Suprachiasmatic Nucleus Brain Slices Research Articles

Functional Peptidomics: Stimulus- and Time-of-Day-Specific Peptide Release in the Mammalian Circadian Clock.

Daily oscillations of brain and body states are under complex temporal modulation by environmental light and the hypothalamic suprachiasmatic nucleus (SCN), the master circadian clock. To better understand mediators of differential temporal modulation, we characterize neuropeptide releasate profiles by nonselective capture of secreted neuropeptides in an optic nerve horizontal SCN brain slice model. Releasates are collected following electrophysiological stimulation of the optic nerve/retinohypothalamic tract under conditions that alter the phase of the SCN activity state. Secreted neuropeptides are identified by intact mass via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found time-of-day-specific suites of peptides released downstream of optic nerve stimulation. Peptide release was modified differentially with respect to time-of-day by stimulus parameters and by inhibitors of glutamatergic or PACAPergic neurotransmission. The results suggest that SCN physiology is modulated by differential peptide release of both known and unexpected peptides that communicate time-of-day-specific photic signals via previously unreported neuropeptide signatures.

Delayed Cryptochrome Degradation Asymmetrically Alters the Daily Rhythm in Suprachiasmatic Clock Neuron Excitability.

Suprachiasmatic nuclei (SCN) neurons contain an intracellular molecular circadian clock and the Cryptochromes (CRY1/2), key transcriptional repressors of this molecular apparatus, are subject to post-translational modification through ubiquitination and targeting for proteosomal degradation by the ubiquitin E3 ligase complex. Loss-of-function point mutations in a component of this ligase complex, Fbxl3, delay CRY1/2 degradation, reduce circadian rhythm strength, and lengthen the circadian period by ∼2.5 h. The molecular clock drives circadian changes in the membrane properties of SCN neurons, but it is unclear how alterations in CRY1/2 stability affect SCN neurophysiology. Here we use male and female Afterhours mice which carry the circadian period lengthening loss-of-function Fbxl3Afh mutation and perform patch-clamp recordings from SCN brain slices across the projected day/night cycle. We find that the daily rhythm in membrane excitability in the ventral SCN (vSCN) was enhanced in amplitude and delayed in timing in Fbxl3Afh/Afh mice. At night, vSCN cells from Fbxl3Afh/Afh mice were more hyperpolarized, receiving more GABAergic input than their Fbxl3+/+ counterparts. Unexpectedly, the progression to daytime hyperexcited states was slowed by Afh mutation, whereas the decline to hypoexcited states was accelerated. In long-term bioluminescence recordings, GABAA receptor blockade desynchronized the Fbxl3+/+ but not the Fbxl3Afh/Afh vSCN neuronal network. Further, a neurochemical mimic of the light input pathway evoked larger shifts in molecular clock rhythms in Fbxl3Afh/Afh compared with Fbxl3+/+ SCN slices. These results reveal unanticipated consequences of delaying CRY degradation, indicating that the Afh mutation prolongs nighttime hyperpolarized states of vSCN cells through increased GABAergic synaptic transmission.SIGNIFICANCE STATEMENT The intracellular molecular clock drives changes in SCN neuronal excitability, but it is unclear how mutations affecting post-translational modification of molecular clock proteins influence the temporal expression of SCN neuronal state or intercellular communication within the SCN network. Here we show for the first time, that a mutation that prolongs the stability of key components of the intracellular clock, the cryptochrome proteins, unexpectedly increases in the expression of hypoexcited neuronal state in the ventral SCN at night and enhances hyperpolarization of ventral SCN neurons at this time. This is accompanied by increased GABAergic signaling and by enhanced responsiveness to a neurochemical mimic of the light input pathway to the SCN. Therefore, post-translational modification shapes SCN neuronal state and network properties.

Urokinase-type plasminogen activator modulates mammalian circadian clock phase regulation in tissue-type plasminogen activator knockout mice.

Glutamate phase shifts the circadian clock in the mammalian suprachiasmatic nucleus (SCN) by activating NMDA receptors. Tissue-type plasminogen activator (tPA) gates phase shifts by activating plasmin to generate m(ature) BDNF, which binds TrkB receptors allowing clock phase shifts. Here, we investigate phase shifting in tPA knockout (tPA-/- ; B6.129S2-Plattm1Mlg /J) mice, and identify urokinase-type plasminogen activator (uPA) as an additional circadian clock regulator. Behavioral activity rhythms in tPA-/- mice entrain to a light-dark (LD) cycle and phase shift in response to nocturnal light pulses with no apparent loss in sensitivity. When the LD cycle is inverted, tPA-/- mice take significantly longer to entrain than C57BL/6J wild-type (WT) mice. SCN brain slices from tPA-/- mice exhibit entrained neuronal activity rhythms and phase shift in response to nocturnal glutamate with no change in dose-dependency. Pre-treating slices with the tPA/uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1), inhibits glutamate-induced phase delays in tPA-/- slices. Selective inhibition of uPA with UK122 prevents glutamate-induced phase resetting in tPA-/- but not WT SCN slices. tPA expression is higher at night than the day in WT SCN, while uPA expression remains constant in WT and tPA-/- slices. Casein-plasminogen zymography reveals that neither tPA nor uPA total proteolytic activity is under circadian control in WT or tPA-/- SCN. Finally, tPA-/- SCN tissue has lower mBDNF levels than WT tissue, while UK122 does not affect mBDNF levels in either strain. Together, these results suggest that either tPA or uPA can support photic/glutamatergic phase shifts of the SCN circadian clock, possibly acting through distinct mechanisms.

Copper chelation and exogenous copper affect circadian clock phase resetting in the suprachiasmatic nucleus in vitro

Light stimulates specialized retinal ganglion cells to release glutamate (Glu) onto circadian clock neurons of the suprachiasmatic nucleus (SCN). Glu resets the phase of the SCN circadian clock by activating N-methyl-d-aspartate receptors (NMDAR) causing either delays or advances in the clock phase, depending on early- or late-night stimulation, respectively. In addition, these Glu-induced phase shifts require tropomyosin receptor kinase B (TrkB) receptor activity. Previous studies show that copper (Cu) released at hippocampal synapses can inhibit NMDAR activity, and application of exogenous Cu likewise inhibits NMDAR activity. We investigated the effects of Cu in acute SCN brain slices prepared from C57BL/6Nhsd adult, male mice using treatments that decrease or increase available Cu levels in vitro and recorded neuronal activity on the following day. When bath-applied for 10min at zeitgeber time (ZT) 16 (where ZT0=lights-on in the donor animal colony), the Cu-specific chelators tetrathiomolybdate (TTM) and bathocuproine disulfonate each induce ∼2.5–3-h phase delays in circadian neuronal activity rhythms, similarly to Glu-induced phase delays. Co-application of 10μM CuCl2, but not 10μM CoCl2 blocks TTM-induced phase delays. Furthermore, TTM causes phase advances when applied at ZT23. At both application times, TTM-induced phase shifts are blocked by NMDA or TrkB receptor antagonists. Surprisingly, bath-application of 10μM Cu alone also induces phase shifts in analogous experiments at ZT16 and ZT23. Inhibiting NMDAR does not block Cu-induced phase shifts. TrkB inhibition blocks Cu-induced phase delays but not phase advances. Thus, increasing and decreasing Cu availability appear to shift the SCN clock phase through different mechanisms, at least at the receptor level. We propose that Cu plays a role in the SCN circadian clock by modulating Glu signaling.

Neuropeptide Y–Induced Phase Shifts of PER2::LUC Rhythms Are Mediated by Long-Term Suppression of Neuronal Excitability in a Phase-Specific Manner

Endogenous circadian rhythms are entrained to the 24-h light/dark cycle by both light and nonphotic stimuli. During the day, nonphotic stimuli, such as novel wheel-induced exercise, produce large phase advances. Neuropeptide Y (NPY) release from the thalamus onto suprachiasmatic nucleus (SCN) neurons at least partially mediates this nonphotic signal. The authors examined the hypothesis that NPY-induced phase advances are accompanied by suppression of PER2 and are mediated by long-term depression of neuronal excitability in a phase-specific manner. First, it was found that NPY-induced phase advances in PER2::LUC SCN cultures are largest when NPY (2.35 µM) is given in the early part of the day (circadian time [CT] 0–6). In addition, PER2::LUC levels in NPY-treated (compared to vehicle-treated) samples were suppressed beginning 6–7 h after treatment. Similar NPY application to organotypic Per1::GFP SCN cultures resulted in long-term suppression of spike rate of green fluorescent protein–positive (GFP+) cells when slices were treated with NPY during the early or middle of the day (zeitgeber time [ZT] 2 or 6), but not during the late day (ZT 10). Furthermore, 1-h bath application of NPY to acute SCN brain slices decreased general neuronal activity measured through extracellular recordings. Finally, NPY-induced phase advances of PER2::LUC rhythms were blocked by latent depolarization with 34.5 mM K+ 3 h after NPY application. These results suggest that NPY-induced phase advances may be mediated by long-term depression of neuronal excitability. This model is consistent with findings in other brain regions that NPY-induced persistent hyperpolarization underlies mechanisms of energy homeostasis, anxiety-related behavior, and thalamocortical synchronous firing. (Author correspondence: klgamble@uab.edu)

Circadian Periods of Sensitivity for Ramelteon on the onset of Running-wheel Activity and the Peak of Suprachiasmatic Nucleus Neuronal Firing Rhythms in C3H/HeN Mice

Ramelteon, an MT1/MT2 melatonin receptor agonist, is used for the treatment of sleep-onset insomnia and circadian sleep disorders. Ramelteon phase shifts circadian rhythms in rodents and humans when given at the end of the subjective day; however, its efficacy at other circadian times is not known. Here, the authors determined in C3H/HeN mice the maximal circadian sensitivity for ramelteon in vivo on the onset of circadian running-wheel activity rhythms, and in vitro on the peak of circadian rhythm of neuronal firing in suprachiasmatic nucleus (SCN) brain slices. The phase response curve (PRC) for ramelteon (90 µg/mouse, subcutaneous [sc]) on circadian wheel-activity rhythms shows maximal sensitivity during the late mid to end of the subjective day, between CT8 and CT12 (phase advance), and late subjective night and early subjective day, between CT20 and CT2 (phase delay), using a 3-day-pulse treatment regimen in C3H/HeN mice. The PRC for ramelteon resembles that for melatonin in C3H/HeN mice, showing the same magnitude of maximal shifts at CT10 and CT2, except that the range of sensitivity for ramelteon (CT8–CT12) during the subjective day is broader. Furthermore, in SCN brain slices in vitro, ramelteon (10 pM) administered at CT10 phase advances (5.6 ± 0.29 h, n = 3) and at CT2 phase delays (−3.2 ± 0.12 h, n = 6) the peak of circadian rhythm of neuronal firing, with the shifts being significantly larger than those induced by melatonin (10 pM) at the same circadian times (CT10: 2.7 ± 0.15 h, n = 4, p < .05; CT2: −1.13 ± 0.08 h, n = 6, p < .001, respectively). The phase shifts induced by both melatonin and ramelteon in the SCN brain slice at either CT10 or CT2 corresponded with the period of sensitivity observed in vivo. In conclusion, melatonin and ramelteon showed identical periods of circadian sensitivity at CT10 (advance) and CT2 (delay) to shift the onset of circadian activity rhythms in vivo and the peak of SCN neuronal firing rhythms in vitro. (Author correspondence: mdubo@buffalo.edu)

Ramelteon Phase Advanced Circadian Rhythms of Neuronal Firing in The Suprachiasmatic Nucleus (SCN) Brain Slice by Activation of Both MT 1 and MT 2 Melatonin (MLT) Receptors

Ramelteon, a high affinity MT1 (Ki: 14 pM) and MT2 (Ki: 104 pM) MLT receptor agonist promotes sleep and phase shifts circadian rhythms (Curr Opinion Inv Drugs 1:–121, 2005). This study determined the MLT receptor type(s) involved in ramelteon-mediated phase shifts of spontaneous circadian rhythms of neuronal firing in SCN brain slices from C3H/HeN mice using single-unit recordings. SCN brain slices from wild type (WT) mice showed a peak of neuronal firing at circadian time (CT) 6.1 ± 0.1 h (n=5) [CT12: activity onset for host mice], which was advanced by ramelteon (10 pM, 10 min) application on day 1 at CT 10 to CT 1.2 ± 0.7 h (n=4, p < 0.0001) on day 2. Ramelteon significantly advanced the peak of neuronal firing in SCN from WT (4.9 ± 0.7 h, n=4), MT1KO (3.4 ± 0.15 h, n=5) and MT2KO (1.6 ± 0.4 h, n=3), but not from MT1MT2KO mice. Ramelteon-mediated phase advance in SCN brain slices from WT mice was MLT receptor specific and resulted from activation of MT2 receptors with a smaller contribution from MT1 receptors. MLT (10 pM), however, induced phase advances of identical magnitude via activation of MT2 receptors in SCN from WT (2.7 ± 0.15h, n=4) and MT1 KO (3 ± 0.1h, n=4) mice, but did not affect phase in SCN from MT2KO or MT1MT2KO mice (Hudson et al., Neuropshychopharmacol 30:, 2005). We conclude that ramelteon uncovered a functional MT1 melatonin receptor in the SCN also involved in phase shifting circadian rhythms. Supported by a Takeda Investigator-Sponsored Research Grant 06-016R.

Disrupted Neuronal Activity Rhythms in the Suprachiasmatic Nuclei of Vasoactive Intestinal Polypeptide-Deficient Mice

Vasoactive intestinal polypeptide (VIP), acting via the VPAC(2) receptor, is a key signaling pathway in the suprachiasmatic nuclei (SCN), the master clock controlling daily rhythms in mammals. Most mice lacking functional VPAC(2) receptors are unable to sustain behavioral rhythms and lack detectable SCN electrical rhythms in vitro. Adult mice that do not produce VIP (VIP/PHI(-/-)) exhibit less severe alterations in wheel-running rhythms, but the effects of this deficiency on the amplitude, phasing, or periodicity of their SCN cellular rhythms are unknown. To investigate this, we used suction electrodes to extracellularly record multiple- and single-unit electrical activity in SCN brain slices from mice with varying degrees of VIP deficiency, ranging from wild-type (VIP/PHI(+/+)) to heterozygous (VIP/PHI(+/-)) and VIP/PHI(-/-) animals. We found decreasing proportions of rhythmic cells in SCN slices from VIP/PHI(+/+) ( approximately 91%, n = 23) through VIP/PHI(-/+) ( approximately 71%, n = 28) to VIP/PHI(-/-) mice (62%; n = 37) and a parallel trend toward decreasing amplitude in the remaining rhythmic cells. SCN neurons from VIP/PHI(-/-) mice exhibited a broad range in the period and phasing of electrical rhythms, concordant with the known alterations in their behavioral rhythms. Further, treatment of VIP/PHI(-/-) slices with a VPAC(2) receptor antagonist significantly reduced the proportion of oscillating neurons, suggesting that VPAC(2) receptors still become activated in the SCN of these mice. The results establish that VIP is important for appropriate periodicity and phasing of SCN neuronal rhythms and suggest that residual VPAC(2) receptor signaling promotes rhythmicity in adult VIP/PHI(-/-) mice.

Brain‐derived neurotrophic factor and neurotrophin receptors modulate glutamate‐induced phase shifts of the suprachiasmatic nucleus

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells. Previous work raised the possibility that brain-derived neurotrophic factor (BDNF) and its high-affinity tropomyosin-related receptor kinase may be important as modulators of this excitatory input into the SCN. In order to test this possibility, we used whole-cell patch-clamp methods to measure spontaneous excitatory currents in mouse SCN neurons. We found that the amplitude and frequency of these currents were increased by BDNF and decreased by the neurotrophin receptor inhibitor K252a. The neurotrophin also increased the magnitude of currents evoked by application of N-methyl-d-aspartate and amino-methyl proprionic acid. Next, we measured the rhythms in action potential discharge from the SCN brain slice preparation. We found that application of K252a dramatically reduced the magnitude of phase shifts of the electrical activity rhythm generated by the application of glutamate. By itself, BDNF caused phase shifts that resembled those produced by glutamate and were blocked by K252a. The results demonstrate that BDNF and neurotrophin receptors can enhance glutamatergic synaptic transmission within a subset of SCN neurons and potentiate glutamate-induced phase shifts of the circadian rhythm of neural activity in the SCN.

Serotonergic pre-treatments block in vitro serotonergic phase shifts of the mouse suprachiasmatic nucleus circadian clock

The suprachiasmatic nucleus (SCN) contains a circadian clock that maintains its time-generating and phase-modulating capacities in vitro. Previous studies report clear differences in the ability of serotonergic stimuli to phase-shift the SCN clock when applied directly to the SCN either in vivo or in vitro: while mice and rat circadian clocks are readily phase-shifted by serotonin (5-HT) or 5-HT agonists applied in vitro, hamster and mice circadian clocks respond inconsistently to 5-HT agonists injected directly into the SCN in vivo. Here we have investigated one possible explanation for these differences: that the SCN isolated in vitro experiences reduced endogenous 5-HT signaling, which increases clock sensitivity to subsequent 5-HT stimulation. For these experiments we treated mouse SCN brain slices with low concentrations of compounds that increase serotonin signaling: 5-HT, a 5-HT agonist (8-OH-DPAT), the 5-HT precursor, l-tryptophan, or the 5-HT re-uptake inhibitor, fluoxetine. Pretreatment with each of these substances completely blocked subsequent phase-shifts induced by mid-subjective day treatment with either 5-HT or 8-OH-DPAT, while they did not block phase-shifts induced by the adenylate cyclase activator, forskolin. Time-course data on l-tryptophan-induced inhibition are consistent with this treatment inducing receptor internalization, while timing of the recovery from inhibition is consistent with receptor reinsertion. Together these data support the hypothesis that SCN clock sensitivity to serotonergic phase modulation is affected by the amount of prior serotonin signaling present in the SCN, and that this signaling alters the density of surface 5-HT receptors on SCN clock neurons.

Effect of MT1 melatonin receptor deletion on melatonin‐mediated phase shift of circadian rhythms in the C57BL/6 mouse

In the mouse suprachiasmatic nucleus (SCN), melatonin activates MT1 and MT2 G-protein coupled receptors, which are involved primarily in inhibition of neuronal firing and phase shift of circadian rhythms. This study investigated the ability of melatonin to phase shift circadian rhythms in wild type (WT) and MT1 melatonin receptor knockout (KO) C57BL/6 mice. In WT mice, melatonin (90 microg/mouse, s.c.) administered at circadian time 10 (CT10; CT12 onset of activity) significantly phase advanced the onset of the circadian activity rhythm (0.60 +/- 0.09 hr, n = 41) when compared with vehicle treated controls (-0.02 +/- 0.07 hr, n = 28) (P < 0.001). In contrast, C57 MT1KO mice treated with melatonin did not phase shift circadian activity rhythms (-0.10 +/- 0.12 hr, n = 42) when compared with vehicle treated mice (-0.12 +/- 0.07 hr, n = 43). Similarly, in the C57 MT1KO mouse melatonin did not accelerate re-entrainment to a new dark onset after an abrupt advance of the dark cycle. In contrast, melatonin (3 and 10 pm) significantly phase advanced circadian rhythm of neuronal firing in SCN brain slices independent of genotype with an identical maximal shift at 10 pm (C57 WT: 3.61 +/- 0.38 hr, n = 3; C57 MT(1)KO: 3.45 +/- 0.11 hr, n = 4). Taken together, these results suggest that melatonin-mediated phase advances of circadian rhythms of neuronal firing in the SCN in vitro may involve activation of the MT2 receptor while in vivo activation of the MT1 and possibly the MT2 receptor may be necessary for the expression of melatonin-mediated phase shifts of overt circadian activity rhythms.

Melatonin desensitizes endogenous MT2 melatonin receptors in the rat suprachiasmatic nucleus: relevance for defining the periods of sensitivity of the mammalian circadian clock to melatonin

The hormone melatonin phase shifts circadian rhythms generated by the mammalian biological clock, the suprachiasmatic nucleus (SCN) of the hypothalamus, through activation of G protein-coupled MT2 melatonin receptors. This study demonstrated that pretreatment with physiological concentrations of melatonin (30-300 pM or 7-70 pg/mL) decreased the number of hMT2 melatonin receptors heterologously expressed in mammalian cells in a time and concentration-dependent manner. Furthermore, hMT2-GFP melatonin receptors heterologously expressed in immortalized SCN2.2 cells or in non-neuronal mammalian cells were internalized upon pretreatment with both physiological (300 pM or 70 pg/mL) and supraphysiological (10 nM or 2.3 ng/mL) concentrations of melatonin. The decrease in MT2 melatonin receptor number induced by melatonin (300 pM for 1 h) was reversible and reached almost full recovery after 8 h; however, after treatment with 10 nM melatonin full recovery was not attained even after 24 h. This recovery process was partially protein synthesis dependent. Furthermore, exposure to physiological concentrations of melatonin (300 pM) for a time mimicking the nocturnal surge (8 h) desensitized functional responses mediated through melatonin activation of endogenous MT2 receptors, i.e., stimulation of protein kinase C (PKC) in immortalized SCN2.2 cells and phase shifts of circadian rhythms of neuronal firing in the rat SCN brain slice. We conclude that in vivo the nightly secretion of melatonin desensitizes endogenous MT2 melatonin receptors in the mammalian SCN thereby providing a temporally integrated profile of sensitivity of the mammalian biological clock to a melatonin signal.

Serotonin phase-shifts the mouse suprachiasmatic circadian clock in vitro

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) receives multiple afferent signals that could potentially modulate its phase. One input, the serotonin (5-HT) projection from the raphe nuclei, has been extensively investigated in rats and hamsters, yet its role(s) in modulating circadian clock phase remains controversial. To expand our investigation of 5-HT modulation of the SCN clock, we investigated the phase-shifting effects of 5-HT and its agonist, (+)8-hydroxy-2-(di- n-propylamino)tetralin (DPAT), when applied to mouse SCN brain slices. 5-HT induced 2–3 h phase advances when applied during subjective day, while non-significant phase shifts were seen after 5-HT application at other times. These phase shifts were completely blocked by the 5-HT antagonist, metergoline. DPAT also induced phase shifts when applied during mid-subjective day, and this effect appeared dose-dependent. Together, these results demonstrate that the mouse SCN, like that of the rat, is directly sensitive to in vitro phase-resetting by 5-HT.

Glutamate Blocks Serotonergic Phase Advances of the Mammalian Circadian Pacemaker through AMPA and NMDA Receptors

The phase of the mammalian circadian pacemaker, located in the suprachiasmatic nucleus (SCN), is modulated by a variety of stimuli, most notably the environmental light cycle. Light information is perceived by the circadian pacemaker through glutamate that is released from retinal ganglion cell terminals in the SCN. Other prominent modulatory inputs to the SCN include a serotonergic projection from the raphe nuclei and a neuropeptide Y (NPY) input from the intergeniculate leaflet. Light and glutamate phase-shift the SCN pacemaker at night, whereas serotonin (5-HT) and NPY primarily phase-shift the pacemaker during the day. In addition to directly phase-shifting the circadian pacemaker, SCN inputs have been shown to modulate the actions of one another. For example, 5-HT can inhibit the phase-shifting effects of light or glutamate applied to the SCN at night, and NPY and glutamate inhibit phase shifts of one another. In this study, we explored the possibility that glutamate can modulate serotonergic phase shifts during the day. For these experiments, we applied various combinations of 5-HT agonists, glutamate agonists, and electrical stimulation of the optic chiasm to SCN brain slices to determine the effect of these treatments on the rhythm of spontaneous neuronal activity generated by the SCN circadian pacemaker. We found that glutamate agonists and optic chiasm stimulation inhibit serotonergic phase advances and that this inhibition involves both AMPA and NMDA receptors. This inhibition by glutamate may be indirect, because it is blocked by both tetrodotoxin and the GABA(A) antagonist, bicuculline.

Neuropeptide Y Activates Protein Kinase C in Hamster Suprachiasmatic Nuclei Brain Slices

The mammalian circadian clock in the suprachiasmatic nuclei (SCN) can be phase-shifted by neuropeptide Y applied in the subjective day. Previous studies suggested that neuropeptide Y might act through a protein kinase C (PKC)-dependent mechanism. We directly measured PKC activity in suprachiasmatic nuclei brain slices following application of neuropeptide Y. PKC activity increased 5 min after neuropeptide Y application, with a return to baseline levels 15 min after application. An initial small decrease in PKC activity 1 min after neuropeptide Y application was also observed after control applications of artificial cerebrospinal fluid. Our results support the hypothesis that phase shifts induced by neuropeptide Y involve activation of PKC.

Do the suprachiasmatic nuclei oscillate in old rats as they do in young ones?

The basis of the decline in circadian rhythms with aging was addressed by comparing the patterns of three behavioral rhythms in young and old rats with the in vitro rhythm of neuronal activity in the suprachiasmatic nuclei (SCN), the primary circadian pacemaker. In some old rats, rhythms of body temperature, drinking, and activity retained significant 24-h periodicities in entraining light-dark cycles; in others, one or two of the rhythms became aperiodic. When these rats were 23-27.5 mo old they were killed, and single-unit firing rates in SCN brain slices were recorded continuously for 30 h. There was significant damping of mean peak neuronal firing rates in old rats compared with young. SCN neuronal activities were analyzed with reference to previous entrained behavioral rhythm patterns of individual rats as well. Neuronal activity from rats with prior aperiodic behavioral rhythms was erratic, as expected. Neuronal activity from rats that were still maintaining significant 24-h behavioral rhythmicity at the time they were killed was erratic in most cases but normally rhythmic in others. Thus there was no more congruence between the behavioral rhythms and the brain slice rhythms than there was among the behavioral rhythms alone. These results, the first to demonstrate aberrant SCN firing patterns and a decrease in amplitude in old rats, imply that aging could either disrupt coupling between SCN pacemaker cells or their output, or cause deterioration of the pacemaking properties of SCN cells.

The organization of the suprachiasmatic circadian pacemaker of the rat and its regulation by neurotransmitters and modulators.

The long-term goal of our research is to understand how cells of the suprachiasmatic nucleus (SCN) are organized to form a 24-hr biological clock, and what roles specific neurotransmitters and modulators play in timekeeping and resetting processes. We have been addressing these questions by assessing the pattern of spontaneous neuronal activity, using extracellular and whole-cell patch recording techniques in long-lived SCN brain slices from rats. We have observed that a robust pacemaker persists in the ventrolateral region of microdissected SCN, and have begun to define the electrophysiological properties of neurons in this region. Furthermore, we are investigating changing sensitivities of the SCN to resetting by exogenous neurotransmitters, such as glutamate, serotonin, and neuropeptide Y, across the circadian cycle. Our findings emphasize the complexity of organization and control of mammalian circadian timing.

Analysis of in vitro glucose utilization in a circadian pacemaker model

An in vitro glucose utilization method, based upon 14C-2-deoxyglucose kinetics in brain slices, has been used to study circadian rhythms in hypothalamic slices containing the suprachiasmatic nucleus (SCN). Spontaneous SCN metabolic activity in vitro is similar to that observed in vivo with higher metabolic rates in subjective daytime and lower rates during subjective night. However, in vitro SCN metabolic activity during late subjective day is above that seen when glucose utilization is measured in vivo, suggesting that an inhibitory influence normally active in vivo is lost during slice isolation. Incubation of slices containing SCN in the presence of TTX exposes a TTX-insensitive component of metabolic activity in early subjective day, supporting prior suggestions that glucose utilization by the circadian oscillator continues in the absence of Na(+)-dependent action potentials. Studies with high Mg2+ concentrations are consistent with the hypothesis that most metabolic activity above the basal level observed with the glucose utilization method is related to synaptic activity. Pharmacological studies of the SCN brain slice model with radiotracers offer potential for analysis of both circadian rhythmicity and neural regulation.