The injury resistant ability of melanopsin-expressing intrinsically photosensitive retinal ganglion cells.

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.

Cell-type specific distribution of chloride transporters in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABA(A) receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl(-) influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K(+)/Cl(-) cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K(+)/Cl(-) cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine-threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl(-) levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.

Serotonergic modulation of retinal input to the mouse suprachiasmatic nucleus mediated by 5-HT1B and 5-HT7 receptors.

Serotonin (5-HT) and 5-HT receptor agonists can modify the response of the mammalian suprachiasmatic nucleus (SCN) to light. It remains uncertain which 5-HT receptor subtypes mediate these effects. The effects of 5-HT receptor activation on optic nerve-mediated input to SCN neurons were examined using whole-cell patch-clamp recordings in horizontal slices of ventral hypothalamus from the male mouse. The hypothesis that 5-HT reduces the effect of retinohypothalamic tract (RHT) input to the SCN by acting at 5-HT1B receptors was tested first. As previously described in the hamster, a mixed 5-HT(1A/1B) receptor agonist, 1-[3-(trifluoromethyl)phenyl]-piperazine hydrochloride (TFMPP), reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by selectively stimulating the optic nerve of wild-type mice. The agonist was negligibly effective in a 5-HT1B receptor knockout mouse, suggesting minimal contribution of 5-HT1A receptors to the TFMPP-induced reduction in the amplitude of the optic nerve-evoked EPSC. We next tested the hypothesis that 5-HT also reduces RHT input to the SCN via activation of 5-HT7 receptors. The mixed 5-HT(1A/7) receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), reduced the evoked EPSC amplitude in both wild-type and 5-HT1B receptor knockout mice. This effect of 8-OH-DPAT was minimally attenuated by the selective 5-HT1A receptor antagonist WAY 100635 but was reversibly and significantly reduced in the presence of ritanserin, a mixed 5-HT(2/7) receptor antagonist. Taken together with the authors' previous ultrastructural studies of 5-HT1B receptors in the mouse SCN, these results indicate that in the mouse, 5-HT reduces RHT input to the SCN by acting at 5-HT1B receptors located on RHT terminals. Moreover, activation of 5-HT7 receptors in the mouse SCN, but not 5-HT1A receptors, also results in a reduction in the amplitude of the optic nerve-evoked EPSC. The findings indicate that 5-HT may modulate RHT glutamatergic input to the SCN through 2 or more 5-HT receptors. The likely mechanism of altered RHT glutamatergic input to SCN neurons is an alteration of photic effects on the SCN circadian oscillator.

5-HT1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus.

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-HT1B receptor-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-HT1B receptors in the mouse SCN using electron microscopic immunocytochemical analysis with 5-HT1B receptor antibodies and whole-cell patch-clamp recordings from SCN neurons in hamster hypothalamic slices. 5-HT1B receptor immunostaining was observed associated with the plasma membrane of retinal terminals in the SCN. 1-[3-(Trifluoromethyl)phenyl]-piperazine HCl (TFMPP), a 5-HT1B receptor agonist, reduced in a dose-related manner the amplitude of glutamatergic EPSCs evoked by stimulating selectively the optic nerve. Selective 5-HT1A or 5-HT7 receptor 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-HT1B receptor knock-out mice. TFMPP inhibited behavioral responses to light in wild-type mice but was ineffective in inhibiting light-induced phase shifts in 5-HT1B receptor knock-out mice. The results indicate that 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1B receptors located on retinal axons in the SCN.

Restoration of circadian behavior by anterior hypothalamic grafts containing the suprachiasmatic nucleus: graft/host interconnections.

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.

Thyrotropin-releasing hormone phase shifts circadian rhythms in hamsters.

The role of thyrotropin-releasing hormone (TRH) in regulating circadian rhythms was investigated by assessing the ability of TRH microinjections into the suprachiasmatic nucleus (SCN) to induce phase shifts in hamster wheel-running behavior. TRH injected into the SCN at 10 and 100 nM doses produced phase advances in wheel-running activity of 18.3 +/- 1.9 and 34.8 +/- 2.9 minutes, respectively, when administered at circadian time (CT) 6. Injections at CT 18 produced no effects. The temporal sensitivity of the SCN to TRH administration was examined by administering TRH at specific circadian times. TRH produced significant phase advances at CT 4, 6, and 8, while no significant changes in wheel-running onset were observed at other CT times. These studies represent the first evidence of TRH's ability to affect circadian function.

Altered circadian rhythmicity in the Wocko mouse, a hyperactive transgenic mutant.

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.

Modulation of IL-1 beta gene expression in the rat CNS during sleep deprivation.

We hypothesize that sleep homeostasis involves, at least in part, the immune system modulator interleukin-1 beta (IL-1 beta). Using the reverse transcription-polymerase chain reaction, IL-1 beta mRNA levels in the rat CNS were evaluated after a period of sleep deprivation. In addition, IL-1 beta gene expression was analyzed before the projected onset of activity and rest phase in free-running animals. No changes in IL-1 beta mRNA were observed in the circadian cycle, but 24 h of sleep deprivation resulted in a 2-fold increase in the level of IL-1 beta mRNA in the hypothalamus and in the brain stem compared with controls (p < 0.0002 and (p < 0.0001 respectively). The alteration in IL-1 beta mRNA levels following sleep deprivation supports the hypothesis that modulation of IL-1 beta gene expression is involved in the sleep homeostatic process.

Mutagenesis and behavioral screening for altered circadian activity identifies the mouse mutant, Wheels.

The molecular processes underlying the generation of circadian behavior in mammals are virtually unknown. To identify genes that regulate or alter circadian activity rhythms, a mouse mutagenesis program was initiated in conjunction with behavioral screening for alterations in circadian period (tau), a fundamental property of the biological clock. Male mice of the inbred BALB/c strain, treated with the potent mutagen N-ethyl-N-nitrosourea were mated with wild-type hybrids. Wheel-running activity of approximately 300 male progeny was monitored for 6-10 weeks under constant dark (DD) conditions. The tau DD of a single mouse (#187) was longer than the population mean by more than three standard deviations (24.20 vs. 23.32 +/- 0.02 h; mean +/- S.E.M.; n = 277). In addition, mouse #187 exhibited other abnormal phenotypes, including hyperactive bi-directional circling/spinning activity and an abnormal response to light. Heterozygous progeny of the founder mouse, generated from outcrossings with wild-type C57BL/6J mice, displayed lengthened tau DD although approximately 20% of the animals showed no wheel-running activity despite being quite active. Under light:dark conditions, all animals displaying circling behavior that ran in the activity wheels exhibited robust wheel-running activity at lights-ON and these animals also showed enhanced wheel-running activity in constant light conditions. The genetic dissection of the complex behavior associated with this mutation was facilitated by the previously described genetic mapping of the mutant locus causing circling behavior, designated Wheels (Whl), to the subcentromeric portion of mouse chromosome 4. In this report, the same locus is shown to be responsible for the abnormal responses to light and presumably for the altered circadian behavior. Characterization of the gene altered in the novel Whl mutation will contribute to understanding the molecular elements involved in mammalian circadian regulation.

Vasoactive intestinal peptide efferent projections of the suprachiasmatic nucleus in anterior hypothalamic transplants: correlation with functional restoration of circadian behavior.

Circadian rhythmicity can be restored by transplantation of fetal anterior hypothalamic (AH) tissue containing the suprachiasmatic nucleus (SCN) into hosts rendered arrhythmic by SCN ablation. However, the nature of the SCN effector pathways mediating functional recovery has remained elusive. To examine implant-derived SCN innervation of the host, AH homografts (hamster-to-hamster) and heterografts (mouse- or rat-to-hamster) were employed and the distribution of vasoactive intestinal peptide (VIP) within the SCN terminal fields was evaluated. A comparison was made between cases where circadian locomotor activity was restored and cases where circadian rhythmicity remained disrupted following AH transplantation. A dense aggregation of VIP neurons and processes was identified in each transplant that restored behavioral rhythmicity in the host. In these cases, SCN-derived VIP fibers were integrated with the host brain and could be identified in host terminal fields typically innervated by SCN-VIP fibers. A correlation was noted between VIP innervation of the host paraventricular thalamic nucleus (PVT) and restoration of circadian rhythmicity. Neither qualitative nor quantitative differences in transplant VIP projections were noted between AH homografts and heterografts. These results demonstrate that SCN VIP neurons in AH transplants send an appropriately restricted set of efferent projections to the host brain and suggest that SCN efferent projections to the PVT may participate in mediating the functional recovery of circadian locomotor activity.

Heterozygosity mapping of partially congenic lines: mapping of a semidominant neurological mutation, Wheels (Whl), on mouse chromosome 4.

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.

Restoration of circadian behavior by anterior hypothalamic heterografts.

The suprachiasmatic nucleus (SCN) of the anterior hypothalamus (AH) is a circadian oscillator and an important component of the mammalian circadian system. To determine whether the SCN is the dominant circadian pacemaker responsible for generating a species-typical characteristic of circadian rhythms [i.e., period length (tau)], neural transplantation was conducted using fetal AH donors of different species and SCN-lesioned (SCNx) hosts. The circadian behavior of each of the three donor species is clearly distinguishable by its species-typical tau. The extent of SCN pacemaker autonomy was assessed by noting whether the period of the restored circadian rhythm following heterograft transplantation was characteristic of the donor or the host, or whether an atypical circadian period was established. Hamsters rendered arhythmic by SCN ablation were implanted with AH tissue from fetal hamsters (E13-E14, homograft controls) or fetal mice or rats (E15-E17). The AH homografts restored circadian activity rhythms with a tau similar to that of intact hamsters, and fetal mouse AH heterografts restored circadian rhythmicity with a tau similar to that of the donor mouse strain. However, fetal rat AH tissue implanted into SCNx hamsters renewed circadian rhythmicity with a period significantly shorter than either the species-typical tau of the rat donor or the hamster host. In both the mouse and rat AH heterograft experiments, immunocytochemical analysis performed with species-specific monoclonal antibodies revealed extensive fiber outgrowth from the implant into the host hypothalamus, evident up to 7 months postimplantation. The rat implants were consistently larger, more fully vascularized and exhibited less necrosis than the implanted mouse tissue. The histological appearance of the grafts, thus, provides no explantation for the difference in efficacy of the grafts to restore species-typical behavior. However, several interpretations are considered that are consistent with the combined behavioral results observed.

Light intensity and splitting in the golden hamster.

Hamster circadian activity rhythms split into two components during prolonged exposure to conditions of constant light (LL). Several aspects of this phenomenon were examined in this study. The frequency of splitting was significantly greater among animals exposed to LL of 100 lux intensity (LL100) compared with animals in LL10. Animals that split had significantly longer free-running periods (tau) compared to nonsplitters and the decrease in tau associated with splitting was highly correlated with the presplit tau. Splitting was also observed under continuous dim light which fluctuated rhythmically from 5-10 lux. Thus, splitting of the circadian rhythm of activity is positively correlated with LL intensity with an LL intensity threshold for the induction of splitting in the range of 3-5 lux.

Time course of fiber outgrowth from fetal anterior hypothalamic heterografts.

Fetal anterior hypothalamic (AH) heterografts can restore circadian rhythmicity to animals rendered arrhythmic following ablation of the suprachiasmatic nucleus (SCN). Behavioral restoration of circadian activity typically begins between two and six weeks post-implantation. The time course of fiber outgrowth from fetal AH heterografts was examined to determine whether neuronal outgrowth from the implants precedes the typically observed effects of such implants upon circadian behavior. Fetal mouse or rat AH tissue containing the SCN was implanted into the third ventricle of SCN-lesioned hamsters. Using species-specific monoclonal antibodies generated against mouse or rat neuronal elements, fiber outgrowth into the host hypothalamus was examined at 2, 4, 7, 14, 30 and 45 days after implantation. Fibers were observed to have emerged from the implant at the earliest time point examined. Four days after surgery, individual fibers had extended up to 0.6 mm into the host neuropil. By 14 days post-implantation, outgrowth from the implant had formed a dense fiber plexus in the host hypothalamus. This observation demonstrates that neuronal integration of the implant with the host brain begins within 48 hours of implantation, and is extensively established well before a restoration of rhythmicity is typically observed. Thus, on the basis of the time course of fiber outgrowth, it is clear that neuronal contact between graft and host may mediate the observed restoration of circadian rhythmicity.

Reversal of the sodium chloride aversion of Fischer 344 rats by chorda tympani nerve transection.

Fischer 344 (F344) rats are atypical in their lack of preference for any concentration of NaCl solution over water. It was hypothesized that abnormal signals mediated by the chorda tympani nerve (CT) could be causally involved in NaCl avoidance by F344 rats. This study assessed whether CT transection would normalize the salt preference of F344 rats. Preference for NaCl solutions (0.6%, 0.8%, and 1.0%) versus water was assessed using two-bottle preference tests. At all concentrations tested, CT-transected animals preferred NaCl solutions to water. This preference differed dramatically from the avoidance of these solutions by controls. These findings are striking, particularly because CT transections have generally failed to significantly affect NaCl preference in other rat strains. The results are consistent with the hypothesis that in F344 rats the avoidance of the taste of NaCl stems from input mediated by the CT.