we analyzed the axonal transport of pseudorabies virus in the presence and absence of pUS9.

A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses., Author summary Neuroinvasive members of the alpha-herpesvirinae include human (i.e. herpes simplex virus type I; HSV-1) and veterinary (i.e. pseudorabies virus; PRV) pathogens that routinely invade the peripheral nervous system of an immunocompetent host in the absence of overt tissue damage. We have identified an essential, and conserved, component of these viruses that directs incoming viral particles into the neural ganglia. Viruses carrying mutations in this protein effector region propagate normally at peripheral sites of inoculation but fail to invade the nervous system by retrograde axonal transport, cannot establish life-long latent infections, and are avirulent. These properties define a promising new class of live-attenuated vaccines that protect from subsequent nervous system invasion and encephalitic disease.

Animals promote their survival by avoiding rapidly approaching objects that indicate threats. In mice, looming-evoked defensive responses are triggered by the superior colliculus (SC) which receives direct retinal inputs. However, the specific neural circuits that begin in the retina and mediate this important behaviour remain unclear. Here we identify a subset of retinal ganglion cells (RGCs) that controls mouse looming-evoked defensive responses through axonal collaterals to the dorsal raphe nucleus (DRN) and SC. Looming signals transmitted by DRN-projecting RGCs activate DRN GABAergic neurons that in turn inhibit serotoninergic neurons. Moreover, activation of DRN serotoninergic neurons reduces looming-evoked defensive behaviours. Thus, a dedicated population of RGCs signals rapidly approaching visual threats and their input to the DRN controls a serotonergic self-gating mechanism that regulates innate defensive responses. Our study provides new insights into how the DRN and SC work in concert to extract and translate visual threats into defensive behavioural responses., Neural circuits underlying innate fear are only partially understood. Huang et al. identify a subset of retinal ganglion cells that project to both the dorsal raphe nucleus and the superior colliculus, and show that these RGCs mediate looming-evoked defensive behaviours in mice.

SummaryMicrotubule transport of herpesvirus capsids from the cell periphery to the nucleus is imperative for viral replication and, in the case of many alphaherpesviruses, transmission into the nervous system. Using the neuroinvasive herpesvirus, pseudorabies virus (PRV), we show that the viral protein 1/2 (VP1/2) tegument protein associates with the dynein/dynactin microtubule motor complex and promotes retrograde microtubule transport of PRV capsids. Functional activation of VP1/2 requires binding to the capsid protein pUL25 or removal of the capsid-binding domain. A proline-rich sequence within VP1/2 is required for the efficient interaction with the dynein/dynactin microtubule motor complex as well as for PRV virulence and retrograde axon transport in vivo. Additionally, in the absence of infection, functionally active VP1/2 is sufficient to move large surrogate cargoes via the dynein/dynactin microtubule motor complex. Thus, VP1/2 tethers PRV capsids to dynein/dynactin to enhance microtubule transport, neuroinvasion, and pathogenesis.

The dorsal raphe nucleus (DRN), the major source of serotonergic input to the forebrain, receives excitatory input from the retina that can modulate serotonin levels and depressive-like behavior. In the Mongolian gerbil, retinal ganglion cells (RGCs) with alpha-like morphological and Y-like physiological properties innervate the DRN with ON DRN-projecting RGCs out numbering OFF DRN-projecting RGCs. The DRN neurons targeted by ON and OFF RGCs are unknown. To explore retino-raphe anatomical organization, retinal afferents labeled with Cholera toxin B were examined for association with the postsynaptic protein PSD-95. Synaptic associations between retinal afferents and DRN serotonergic and GABAergic neurons were observed. To explore retino-raphe functional organization, light-evoked c-fos expression was examined. Light significantly increased the number of DRN serotonergic and GABAergic cells expressing c-Fos. When ON RGCs were rendered silent while enhancing the firing rate of OFF RGCs, c-Fos expression was greatly increased in DRN serotonergic neurons suggesting that OFF DRN-projecting RGCs predominately activate serotonergic neurons whereas ON DRN-projecting RGCs mainly target GABAergic neurons. Direct glutamatergic retinal input to DRN 5-HT neurons contributes to the complex excitatory drive regulating these cells. Light, via the retinoraphe pathway can modify DRN 5-HT neuron activity which may play a role in modulating affective behavior.

In order to resolve the location and activity of submicroscopic viruses in living cells, viral proteins are often fused to fluorescent proteins (FPs) and visualized by microscopy. In this study, we describe the fusion of FPs to three proteins of pseudorabies virus (PRV) that allowed imaging of capsids in living cells. Included in this study are the first recombinant PRV strains expressing FP–pUL25 fusions based on a design applied to herpes simplex virus type 1 by Homa and colleagues. The properties of each reporter virus were compared in both in vitro and in vivo infection models. PRV strains expressing FP–pUL25 and FP–pUL36 preserved wild-type properties better than traditional FP–pUL35 isolates in assays of plaque size and virulence in mice. The utility of these strains in studies of axon transport, nuclear dynamics and viral particle composition are documented.

Purpose A retinal projection into the dorsal raphe nucleus (DRN), namely, the retino-raphe projection, exists in many species. The rat is one of several species in which a retino-raphe projection has been described; however, the retinal ganglion cell (RGC) types that contribute to this pathway are unknown. Methods Retrograde tracing via cholera toxin subunit B (CTB) was used to reveal DRN-projecting RGCs in rats, combined with intracellular injection in vitro, melanopsin immunostaining in whole-mounted retinas, and serotonin immunostaining to define the DRN. We modified methods of CTB injection into DRN used previously in order to avoid possible contamination with other retinorecipient regions, particularly the superior colliculus (SC). Results The majority of DRN-projecting RGCs showed alpha-like morphology, and some CTB-positive RGCs were colabeled with melanopsin. Approximately 80% of the total population of CTB-labeled DRN-projecting RGCs was alpha-like cells including ON alpha cells and OFF alpha cells; these alpha-like cells were melanopsin immunonegative. Approximately 10% of the remaining DRN-projecting RGCs were melanopsin immunopositive, in which the M1 subtype of intrinsically photosensitive retinal ganglion cell (ipRGC) provided the dominant projection of ipRGCs into DRN, with only few non-M1 ipRGCs involved. The DRN-projecting ipRGCs could be retrogradely labeled following tracer injection into all rostrocaudal aspects of the DRN. Conclusions Both conventional RGCs with alpha-like morphology and melanopsin-expressing ipRGCs project into the rat DRN. Approximately 10% of DRN-projecting RGCs were colabeled with melanopsin, and the majority of these were the M1 subtype of ipRGCs. An ipRGC component of the retino-raphe projection may contribute to a sustained light-mediated modulation of DRN serotonin release.

Neuroinvasive herpesviruses display a remarkable propensity to enter the nervous system of healthy individuals in the absence of obvious trauma at the site of inoculation. We document a repurposing of cellular ubiquitin during infection to switch the virus between two invasive states. The states act sequentially to defeat consecutive host barriers of the peripheral nervous system and together promote the potent neuroinvasive phenotype. The first state directs virus access to nerve endings in peripheral tissue, whereas the second delivers virus particles within nerve fibers to the neural ganglia. Mutant viruses locked in either state remain competent to overcome the corresponding barrier but fail to invade the nervous system. The herpesvirus "ubiquitin switch" may explain the unusual ability of these viruses to routinely enter the nervous system and, as a consequence, their prevalence in human and veterinary hosts.

Neurotropic herpesviruses exit the peripheral nervous system and return to exposed body surfaces following reactivation from latency. The pUS9 protein is a critical viral effector of the anterograde axonal transport that underlies this process. We recently reported that while pUS9 increases the frequency of sorting of newly assembled pseudorabies virus particles to axons from the neural soma during egress, subsequent axonal transport of individual virus particles occurs with wild-type kinetics in the absence of the protein. Here, we examine the role of a related pseudorabies virus protein, pUL56, during neuronal infection. The findings indicate that pUL56 is a virulence factor that supports virus dissemination in vivo, yet along with pUS9, is dispensable for axonal transport.

Ca2+channel β subunits regulate cell-surface expression and gating of voltage-dependent Ca2+channel α1 subunits. Based on primary sequence comparisons, β subunits are predicted to be modular structures composed of five domains (A–E) that are related to the large family of membrane-associated guanylate kinase proteins. The crystal structure of the β subunit core B–D domains has been reported recently; however, little is known about the structures of the A and E domains. The N-terminal A domain differs among the four subtypes of Ca2+channel β subunits (β1–β4) primarily as the result of two duplications of an ancestral gene containing multiple alternatively spliced exons. At least nine A domain sequences can be generated by alternative splicing. In this report, we focus on one A domain sequence, the highly conserved β4aA domain. We solved its three-dimensional structure and show that it is expressed in punctate structures throughout the molecular layer of the cerebellar cortex. We also demonstrate that it does not participate directly in Cav2.1 Ca2+channel gating but serves as a binding site in protein–protein interactions with synaptotagmin I and the LC2 domain of microtubule-associated protein 1A. With respect to β4subunits, the interactions are specific for the β4asplice variant, because they do not occur with the β4bA domain. These results have strong bearing on our current understanding of the structure of alternatively spliced Ca2+channel β subunits and the cell-specific roles they play in the CNS.

Mammalian alphaherpesviruses normally establish latent infections in ganglia of the peripheral nervous system in their natural hosts. Occasionally, however, these viruses spread to the central nervous system (CNS), where they cause damaging, often fatal, infections. Attenuated alphaherpesvirus derivatives have been used extensively as neuronal circuit tracers in a variety of animal models. Their circuit-specific spread provides a unique paradigm to study the local and global CNS response to infection. Thus, we systematically analyzed the host gene expression profile after acute pseudorabies virus (PRV) infection of the CNS using Affymetrix GeneChip technology. Rats were injected intraocularly with one of three selected virulent and attenuated PRV strains. Relative levels of cellular transcripts were quantified from hypothalamic and cerebellar tissues at various times postinfection. The number of cellular genes responding to infection correlated with the extent of virus dissemination and relative virulence of the PRV strains. A total of 245 out of 8,799 probe sets, corresponding to 182 unique cellular genes, displayed increased expression ranging from 2- to more than 100-fold higher than in uninfected tissue. Over 60% thereof were categorized as immune, proinflammatory, and other cellular defense genes. Additionally, a large fraction of infection-induced transcripts represented cellular stress responses, including glucocorticoid- and redox-related pathways. This is the first comprehensive in vivo analysis of the global transcriptional response of the mammalian CNS to acute alphaherpesvirus infection. The differentially regulated genes reported here are likely to include potential diagnostic and therapeutic targets for viral encephalitides and other neurodegenerative or neuroinflammatory diseases.

The hypothalamic suprachiasmatic nucleus (SCN) is a circadian oscillator that receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain. Activation of presynaptic serotonin 1B (5-HT1B) receptors on retinal terminals in the SCN inhibits retinohypothalamic neurotransmission and light-induced behavioral phase shifts. To assess the role of 5-HT1B receptors in photic entrainment, 5-HT1B receptor knockout (5-HT1B KO) and wild-type (WT) mice were maintained in non-24 h L:D cycles (T cycles). WT mice entrained to T = 21 h and T = 22 h cycles, whereas 5-HT1B KO animals did not. 5-HT1B KO animals did entrain to T = 23 h and T = 26 h cycles, although their phase angle of entrainment was altered compared to WT animals. 5-HT1BKO mice were significantly more phase delayed under T = 23 h conditions and significantly more phase advanced under T = 26 h conditions compared to WT mice. When 5-HT1B KO mice were housed in a T = 23 h short-day photoperiod (9.5L:13.5D), the delayed phase angle of entrainment was more pronounced. Light-induced phase shifts were reduced in 5-HT1B KO mice, consistent with their behavior in T cycles, suggesting an attenuated response to light. Based on previous work, this attenuated response to light might not have been predicted but can be explained by consideration of GABAergic mechanisms within the SCN. Phase-delayed circadian rhythms during the short days of winter are characteristic of patients suffering from seasonal affective disorder, and 5-HT has been implicated in its pathophysiology. The 5-HT1B KO mouse may be useful for investigating the altered entrainment evident during this serious mood disorder.

The suprachiasmatic nucleus (SCN) receives a dense serotonergic innervation that modulates photic input to the SCN via serotonin 1B (5-HT1B) presynaptic receptors on retinal glutamatergic terminals. However, the majority of 5-HT1B binding sites in the SCN are located on nonretinal terminals and most axonal terminals in the SCN are GABAergic. We therefore tested the hypothesis that 5-HT1B receptors might also be located on SCN GABAergic terminals by examining the effects of the highly selective 5-HT1B receptor agonist CP-93,129 on SCN miniature inhibitory postsynaptic currents (mIPSCs). Whole cell patch-clamp recordings of mIPSCs were obtained from rat and mouse SCN neurons in hypothalamic slices. Using CsCl-containing microelectrodes with QX314, we isolated mPSCs that were sensitive to the GABAA receptor antagonist, bicuculline. Bath application of CP-93,129 (1 μM) decreased the frequency of mIPSCs by an average of 22% ( n = 7) in rat SCN neurons and by an average of 30% ( n = 8) in mouse SCN neurons with no clear effect on mIPSC amplitude. In mice lacking functional 5-HT1B receptors, CP-93,129 (1 μM) had no clear effect on the frequency or the amplitude of mIPSCs recorded in any of the cells tested ( n = 4). The decrease in the frequency of mIPSCs of SCN neurons produced by the selective 5-HT1B receptor agonist CP-93,129 is consistent with the interpretation that 5-HT1B receptors are located on GABA terminals in the SCN and that 5-HT inhibits GABA release via a 5-HT1B presynaptic receptor-mediated mechanism.

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. © 2004 Wiley-Liss, Inc.

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 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.

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.

Serotonin (5-HT) can act presynaptically at 5-HT1B receptors on retinal terminals in the suprachiasmatic nucleus (SCN) to inhibit glutamate release, thereby modulating the effects of light on circadian behavior. 5-HT1B receptor agonists (1) inhibit light-inducedphase shifts of circadian activity rhythms, (2) attenuate light-induced Fos expression in the SCN, and (3) reduce the amplitude of optic nerve–evoked excitatory postsynaptic currents in SCN neurons in vitro. To determine whether functional disruption of the 5-HT1B presynaptic receptors would result in an amplified response of the SCN to light, the period ([.tau]) of the circadian rhythm of wheel-running activity was estimated under several different conditions in 5-HT1B receptor knockout (KO) mice and genetically matched wild- typeanimals. Under constant light (LL) conditions, the [.tau] of 5-HT1B receptor KO mice was significantly greater than the [.tau] of wild-type mice. Aquantitative analysis of the wheel-running activity revealed no differences between wild-type and KO mice in either total activity or the temporal distribution of activity under LL conditions, suggesting that the observed increase in [.tau] was not a function of reduced activity. Under constant dark conditions, the period of the circadian rhythm of wheel-running activity of wild-type and 5-HT1B receptor KO mice was similar. In addition, no differences were noted between wild-type and 5-HT1Breceptor KO mice in the rate of reentrainment toa6h phase advance in the 12:12 light:dark cycle or in phase shifts in response to a 10 min light pulse presented at circadian time 16. The enhanced response of the SCN circadian clock of the 5- HT1B receptor KO mice to LLconditions is consistent with the hypothesis that the endogenous activation of 5-HT1B presynaptic receptors modulates circadian behavior by attenuating photic input to the SCN.

Neurons in the mammalian retina expressing the photopigment melanopsin have been identified as a class of intrinsically photosensitive retinal ganglion cells (ipRGCs). This discovery more than a decade ago has opened up an exciting new field of retinal research, and following the initial identification of photosensitive ganglion cells, several subtypes have been described. A number of studies have shown that ipRGCs subserve photoentrainment of circadian rhythms. They also influence other non-image forming functions of the visual system, such as the pupillary light reflex, sleep, cognition, mood, light aversion and development of the retina. These novel photosensitive neurons also influence form vision by contributing to contrast detection. Furthermore, studies have shown that ipRGCs are more injury-resistant following optic nerve injury, in animal models of glaucoma, and in patients with mitochondrial optic neuropathies, i.e., Leber’s hereditary optic neuropathy and dominant optic atrophy. There is also an indication that these cells may be resistant to glutamate-induced excitotoxicity. Herein we provide an overview of ipRGCs and discuss the injury-resistant character of these neurons under certain pathological and experimental conditions.

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. © 2001 Wiley-Liss, Inc.

Physiological properties of central nervous system neurons infected with a pseudorabies virus were examined in vitro by using whole-cell patch-clamp techniques. A strain of pseudorabies virus (PRV 152) isogenic with the Bartha strain of PRV was constructed to express an enhanced green fluorescent protein (EGFP) from the human cytomegalovirus immediate early promoter. Unilateral PRV 152 injections into the vitreous body of the hamster eye transsynaptically infected a restricted set of retinorecipient neurons including neurons in the hypothalamic suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) of the thalamus. Retinorecipient SCN neurons were identified in tissue slices prepared for in vitro electrophysiological analysis by their expression of EGFP. At longer postinjection times, retinal ganglion cells in the contralateral eye also expressed EGFP, becoming infected after transsynaptic uptake and retrograde transport from infected retinorecipient neurons. Retinal ganglion cells that expressed EGFP were easily identified in retinal whole mounts viewed under epifluorescence. Whole-cell patch-clamp recordings revealed that the physiological properties of PRV 152-infected SCN neurons were within the range of properties observed in noninfected SCN neurons. Physiological properties of retinal ganglion cells also appeared normal. The results suggest that PRV 152 is a powerful tool for the transsynaptic labeling of neurons in defined central nervous system circuits that allows neurons to be identified in vitro by their expression of EGFP, analyzed electrophysiologically, and described in morphological detail.

In this review, we describe six lines of evidence that reveal a modulatory role for serotonin (5-HT) in the regulation of the response of suprachiasmatic nucleus (SCN) neurons to retinal illuminati...

Destruction of the hypothalamic suprachiasmatic nucleus (SCN) disrupts circadian behavior. Transplanting SCN tissue from fetal donors into SCN-lesioned recipients can restore circadian behavior to the arrhythmic hosts. In the transplantation model employing fetal hamster donors and SCN-lesioned hamsters as hosts, the period of the restored circadian behavior is hamster-typical. However, when fetal rat anterior hypothalamic tissue containing the SCN is implanted into SCN-lesioned rats, the period of the restored circadian rhythm is only rarely typical of that of the intact rat. The use of an anterior hypothalamic heterograft model provides new approaches to donor specificity of restored circadian behavior and with the aid of species-specific markers, provides a means for assessing connectivity between the graft and the host. Using an antibody that stains rat and mouse neuronal tissue but not hamster neurons, it has been demonstrated that rat and mouse anterior hypothalamic heterografts containing the SCN send numerous processes into the host (hamster) neuropil surrounding the graft, consistent with graft efferents reported in other hypothalamic transplantation models in which graft and host tissue can be differentiated (i.e., Brattleboro rat and hypogonadal mouse). Moreover, SCN neurons within anterior hypothalamic grafts send an appropriately restricted set of efferent projections to the host brain which may participate in the functional recovery of circadian locomotor activity.

Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image-forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several central nervous system responses, including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. New sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod- and cone-mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses, including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions.

The suprachiasmatic nucleus (SCN) is a circadian oscillator and a critical component of the mammalian circadian system. It receives afferents from the retina and the mesencephalic raphe. Retinal afferents mediate photic entrainment of the SCN, whereas the serotonergic afferents originating from the midbrain modulate photic responses in the SCN; however, the serotonin (5HT) receptor subtypes in the SCN responsible for these modulatory effects are not well characterized. In this study, we tested the hypothesis that 5HT1Breceptors are located presynaptically on retinal axon terminals in the SCN and that activation of these receptors inhibits retinal input.The 5HT1Breceptor agonists TFMPP and CGS 12066A, administered systemically, inhibited light-induced phase shifts of the circadian activity rhythm in a dose-dependent manner at phase delay and phase advance time points. This inhibition was not affected by previous systemic application of either the selective 5HT1Areceptor antagonist (+)WAY 100135 or by the 5HT2receptor antagonist mesulergine, whereas pretreatment with the nonselective 5HT1antagonist methiothepin significantly attenuated the effect of TFMPP. TFMPP also produced a dose-dependent reduction in light-stimulated Fos expression in the SCN, although a small subset of cells in the dorsolateral aspect of the caudal SCN were TFMPP-insensitive. TFMPP (1 mm) infused into the SCN produced complete inhibition of light-induced phase advances. Finally, bilateral orbital enucleation reduced the density of SCN 5HT1Breceptors as determined using [125I]-iodocyanopindolol to define 5HT1Bbinding sites. These results are consistent with the interpretation that 5HT1Breceptors are localized presynaptically on retinal terminals in the SCN and that activation of these receptors by 5HT1Bagonists inhibits retinohypothalamic input.

The outer segments of vertebrate retinal photoreceptors undergo periodic shedding of membrane from their distal tips. This circadian rhythm of disk shedding persists with a period of about 24 h in the absence of external time cues. A circadian oscillator controlling photoreceptor disk shedding may exist in the eye, but in addition, the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) may also influence ocular rhythms including that of disk shedding. The tau mutation directly affects the SCN, and shortens the period of locomotor activity from 24 h in wild-type hamsters to 20 h in homozygous mutants. Here we show that homozygous tau-mutant hamsters in a 20-h light/dark cycle exhibit a 20-h oscillation in the rate of disk shedding, with peak phagosome numbers in the retinal pigmented epithelium occurring just after light onset. The numbers of phagosomes are significantly elevated from mid-dark levels prior to light onset, indicating that the disk shedding cycle anticipates dawn. Under conditions of constant darkness, the disk shedding rhythm in tau-mutant hamsters persists with a period of approximately 20 h. These results indicate that a rhythm of retinal photoreceptor outer segment disk shedding exists in the hamster eye, and that the period of this rhythm is shortened by the tau mutation.

The lateral geniculate complex innervates the hypothalamic suprachiasmatic nucleus (SCN). The location of neurons in the cat ventral lateral geniculate nucleus (vLGN) that give rise to the geniculohypothalamic tract has not been described. In this study, retrogradely labeled neurons were noted throughout the rostrocaudal extent of the medial vLGN following tracer injection into the SCN region. In addition, neuropeptide Y immunoreactive processes were also observed in the vLGN in this same medial zone and in the SCN. The data suggest that the medial zone of the cat vLGN may be homologous to the rodent intergeniculate leaflet (IGL).

Induced changes in the level of daily activity can alter the period of the mammalian circadian clock. In this report, we examined the period of the circadian rhythm of wheel-running activity in a transgenic neurological mouse mutant, Wocko. Wocko mice display a dominant behavioral phenotype that consists of hyperactivity, circling and head tossing. The period of the circadian rhythm of wheel-running activity in constant dark conditions was significantly shorter in mice expressing the Wocko mutation than in their normal littermates. Total activity, monitored by the interruption of an array of infrared beams, was significantly elevated in Wocko mice. These findings support the view that spontaneous exercise can modulate the circadian timekeeping system.

Light is a powerful modulator of higher-order cognitive processes such as mood but it remains unclear which neural circuits mediate the impact of light on affective behavior. We found that light deprivation produces a depressive-like behavioral state that is reversed by activation of direct retinal signals to the serotonergic dorsal raphe nucleus (DRN) in a manner equivalent to treatment with the selective serotonin reuptake inhibitor fluoxetine. Surprisingly, the DRN-projecting retinal ganglion cells (RGCs) are indistinguishable from the classic alpha/Y-like RGC type that contributes to image-forming visual pathways. Silencing RGC firing or specific immunotoxin ablation of DRN-projecting RGCs increased depressive-like behavior and reduced serotonin levels in the DRN. Serotonin has a key role in the pathophysiology of depression, and these results demonstrate that retino-raphe signals modulate DRN serotonergic tone and affective behavior., link_to_OA_fulltext

We identified a semidominant, chemically induced, mouse mutation with a complex array of abnormal behaviors including bidirectional circling and hyperactivity, abnormal circadian rhythmicity and abnormal responses to light. In this report, we genetically and phenotypically characterized the circling/waltzing component of the abnormal behavior. We mapped the locus controlling this trait by heterozygosity mapping of partially congenic lines carrying the mutagenized chromosome outcrossed to different inbred strains for three generations. Analysis of 68 PCR-based markers in 13 affected individuals indicated that the mutant locus, named Wheels (Whl), resides in the subcentromeric portion of mouse chromosome 4. The statistical evaluation of data obtained by heterozygosity mapping validates this efficient mapping approach. Further characterization of the Whl mutation demonstrated that Whl/Whl homozygotes die during embryonic life and that the penetrance of circling behavior depends on genetic background. Morphological analysis of the inner ears of Whl/+ mice revealed a variable number of abnormalities in the sensory and nonsensory portions of their semicircular canals. Abnormalities ranged from slight atrophy of one or more cristae to complete absence of the lateral crista and canal. The molecular characterization of the gene disrupted in the Whl mutation will provide insight into developmental mechanisms involved in inner ear formation.

The canonical flow of visual signals proceeds from outer to inner retina (photoreceptors → bipolar cells → ganglion cells). However, melanopsin-expressing ganglion cells are photosensitive and functional sustained light signaling to retinal dopaminergic interneurons persists in the absence of rods and cones. Here we show that the sustained-type light response of retinal dopamine neurons requires melanopsin and that the response is mediated by AMPA-type glutamate receptors, defining a retrograde retinal visual signaling pathway that fully reverses the usual flow of light signals in retinal circuits.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond to light in the absence of all rod and cone photoreceptor input. The existence of these ganglion cell photoreceptors, although predicted from observations scattered over many decades, was not established until it was shown that a novel photopigment, melanopsin, was expressed in retinal ganglion cells of rodents and primates. Phototransduction in mammalian ipRGCs more closely resembles that of invertebrate than vertebrate photoreceptors and appears to be mediated by transient receptor potential channels. In the retina, ipRGCs provide excitatory drive to dopaminergic amacrine cells and ipRGCs are coupled to GABAergic amacrine cells via gap junctions. Several subtypes of ipRGC have been identified in rodents based on their morphology, physiology and expression of molecular markers. ipRGCs convey irradiance information centrally via the optic nerve to influence several functions including photoentrainment of the biological clock located in the hypothalamus, the pupillary light reflex, sleep and perhaps some aspects of vision. In addition, ipRGCs may also contribute irradiance signals that interface directly with the autonomic nervous system to regulate rhythmic gene activity in major organs of the body. Here we review the early work that provided the motivation for searching for a new mammalian photoreceptor, the ground-breaking discoveries, current progress that continues to reveal the unusual properties of these neuron photoreceptors, and directions for future investigation.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond to light in the absence of all rod and cone photoreceptor input. The existence of these ganglion cell photoreceptors, although predicted from observations scattered over many decades, was not established until it was shown that a novel photopigment, melanopsin, was expressed in retinal ganglion cells of rodents and primates. Phototransduction in mammalian ipRGCs more closely resembles that of invertebrate than vertebrate photoreceptors and appears to be mediated by transient receptor potential channels. In the retina, ipRGCs provide excitatory drive to dopaminergic amacrine cells and ipRGCs are coupled to GABAergic amacrine cells via gap junctions. Several subtypes of ipRGC have been identified in rodents based on their morphology, physiology and expression of molecular markers. ipRGCs convey irradiance information centrally via the optic nerve to influence several functions including photoentrainment of the biological clock located in the hypothalamus, the pupillary light reflex, sleep and perhaps some aspects of vision. In addition, ipRGCs may also contribute irradiance signals that interface directly with the autonomic nervous system to regulate rhythmic gene activity in major organs of the body. Here we review the early work that provided the motivation for searching for a new mammalian photoreceptor, the ground-breaking discoveries, current progress that continues to reveal the unusual properties of these neuron photoreceptors, and directions for future investigation.