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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.

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.

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.

A small number (2+]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+]iwas 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+]iin light-stimulated ipRGCs.

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.

The suprachiasmatic nucleus (SCN) receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain, and serotonin (5-HT) can modify the response of the SCN circadian oscillator to light. 5-HT1Breceptor-mediated presynaptic inhibition has been proposed as one mechanism by which 5-HT modifies retinal input to the SCN (Pickard et al., 1996). This hypothesis was tested by examining the subcellular localization of 5-HT1Breceptors in the mouse SCN using electron microscopic immunocytochemical analysis with 5-HT1Breceptor antibodies and whole-cell patch-clamp recordings from SCN neurons in hamster hypothalamic slices. 5-HT1Breceptor immunostaining was observed associated with the plasma membrane of retinal terminals in the SCN. 1-[3-(Trifluoromethyl)phenyl]-piperazine HCl (TFMPP), a 5-HT1Breceptor agonist, reduced in a dose-related manner the amplitude of glutamatergic EPSCs evoked by stimulating selectively the optic nerve. Selective 5-HT1Aor 5-HT7receptor antagonists did not block this effect. Moreover, in cells demonstrating an evoked EPSC in response to optic nerve stimulation, TFMPP had no effect on the amplitude of inward currents generated by local application of glutamate. The effect of TFMPP on light-induced phase shifts was also examined using 5-HT1Breceptor knock-out mice. TFMPP inhibited behavioral responses to light in wild-type mice but was ineffective in inhibiting light-induced phase shifts in 5-HT1Breceptor knock-out mice. The results indicate that 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1Breceptors located on retinal axons in the SCN.