Light pulse duration differentially regulates mouse locomotor suppression and phase shifts.

Brief exposure of mice to nocturnal light causes circadian rhythm phase shifts, simultaneously inducing locomotor suppression, a drop in body temperature, and associated sleep. The exact nature of the relationship between these light-induced responses is uncertain, although locomotor suppression and phase shift magnitudes are related to stimulus irradiance. Whether stimulus duration has similar effects is less clear. Here, the relationship between stimulus duration and response magnitude was evaluated further using 100 µW/cm(2) white light-emitting diode pulses administered for 30, 300, 1200, or 3000 sec. The results show that, in general, shorter pulses yielded smaller responses and larger pulses yielded larger responses. However, the 300-sec pulse failed to augment locomotor suppression compared with the effect of a 30-sec pulse (44.7 ± 4.8 vs 40.6 ± 2.0 min) but simultaneously induced much larger phase shifts (1.28 ± 0.20 vs 0.52 ± 0.11 h). The larger phase shifts induced by the 300-sec stimulus did not differ from those induced by either the 1200- or 3000-sec pulses (1.43 ± 0.10 and 1.30 ± 0.17 h, respectively). The results demonstrate differential photic regulation of the two response types. Pulses ranging from 300 to 3000 sec produce equal phase shifts (present data); pulses ranging from 30 to 600 sec produce equal locomotor suppression levels. Greater suppression can occur additively in response to pulses of 1200 sec or more (present data), but this is not true for phase shifts. Nocturnal light appears to trigger a fixed duration event, locomotor suppression, or phase shift, with the latter followed by a light-refractory interval during which locomotor suppression can additively increase. The results also provide further support for the view that temporal integration of photic energy applies, at best, across a limited set of stimulus durations for both light-induced locomotor suppression/sleep and phase shift regulation.

Retinofugal projections in the mouse.

The laboratory mouse is increasingly a subject for visual system investigation, but there has been no comprehensive evaluation of this species' visual projections. Here, projections were visualized and mapped following intraocular injection of cholera toxin B subunit. Tissue was processed using standard procedures applied to 30 µm free-floating sections with diaminobenzidine as the chromogen. The mouse retina projects to ~46 brain regions, including 14 not previously described in this species. These include two amygdaloid nuclei, the horizontal limb of the diagonal band, the paraventricular hypothalamic nucleus, several visual thalamic nuclei, the paranigral nucleus, several pretectal nuclei, and the dorsal cortex of the inferior colliculus. Dense retinal patches were also observed in a narrow portion of the ipsilateral intermediate layer of the superior colliculus. The superior fasciculus of the accessory optic tract, which innervates the medial terminal nucleus, was also determined to be a terminal zone throughout its length. The results are compared with previous descriptions of projections from mouse intrinsically photoreceptive retinal ganglion cells, and with data from the hamster, Nile grass rat, and laboratory rat. The retinal projection patterns are similar in all four species, although there are many differences with respect to the details. The specific visual functions of most retinorecipient areas are unknown, but there is substantial convergence of retinal projections onto regions concerned with olfaction and audition.

Brief light stimulation during the mouse nocturnal activity phase simultaneously induces a decline in core temperature and locomotor activity followed by EEG-determined sleep.

Light exerts a variety of effects on mammals. Unexpectedly, one of these effects is the cessation of nocturnal locomotion and the induction of behavioral sleep (photosomnolence). Here, we extend the initial observations in several ways, including the fundamental demonstration that core body temperature (T(c)) drops substantially (about 1.5°C) in response to the light stimulation at CT15 or CT18 in a manner suggesting that the change is a direct response to light rather than simply a result of the locomotor suppression. The results show that 1) the decline of locomotion and T(c) begin soon after nocturnal light stimulation; 2) the variability in the magnitude and onset of light-induced locomotor suppression is very large, whereas the variability in T(c) is very small; 3) T(c) recovers from the light-induced decline in advance of the recovery of locomotion; 4) under entrained and freerunning conditions, the daily late afternoon T(c) increase occurs in advance of the corresponding increase in wheel running; and 5) toward the end of the subjective night, the nocturnally elevated T(c) persists longer than does locomotor activity. Finally, EEG measurements confirm light-induced sleep and, when T(c) or locomotion was measured, show their temporal association with sleep onset. Both EEG- and immobility-based sleep detection methods confirm rapid induction of light-induced sleep. The similarities between light-induced loss of locomotion and drop in T(c) suggest a common cause for parallel responses. The photosomnolence response may be contingent upon both the absence of locomotion and a simultaneous low T(c).

Neurons identified by NeuN/Fox-3 immunoreactivity have a novel distribution in the hamster and mouse suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) has several structural characteristics and cell phenotypes shared across species. Here, we describe a novel feature of SCN anatomy that is seen in both hamster and mouse. Frozen sections through the SCN were obtained from fixed brains and stained for the presence of immunoreactivity to neuronal nuclear protein (NeuN-IR) using a mouse monoclonal antibody which is known to exclusively identify neurons. NeuN-IR did not identify all SCN neurons as medial NeuN-IR neurons were generally not present. In the hamster, NeuN-IR cells are present rostrally, scattered in the dorsal half of the nucleus. More caudally, the NeuN-IR cells are largely, but not exclusively, scattered inside the lateral and dorsolateral border. At mid- to mid-caudal SCN levels, a dense group of NeuN-IR cells extends from the dorsolateral border ventromedially to encompass the central subnucleus of the SCN (SCNce). The pattern is similar in the mouse SCN. NeuN-IR does not co-localize with either cholecystokinin- or vasoactive intestinal polypeptide, but does with vasopressin-IR in the caudal SCN. In the hamster SCNce, numerous cells contain both calbindin- and NeuN-IR. The distribution of NeuN-IR cells in the SCN is unique, especially with regard to its generally lateral location through the length of the nucleus. The distribution of NeuN-IR cells is not consistent with most schemas representing SCN organization or with terminology referring to its widely accepted subdivisions. NeuN has recently been identified as Fox-3 protein. Its function in the SCN is not known, nor is it known why a large proportion of SCN cells do not contain NeuN-IR.

Two components of nocturnal locomotor suppression by light.

In nocturnal rodents, millisecond light ("flash") stimuli can induce both a large circadian rhythm phase shift and an associated state change from highly active to quiescence followed by behavioral sleep. Suppression of locomotion ("negative masking") is an easily measured correlate of the state change. The present mouse studies used both flashes and longer light stimuli ("pulses") to distinguish initiation from maintenance effects of light on locomotor suppression and to determine whether the locomotor suppression exhibits temporal integration as is thought to be characteristic of phase shift responses to pulse, but not flash, stimuli. In experiment 1, locomotor suppression increased with irradiance (0.01-100 microW/cm( 2)), in accordance with previous reports. It also increased with stimulus duration (3-3000 sec), but interpretation of this result is complicated by the ability of light to both initiate and maintain locomotor suppression. In experiment 2, an irradiance response curve was determined using a stimulus series of 10 flashes, 2 msec each, with total flash energy varying from 0.0025 to 110.0 J/m(2). This included a test for temporal integration in which the effects of two equal energy series of flashes that differed in the number of flashes per series (10 vs 100), were compared. The 10 flash series more effectively elicited locomotor suppression than the 100 flash series, a result consistent with prior observations involving flash-induced phase shifts. In experiment 3, exposure of mice to an 11-h light stimulus yielded irradiance-dependent locomotor suppression that was maintained for the entire stimulus duration by a 100-microW/cm(2) stimulus. Light has the ability to initiate a time-limited (30-40 min) interval of locomotor suppression (initiation effect) that can be extended by additional light (maintenance effect). Temporal integration resembling that seen in phase-shifting responses to light does not exist for either phase shift or locomotor suppression responses to flashes or for locomotor suppression responses to light pulses. The authors present an alternative interpretation of data thought to demonstrate temporal integration in the regulation of phase shift responses to light pulses.

Endocannabinoids in the intact retina: 3 H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide.

There is much evidence for an endocannabinoid system in the retina. However, neither the distribution of endocannabinoid uptake, the regulation of endocannabinoid levels, nor the role of endocannabinoid metabolism have been investigated in the retina. Here we focused on one endocannabinoid, anandamide (AEA), and its major hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), in the goldfish retina. Immunoblots of FAAH immunoreactivity (IR) in goldfish retina, brain and rat retina, and brain homogenates showed a single band at 61 kDa that was blocked by preadsorption with peptide antigen. Specific FAAH IR (blocked by preadsorption) was most prominent over Müller cells and cone inner segments. Weaker label was observed over some amacrine cells, rare cell bodies in the ganglion cell layer, and in four lamina in the inner plexiform layer. FAAH activity assays showed that goldfish-retinal and brain homogenates hydrolyzed AEA at rates comparable to rat brain homogenate, and the hydrolysis was inhibited by methyl arachidonyl fluorophosphonate (MAFP) and N-(4 hydroxyphenyl)-arachidonamide (AM404), with IC(50)s of 21 nM and 1.5 microM, respectively. Cellular 3H-AEA uptake in the intact retina was determined by in vitro autoradiography. Silver-grain accumulation at 20 degrees C was most prominent over cone photoreceptors and Müller cells. Uptake was significantly reduced when retinas were incubated at 4 degrees C, or preincubated with 100 nM MAFP or 10 microM AM404. There was no differential effect of blocking conditions on the distribution of silver grains over cones or Müller cells. The codistribution of FAAH IR and 3H-AEA uptake in cones and Müller cells suggests that the bulk clearance of AEA in the retina occurs as a consequence of a concentration gradient created by FAAH activity. We conclude that endocannabinoids are present in the goldfish retina and underlay the electrophysiological effects of cannabinoid ligands previously shown on goldfish cones and bipolar cells.

Vanilloid receptor like 1 (VRL1) immunoreactivity in mammalian retina: colocalization with somatostatin and purinergic P2X1 receptors.

The distribution of vanilloid receptor like1 immunoreactivity (VRL1-IR) in the retinas of rat, cat, and monkey was studied by single- and double-labeling immunocytochemistry. The patterns were similar for all three species in that VRL1-IR was most prominent in the inner plexiform layer, with scattered compact projections to the outer plexiform layer (OPL). VRL1-immunoreactive cell bodies were present throughout the rat retina, represented by amacrine cells in the inner nuclear layer and ganglion cell layer (GCL). In cat and monkey retinas, VRL1-immunoreactive cell bodies were restricted to the GCL in the inferior retina. Occasional cell bodies were associated with retinal blood vessels, but their identity as pericytes, glia, or neurons is uncertain. All VRL1-immunoreactive cells and processes colocalized with somatostatin and purinergic P2X1 receptor-IR but not with tyrosine hydroxylase-IR. VRL1-immunoreactive processes in the OPL did not label with antisera against synaptic vesicle 2 (SV2), suggesting that they were dendritic and did not derive from interplexiform cells. However, VRL1-immunoreactive processes in the far periphery toward the pars plana labeled for SV2, suggesting that these processes were presynaptic. The VRL1-immunoreactive cell bodies in the monkey GCL were not calbindin-immunoreactive, demonstrating that they were not displaced H2 horizontal cells. The VRL1-immunoreactive cells in cat and monkey could represent biplexiform and/or associational ganglion cells that receive input in the OPL throughout the retina and direct output to the far periphery. The presence of P2X1 receptors and vanilloid receptor like 1 protein on somatostatin-containing neurons in mammalian retina adds to the growing complexity regarding the chemical control of retinal function that is likely to include the microcirculation.

Evidence against the presence of an anandamide transporter.

On the basis of temperature dependency, saturability, selective inhibition, and substrate specificity, it has been proposed that an anandamide transporter exists. However, all of these studies have examined anandamide accumulation at long time points when downstream effects such as metabolism and intracellular sequestration are operative. In the current study, we have investigated the initial rates (<1 min) of anandamide accumulation in neuroblastoma and astrocytoma cells in culture and have determined that uptake is not saturable with increasing concentrations of anandamide. However, anandamide hydrolysis, after uptake in neuroblastoma cells, was saturable at steady-state time points (5 min), suggesting that fatty acid amide hydrolase (FAAH) may be responsible for observed saturation of uptake at long time points. In general, arvanil, olvanil, and N-(4-hydroxyphenyl)arachidonylamide (AM404) have been characterized as transport inhibitors in studies using long incubations. However, we found these "transport inhibitors" did not inhibit anandamide uptake in neuroblastoma and astrocytoma cells at short time points (40 sec or less). Furthermore, we confirmed that these inhibitors in vitro were actually inhibitors of FAAH. Therefore, the likely mechanism by which the transport inhibitors raise anandamide levels to exert pharmacological effects is by inhibiting FAAH, and they should be reevaluated in this context. Immunofluorescence has indicated that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, and this finding is consistent with our observed kinetics of anandamide hydrolysis. In summary, these data suggest that anandamide uptake is a process of simple diffusion. This process is driven by metabolism and other downstream events, rather than by a specific membrane-associated anandamide carrier.