J. David Glass

Serotonergic inhibition of light-induced fos protein expression and extracellular glutamate in the suprachiasmatic nuclei

The present experiments were undertaken to explore a role for serotonin (5-HT) in modulating photic signal transduction and extracellular glutamate (Glu) concentration in the suprachiasmatic nuclei (SCN) of the Syrian hamster. Pretreatment with an i.p. injection of the serotonergic, quipazine, caused a marked decrease in the number of SCN cells expressing Fos protein-like immunoreactivity (Fos-LI) induced by a light pulse delivered during the latter part of the dark phase (7 h after lights-off; 55.6 +/- 7.5% of vehicle controls, P < 0.004). This effect of quipazine was dose-dependent and was limited principally to the ventrolateral region of the SCN. In a likewise manner, intra-SCN microinjection of quipazine inhibited light-induced Fos-LI in the ventrolateral SCN, indicating that the suppressive action of quipazine is centered in the SCN. In a separate experiment, localized perfusion of the SCN region with 5-HT using the microdialysis technique caused a significant reduction in the extracellular concentration of Glu. The effect was greater during the dark phase, compared to the light phase of the day-night cycle (60.7 +/- 6.8% vs. 39.3 +/- 6.8% maximal suppression, respectively; P < 0.05). Similar localized application of quipazine also decreased extracellular Glu (48.0 +/- 6.1% maximal suppression; P < 0.05). Collectively, these results are evidence for a serotonergic modulation of retinohypothalamic input in the SCN, which could involve a presynaptic inhibition of Glu release.

Dorsal raphe nuclear stimulation of SCN serotonin release and circadian phase-resetting

Serotonin (5-HT) is strongly implicated in the regulation of mammalian circadian rhythms. However, little is known of the functional relationship between the circadian clock located in the suprachiasmatic nucleus (SCN) and its source of serotonergic innervation, the midbrain raphe nuclei. In previous studies, we reported that electrical stimulation of the dorsal or median raphe nuclei (DRN and MRN, respectively) induced 5-HT release in the SCN. Notably, DRN- but not MRN-stimulated 5-HT release was blocked by the 5-HT(1,2,7) antagonist, metergoline, suggesting that the DRN signals to the SCN indirectly via the activation of a 5-HT-responsive multisynaptic pathway. In the present study, pretreatment with the 5-HT(2,7) antagonist, ritanserin, also significantly inhibited DRN-electrically stimulated SCN 5-HT release. However, pretreatment with the 5-HT(1A) antagonist, NAN-190, or the 5-HT(2) antagonists ketanserin and cinanserin had little suppressive effect on this DRN-stimulated 5-HT release. In complementary behavioral trials, electrical stimulation of the DRN during subjective midday caused a 1.3-h advance in the free-running circadian activity rhythm under constant darkness, which was inhibited by metergoline. Collectively, these results are evidence that: (1) DRN-stimulated 5-HT release in the SCN requires the activation of an intermediate target with receptors having 5-HT(7) pharmacological characteristics; (2) electrical stimulation of the DRN induces phase-resetting of the circadian activity rhythm; and (3) activation of 5-HT receptors is necessary for this DRN-stimulated circadian phase-resetting. In view of the dynamic changes in DRN neuronal activity incumbent with the daily sleep-activity cycle, and its functional linkages to the SCN and intergeniculate leaflet, the DRN could serve to provide behavioral/arousal state information to various sites comprising the brain circadian system.

Modulation of light-induced C-Fos expression in the suprachiasmatic nuclei by 5-HT1A receptor agonists

In previous studies, we showed that light-induced Fos protein expression in the ventrolateral SCN is markedly inhibited by the nonselective serotonergic, quipazine. The present experiments were undertaken to characterize the effects of various serotonin (5-HT) receptor ligands on photic signalling in the SCN. The extent of expression of light-induced Fos-like immunoreactivity (Fos-LI) in the SCN was used as a marker for this response. Exposure of hamsters to a light pulse delivered during the latter part of the dark phase (7 h after lights-off; LD 14:10) elicited an intense expression of Fos-LI in nuclei of cells situated principally in the ventrolateral region of the SCN. Pretreatment with an i.p. injection of the 5-HT1A receptor agonists, 8-OH-DPAT or buspirone, 30 min before the light pulse significantly inhibited the photic expression of Fos-LI (maximal suppression 45.7 +/- 8.1 and 43.0 +/- 1.3%, respectively, both P < 0.01 vs. vehicle controls). Treatment with the 5-HT1A receptor antagonist, NAN-190, administered 15 min before 8-OH-DPAT injection prevented the inhibitory effect of 8-OH-DPAT (100.9 +/- 6.0% vs. controls, P > 0.9). Pretreatment with the 5-HT1B receptor agonist, TFMPP, caused a small but significant suppression of Fos-LI (14.8 +/- 3.5% vs. controls, P < 0.05). In contrast to the significant 5-HT1 receptor agonist effects, pretreatment with 5-HT2 or 5-HT3 receptor agonists, alpha-methyl-5-HT and 1-phenylbiguanide had little suppressive effect on Fos-LI (7.9 +/- 2.1 and 13.0 +/- 5.0% suppression, respectively, both P > 0.1 vs. controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonergic inhibition of extracellular glutamate in the suprachiasmatic nuclear region assessed using in vivo brain microdialysis

In previous studies, we showed that localized perfusion of the SCN region with serotonin (5-HT) or the non-selective serotonergic, quipazine, using the microdialysis technique significantly reduced the extracellular concentration of the excitatory amino acid (EAA), glutamate. The present investigation was undertaken to extend these findings by characterizing the effects of various classes of 5-HT receptor ligands on the extracellular glutamate concentration in the SCN. Localized SCN application or i.p. injection of the 5-HT1A receptor agonist, 8-OH-DPAT, during the dark phase (6 h after lights-off) significantly reduced the extracellular glutamate concentration in the SCN region from baseline levels (38.7 +/- 8.7 and 53.4 +/- 11.2%, respectively, of pretreatment values; P < 0.05). The effect of systemically applied 8-OH-DPAT was abolished by i.p. injection of the 5-HT1A receptor antagonist, NAN-190, administered 20 min before the 8-OH-DPAT. Localized perfusion of the SCN with the 5-HT1B receptor agonist, TMFPP, also reduced extracellular glutamate but to a lesser degree than 8-OH-DPAT (80.1 +/- 3.9% of pretreatment levels; P < 0.05). This effect was prevented by i.p. injection of the non-selective 5-HT receptor antagonist, metergoline 20 min before TFMPP perfusion. Localized perfusion of the SCN region with the 5-HT2 and 5-HT3 receptor agonists, alpha-methyl 5-HT and 1-phenylbiguanide, respectively, had little effect on extracellular glutamate (both P > 0.1 vs. baseline). Systemic treatment with NAN-190 alone had little effect on extracellular glutamate, however, similar treatments with metergoline or the 5-HT2 receptor antagonist, ritanserin, induced significant increases extracellular glutamate levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic Ethanol Disrupts Circadian Photic Entrainment and Daily Locomotor Activity in the Mouse

BACKGROUND Chronic ethanol abuse is associated with disrupted circadian rhythms and sleep. Ethanol administration impairs circadian clock phase-resetting, suggesting a mode for the disruptive effect of alcohol abuse on circadian timing. Here, we extend previous studies to explore the effects of chronic forced ethanol on photic phase-resetting, photic entrainment, and daily locomotor activity patterns in C57BL/6J mice. METHODS First, microdialysis was used to characterize the circadian patterns of ethanol uptake in the suprachiasmatic (SCN) circadian clock and correlate this with systemic ethanol levels and episodic drinking of 10 or 15% ethanol. Second, the effects of chronic forced ethanol drinking and withdrawal on photic phase-delays of the circadian activity rhythm were assessed. Third, the effects of chronic ethanol drinking on entrainment to a weak photic zeitgeber (1 minute of 25 lux intensity light per day) were assessed. This method was used to minimize any masking actions of light that could mask ethanol effects on clock entrainment. RESULTS Peak ethanol levels in the SCN and periphery occurred during the dark phase and coincided with the time when light normally induces phase-delays in mice. These delays were dose-dependently inhibited by chronic ethanol and its withdrawal. Chronic ethanol did not impede re-entrainment to a shifted light cycle but affected entrainment under the weak photic zeitgeber and disrupted the daily pattern of locomotor activity. CONCLUSIONS These results confirm that chronic ethanol consumption and withdrawal markedly impair circadian clock photic phase-resetting. Ethanol also disturbs the temporal structure of nighttime locomotor activity and photic entrainment. Collectively, these results suggest a direct action of ethanol on the SCN clock.

Chronic ethanol attenuates circadian photic phase resetting and alters nocturnal activity patterns in the hamster

Acute ethanol (EtOH) administration impairs circadian clock phase resetting, suggesting a mode for the disruptive effect of alcohol abuse on human circadian rhythms. Here, we extend this research by characterizing the chronobiological effects of chronic alcohol consumption. First, daily profiles of EtOH were measured in the suprachiasmatic nucleus (SCN) and subcutaneously using microdialysis in hamsters drinking EtOH. In both cases, EtOH peaked near lights-off and declined throughout the dark-phase to low day-time levels. Drinking bouts preceded EtOH peaks by approximately 20 min. Second, hamsters chronically drinking EtOH received a light pulse during the late dark phase [Zeitgeber time (ZT) 18.5] to induce photic phase advances. Water controls had shifts of 1.2 +/- 0.2 h, whereas those drinking 10% and 20% EtOH had much reduced shifts (0.5 +/- 0.1 and 0.3 +/- 0.1 h, respectively; P < 0.001 vs. controls). Third, incremental decreases in light intensity (270 lux to 0.5 lux) were used to explore chronic EtOH effects on photic entrainment and rhythm stability. Activity onset was unaffected by 20% EtOH at all light intensities. Conversely, the 24-h pattern of activity bouts was disrupted by EtOH under all light intensities. Finally, replacement of chronic EtOH with water was used to examine withdrawal effects. Water controls had photic phase advances of 1.1 +/- 0.3 h, while hamsters deprived of EtOH for 2-3 days showed enhanced shifts (2.1 +/- 0.3 h; P < 0.05 vs. controls). Thus, in chronically drinking hamsters, brain EtOH levels are sufficient to inhibit photic phase resetting and disrupt circadian activity. Chronic EtOH did not impair photic entrainment; however, its replacement with water potentiated photic phase resetting.

On the intrinsic regulation of neuropeptide Y release in the mammalian suprachiasmatic nucleus circadian clock

Timing of the circadian clock of the suprachiasmatic nucleus (SCN) is regulated by photic and non‐photic inputs. Of these, neuropeptide Y (NPY) signaling from the intergeniculate leaflet (IGL) to the SCN plays a prominent role. Although NPY is critical to clock regulation, neither the mechanisms modulating IGL NPY neuronal activity nor the nature of regulatory NPY signaling in the SCN clock are understood, as NPY release in the SCN has never been measured. Here, microdialysis procedures for in vivo measurement of NPY were used in complementary experiments to address these questions. First, neuronal release of NPY in the hamster SCN was rhythmic under a 14L : 10D photocycle, with the acrophase soon after lights‐on and the nadir at midday. No rhythmic fluctuation in NPY occurred under constant darkness. Second, a behavioral phase‐resetting stimulus (wheel‐running at midday that induces IGL serotonin release) acutely stimulated SCN NPY release. Third, bilateral IGL microinjection of the serotonin agonist, (±)‐2‐dipropyl‐amino‐8‐hydroxyl‐1,2,3,4‐tetrahydronapthalene (8‐OH‐DPAT) (another non‐photic phase‐resetting stimulant), at midday enhanced SCN NPY release. Conversely, similar application of the serotonin antagonist, metergoline, abolished wheel‐running‐induced SCN NPY release. IGL microinjection of the GABA agonist, muscimol, suppressed SCN NPY release. These results support an intra‐IGL mechanism whereby behavior‐induced serotonergic activity suppresses inhibitory GABAergic transmission, promoting NPY activity and subsequent phase resetting. Collectively, these results confirm IGL‐mediated NPY release in the SCN and verify that its daily rhythm of release is dependent upon the 14L : 10D photocycle, and that it is modulated by appropriately‐timed phase‐resetting behavior, probably mediated by serotonergic activation of NPY units in the IGL.

Acute ethanol impairs photic and nonphotic circadian phase resetting in the Syrian hamster

Disrupted circadian rhythmicity is associated with ethanol (EtOH) abuse, yet little is known about how EtOH affects the mammalian circadian clock of the suprachiasmatic nucleus (SCN). Clock timing is regulated by photic and nonphotic inputs to the SCN involving glutamate release from the retinohypothalamic tract and serotonin (5-HT) from the midbrain raphe, respectively. Our recent in vitro studies in the SCN slice revealed that EtOH blocks photic phase-resetting action of glutamate and enhances the nonphotic phase-resetting action of the 5-HT1A,7 agonist, 8-OH-DPAT. To explore the basis of these effects in the whole animal, we used microdialysis to characterize the pharmacokinetics of intraperitoneal injection of EtOH in the hamster SCN extracellular fluid compartment and then studied the effects of such EtOH treatment on photic and serotonergic phase resetting of the circadian locomotor activity rhythm. Peak EtOH levels (approximately 50 mM) from a 2 g/kg injection occurred within 20-40 min with a half-life of approximately 3 h. EtOH treatment dose-dependently attenuated photic phase advances but had no effect on phase delays and, contrary to in vitro findings, markedly attenuated 8-OH-DPAT-induced phase advances. In a complementary experiment using reverse microdialysis to deliver a timed SCN perfusion of EtOH during a phase-advancing light pulse, the phase advances were blocked, similar to systemic EtOH treatment. These results are evidence that acute EtOH significantly affects photic and nonphotic phase-resetting responses critical to circadian clock regulation. Notably, EtOH inhibition of photic signaling is manifest through direct action in the SCN. Such actions could underlie the disruption of circadian rhythmicity associated with alcohol abuse.

Short-term exposure to constant light promotes strong circadian phase-resetting responses to nonphotic stimuli in Syrian hamsters

Behavioral (nonphotic) stimuli can shift circadian rhythms by serotonin (5‐HT) and/or neuropeptide Y (NPY) inputs to the suprachiasmatic nucleus (SCN) circadian clock. Based on the idea that behavioral phase resetting is modulated by endogenous changes in postsynaptic sensitivity to such transmitters, hamsters were exposed to constant light (LL; ∼ 250 lx) for 1–3 days, which suppresses locomotor activity and eliminates the daily rhythm of SCN 5‐HT release measured by microdialysis. Groups subjected to brief LL or maintained under a light/dark cycle (LD) received phase‐resetting treatments with the 5‐HT1A,7 agonist (±)‐2‐dipropyl‐amino‐8‐hydroxyl‐1,2,3,4‐tetrahydronapthalene (8‐OH‐DPAT) or sleep deprivation (SD). Animals were released to constant darkness at the start of the treatments. Phase advances to 8‐OH‐DPAT and SD during the day were 11 and 3 h for LL vs. 2 and 1 h for LD, respectively. Phase delays during the night were −12 and −5 h for LL vs. no responses for LD, respectively. Phase‐transition curves for both LL treatments had slopes approximating 0, indicative of Type 0 phase resetting. For all treatments, the degree of locomotor suppression by LL was not correlated with the phase shift magnitude. Re‐establishing locomotor activity by overnight food deprivation did not prevent potentiated shifting to SD. However, re‐establishing peak extracellular 5‐HT levels by intra‐SCN 5‐HT reverse microdialysis perfusion in LL did significantly reduce potentiated 8‐OH‐DPAT phase advances. Constant light also enhanced intra‐SCN NPY‐induced phase advances during the day (6 vs. 2 h for LD). These results suggest that LL promotes Type 0 phase resetting by supersensitizing 5‐HT and/or NPY postsynaptic responses and possibly by attenuating the amplitude of the circadian pacemaker, thus enhancing circadian clock resetting nonspecifically.

Expression of immunoreactive polysialylated neural cell adhesion molecule in the suprachiasmatic nucleus

Light-microscopic and immunoblot immunochemical procedures were used to study the distribution and biochemical characteristics of neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) in the suprachiasmatic nuclei (SCN) of the adult Siberian hamster. In the adult brain PSA-NCAM is located in regions capable of undergoing morphological rearrangements and thus is generally considered to be an indicator of neural plasticity. Immunostaining for PSA in the Siberian hamster SCN (using a monoclonal antibody against the alpha 2,8-linked PSA of NCAM) was evident throughout the rostrocaudal axis of the SCN, with the most intense reaction in the ventrolateral region. Immunoreactivity was present in the neuropil, which delineated groups of cells with unstained cytoplasm. Many of the SCN cells were aggregated into cords or clusters. The optic chiasm was free from label, except for short processes apparently extending from the densely stained neuropil in the ventrolateral SCN. Immunoreactivity was abolished by preincubating sections in an endoneuraminidase (endo-N) or by preincubation of the primary antibody with PSA-NCAM. Immunostaining of the nonsialylated NCAM polypeptide was also limited to the neuropil, but this was more diffuse and less regionally specific than PSA staining. The ventral SCN exhibited the most intense labeling for NCAM. Immunoblot analyses revealed the immunoreactive PSA-NCAM as a broad band migrating between apparent molecular weights in the range of 150-300 kD, which was ablated by treatment of SCN tissue extract with endo-N. This electrophoretic migration pattern of PSA-NCAM from the SCN region was similar to that seen in several other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)