No abstract

Serotonin (5-HT) has been strongly implicated in the regulation of the mammalian circadian clock located in the suprachiasmatic nuclei (SCN); however, its role in behavioral (nonphotic) circadian phase resetting remains elusive. Central to this issue are divergent lines of evidence that the SCN may, or may not, be a target for the phase-resetting effects of 5-HT. We have addressed this question using a novel reverse-microdialysis approach for timed perfusions of serotonergic and other agents to the Syrian hamster SCN with durations equivalent to the increases in in vivo 5-HT release during phase-resetting behavioral manipulations. We found that 3 hr perfusions of the SCN with either 5-HT or the 5-HT 1A,7 receptor agonist 2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydro-naphthalene (8-OH-DPAT) at midday advanced the phase of the free-running circadian rhythm of wheel-running assessed using an Aschoff type II procedure. Phase shifts induced by 8-OH-DPAT were enhanced more than threefold by pretreatment with the 5-HT synthesis inhibitor para-chlorophenylalanine. Phase advances induced by SCN 8-OH-DPAT perfusion were significantly inhibited by the 5-HT 2,7 receptor antagonist ritanserin and by the more selective 5-HT 7 receptor antagonist DR4004, implicating the 5-HT 7 receptor in mediating this phase resetting. Concurrent exposure to light during the 8-OH-DPAT perfusion abolished the phase advances. Furthermore, coperfusion of the SCN with TTX, which blocked in vivo 5-HT release, did not suppress intra-SCN 8-OH-DPAT-induced phase advances. These results indicate that 5-HT 7 receptor-mediated phase resetting in the SCN is markedly influenced by the degree of postsynaptic responsiveness to 5-HT and by photic stimulation. Finally, 5-HT may act directly on SCN clock cells to induce in vivo nonphotic phase resetting.

Tulp1 is a protein of unknown function exclusive to rod and cone photoreceptor cells. Mutations in the gene cause autosomal recessive retinitis pigmentosa in humans and photoreceptor degeneration in mice. In tulp1−/− mice, rod and cone opsins are mislocalized, and rhodopsin-bearing extracellular vesicles accumulate around the inner segment, indicating that Tulp1 is involved in protein transport from the inner segment to the outer segment. To investigate this further, we sought to define which outer segment transport pathways are Tulp1-dependent. We used immunohistochemistry to examine the localization of outer segment proteins in tulp1−/− photoreceptors, prior to retinal degeneration. We also surveyed the condition of inner segment organelles and rhodopsin transport machinery proteins. Herein, we show that guanylate cyclase 1 and guanylate cyclase activating proteins 1 and 2 are mislocalized in the absence of Tulp1. Furthermore, arrestin does not translocate to the outer segment in response to light stimulation. Additionally, data from the tulp1−/− retina adds to the understanding of peripheral membrane protein transport, indicating that rhodopsin kinase and transducin do not co-transport in rhodopsin carrier vesicles and phosphodiesterase does not co-transport in guanylate cyclase carrier vesicles. These data implicate Tulp1 in the transport of selective integral membrane outer segment proteins and their associated proteins, specifically, the opsin and guanylate cyclase carrier pathways. The exact role of Tulp1 in outer segment protein transport remains elusive. However, without Tulp1, two rhodopsin transport machinery proteins exhibit abnormal distribution, Rab8 and Rab11, suggesting a role for Tulp1 in vesicular docking and fusion at the plasma membrane near the connecting cilium.

PURPOSE Mutations in the photoreceptor-specific tubby-like protein 1 (TULP1) underlie a form of autosomal recessive retinitis pigmentosa. To investigate the role of Tulp1 in the photoreceptor synapse, the authors examined the presynaptic and postsynaptic architecture and retinal function in tulp1−/− mice METHODS The authors used immunohistochemistry to examine tulp1−/− mice before retinal degeneration and made comparisons with wild-type (wt) littermates and retinal degeneration 10 (rd10) mice, another model of photoreceptor degeneration that has a comparable rate of degeneration. Retinal function was characterized with the use of electroretinography. RESULTS In wt mice, Tulp1 is localized to the photoreceptor synapse. In the tulp1−/− synapse, the spatial relationship between the ribbon-associated proteins Bassoon and Piccolo are disrupted, and few intact ribbons are present. Furthermore, bipolar cell dendrites are stunted. Comparable abnormalities are not seen in rd10 mice. The leading edge of the a-wave had normal kinetics in tulp1−/− mice but reduced gain in rd10 mice. The b-wave intensity-response functions of tulp1−/− mice are shifted to higher intensities than in wt mice, but those of rd10 mice are not. CONCLUSIONS Photoreceptor synapses and bipolar cell dendrites in tulp1−/− mice display abnormal structure and function. A malformation of the photoreceptor synaptic ribbon is likely the cause of the dystrophy in bipolar cell dendrites. The association of early-onset, severe photoreceptor degeneration preceded by synaptic abnormalities appears to represent a phenotype not previously described. Not only is Tulp1 critical for photoreceptor function and survival, it is essential for the proper development of the photoreceptor synapse.