Angiotensin II regulates the activity of mouse suprachiasmatic nuclei neurons

Neuroscience 154 (2008) 839 – 847

ANGIOTENSIN II REGULATES THE ACTIVITY OF MOUSE SUPRACHIASMATIC NUCLEI NEURONS T. M. BROWN, E. McLACHLAN AND H. D. PIGGINS*

reviews). Circadian rhythms are generated in individual SCN neurons via positive and negative feedback/forward loops involving transcription and translation of so-called clock genes (Hastings, 2000; Reppert and Weaver, 2002; Ko and Takahashi, 2006). Neurochemical and electrical signaling between SCN neurons is necessary for these individual cellular clocks to coordinate their activities and maintain robust oscillations (see Kuhlman and McMahon, 2006; Morin and Allen, 2006; Brown and Piggins, 2007 for reviews). SCN neurons synthesize a number of different neuropeptides including arginine vasopressin, vasoactive intestinal polypeptide, gastrin-releasing peptide, and angiotensin II (ANGII) (Abrahamson and Moore, 2001; Piggins et al., 2002; Antle and Silver, 2005; Morin and Allen, 2006). Of these, vasoactive intestinal polypeptide, gastrin-releasing peptide, and arginine vasopressin have been extensively investigated for their functions in the SCN (Piggins and Cutler, 2003; Vosko et al., 2007; Ingram et al., 1998). By contrast, there are few functional investigations of ANGII in circadian timekeeping, and very little is known about the neurophysiological actions of this neuropeptide in the SCN. ANGII acts at two different receptor subtypes; AT1 (of which there are two isoforms AT1A and AT1B) and AT2 (Murphy et al., 1991; Iwai and Inagami, 1992; Kambayashi et al., 1993). The mouse SCN contain only the AT1 subtype (Johren et al., 1997). The AT1 receptor couples to multiple G proteins, initiating diverse signal transduction pathways including activation of extracellular regulated kinases 1 and 2 (Higuchi et al., 2007) and modulating various ion-channels, depolarizing neurons (Cato and Toney, 2005; Latchford and Ferguson, 2005). In the periphery, AT1 receptor activation by ANGII increases circadian amplitude of expression of the clock gene Per2 (Nonaka et al., 2001), suggesting that this neuropeptide can regulate the molecular clock. The current study used extracellular and whole-cell voltage and current clamp recordings in mouse brain slices to determine the effect of ANG II on SCN neuronal activity. Since GABA is contained in most, if not all, SCN peptidergic neurons (Moore and Speh, 1993), and virtually all cells in these nuclei receive GABAA receptor-mediated synaptic input (Kim and Dudek, 1992; Itri and Colwell, 2003; Itri et al., 2004) we also assessed if GABAergic signaling is recruited by ANGII in the SCN. We report that ANGII dose-dependently regulates SCN neuronal activity by directly depolarizing cells and increasing action potential discharge. This action of ANGII leads to increased GABA

Faculty of Life Sciences, 1.124 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK

Abstract—Neuropeptide signaling plays key roles in coordinating cellular activity within the suprachiasmatic nuclei (SCN), site of the master circadian oscillator in mammals. The neuropeptide angiotensin II (ANGII) and its cognate receptor AT1, are both expressed by SCN cells, but unlike other SCN neurochemicals, very little is known about the cellular actions of ANGII within this circadian clock. We used multi-electrode, multiunit, extracellular electrophysiology, coupled with whole-cell voltage and current clamp techniques to investigate the actions of ANGII in mouse SCN slices. ANGII (0.001–10 ␮M) dose dependently stimulated and inhibited extracellularly recorded neuronal discharge in many SCN neurons (⬃60%). Both actions were blocked by pre-treatment with the AT1 receptor antagonist ZD7155 (0.03 ␮M), while suppressions but not activations were prevented by pre-treatment with the GABAA receptor antagonist bicuculline (20 ␮M). AT1 receptor blockade itself suppressed discharge in a subset (⬃30%) of SCN neurons, and this action was not blocked by bicuculline. In voltage-clamped SCN neurons (ⴚ70 mV), AT1 receptor activation dose-dependently enhanced the frequency of action potential-driven, GABAA receptor-mediated currents, but did not alter their responses to exogenously applied GABA. In current-clamped SCN neurons perfused with tetrodotoxin, ANGII induced a membrane depolarization with a concomitant decrease in input resistance. In conclusion we show that AT1 receptor activation by ANGII depolarizes SCN neurons and stimulates action potential firing, leading to increased GABA release in the mouse SCN. Additionally we provide the first evidence that endogenous AT1 receptor signaling tonically regulates the activities of some SCN neurons. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: circadian, GABA, hypothalamus, neuropeptide, patch-clamp, extracellular.

The suprachiasmatic nuclei (SCN) of the hypothalamus are the main center for the generation and regulation of circadian rhythms in mammals (see Rusak and Zucker, 1979; Hastings et al., 2003; Guilding and Piggins, 2007 for *Corresponding author. Tel: ⫹44-0-161-275-3897; fax: ⫹44-0-161275-3938. E-mail address: [email protected] (H. D. Piggins). Abbreviations: aCSF, artificial cerebrospinal fluid; ANGII, angiotensin II; CED, Cambridge Electronic Design; EGTA, ethyleneglycol-bis-(␤aminoethyl ether) N,N,N=,N=-tetraacetic acid; Hepes, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid; IPSC, inhibitory postsynaptic current; MUA, multiunit activity; NMDA, N-methyl-D-aspartate; SCN, suprachiasmatic nuclei; TTX, tetrodotoxin; ZT, Zeitgeber time.

0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.03.068

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release within the SCN, partly counterbalancing the stimulatory effects of this peptide.

EXPERIMENTAL PROCEDURES Animals Adult male C57Bl6/j mice (8 –12 weeks old; initially purchased at 5– 6 weeks of age from Harlan Olac, Bicester, UK) were housed under a 12-h light/dark cycle (lights on 8.00 am– 8:00 pm), at an ambient temperature of 22⫾1 °C. A subset of animals was maintained under identical conditions except for a shifted light dark cycle (lights-on 10 pm–10 am) to facilitate projected night experiments. Food and water were available ad libitum. Zeitgeber time (ZT) 0 was defined as lights-on and ZT12 as lights-off. Animals were maintained under these conditions for ⬎2 weeks prior to experimental protocols. All scientific procedures were carried out in accordance with the UK Animal (Scientific Procedures) Act 1986 and conformed to international guidelines on the ethical use of animals. Every effort was taken to minimize the number of animals used and their suffering.

Slice preparation Slices were prepared during the early (ZT 1–2) or late (ZT 10 –12) subjective day and maintained using methods similar to those described earlier (Brown et al., 2005). Mice were culled by cervical dislocation and decapitation, the brain was removed and placed in 4 °C artificial cerebrospinal fluid (aCSF: pH 7.4) of composition (in mM): NaCl 124, KCl 2.2, KH2PO4 1.2, CaCl2 2.5, MgSO4 1.0, NaHCO3 25.5, D-glucose 10, ascorbic acid 1.14. Coronal brain sections (350 ␮m thick) were cut using a vibroslicer (Campden Instruments, Leicester, UK) and transferred to the recording chamber and equilibrated for ⬃1 h before the start of electrophysiological experiments. Throughout the dissection procedure aCSF was bubbled with 95% O2/5% CO2.

Extracellular recordings Slices were maintained at 34⫾1 °C in an submerged recording chamber (PDMI-2; Harvard Apparatus, Eddenbridge, UK), continuously perfused with oxygenated aCSF at ⬃1.5 ml/min. Extracellular multiunit activity (MUA) was recorded from dorsal and ventral portions of the SCN via two aCSF-filled suction electrodes constructed as previously described (Brown et al., 2006). The SCN multiunit signal was differentially amplified (⫻20,000) and bandpass filtered (300 –3000 Hz) via a Neurolog system (Digitimer, Welwyn Garden City, UK), digitized (25,000 Hz) using a micro 1401 mkII interface (Cambridge Electronic Design (CED), Cambridge, UK) and recorded on a PC running Spike2 version 6 software (CED). Using Spike2 software, single unit activity was discriminated from these MUA recordings offline as previously described (Brown et al., 2005, 2006). Briefly, single units were discriminated on the basis of waveform shape, principal components-based clustering, and the presence of a clear refractory period in an interspike interval histogram. Using these criteria we were able to successfully isolate up to eight single units from each dual-electrode recording. Since the responses of SCN neurons to test compounds did not vary according to the anatomical subregion in which the cell was located, data for all cells tested were pooled. Slices received three to six different drug treatments of 5 min duration over a ⬃6 h recording session with at least 40 min between each treatment. In some experiments slices were continuously perfused with 20 ␮M bicuculline or 0.03 ␮M ZD7155 while test compounds were applied.

Patch clamp recordings Slices were maintained at room temperature (⬃25 °C) in an submerged recording chamber (ALA Scientific Instruments; NY, USA) fixed to the stage of an upright microscope (BX51WI; Olympus, Southall, UK), continuously perfused with oxygenated aCSF at ⬃2 ml/min. Electrodes (resistance 4 – 8 M⍀) were pulled from borosilicate glass (1.5 mm OD) on a horizontal puller (P-97; Sutter Instruments, CA, USA) and filled with an internal solution containing (mM): K-gluconate 120, KCl 20, MgCl2 2, K2ATP 2, NaGTP 0.5, Hepes 20, EGTA 0.5 (pH: 7.3 osmolality: 300 mOsm). Whole cell voltage-clamp recordings were obtained via established protocols (Itri and Colwell, 2003; Itri et al., 2004) using an Axon Instruments 200B amplifier filtering at 1 kHZ, digitized (10 kHZ) via Digidata 1440a and monitored on-line with pCLAMP Version 10.0 (Molecular Devices, CA, USA). Cells were approached with slight positive pressure, pipette offset was corrected, then the pipette was lowered onto the cell membrane. A high-resistance seal was formed (2–10 G⍀) by applying negative pressure and a second pulse of negative pressure was used to rupture the membrane. Spontaneous currents were recorded from cells at holding potential of ⫺70 mV. The series and input resistances were monitored at the beginning and end of the experiment by checking the cell membrane response to small pulses in the passive potential range. Data were discounted from further analysis if the series resistance was ⬎40 M⍀ or had changed significantly (⬎20%) during the experiment. Postsynaptic currents were recorded in gap-free mode and analyzed offline with pClamp software (Version 10.0) using the following criteria: threshold ⫺6 pA; pretrigger length 3 ms; min allowed duration 6 ms; max allowed duration 150 ms. Test compounds were bath applied for 2 min. In experiments utilizing ZD7155, the antagonist was bath applied starting 5 min before the addition of ANGII and ending 5 min after ANGII washout. In some experiments tetrodotoxin (TTX: 1 ␮M) was included in the perfusion media throughout the experiment. In another set of experiments, 1 s pulses of GABA (100 ␮M) were applied each min using a pressure driven perfusion system (ALA Scientific Instruments, Westbury, NY, USA) through a micromanifold positioned close to the recorded cell. Current-clamp recordings were performed using similar equipment and methodology to voltage clamp, but with an npi BA-03X amplifier (npi Electronic, Tamm, Germany). TTX was present throughout the recording session and ANGII was applied for 5 min epoch after establishing a stable membrane potential and input resistance.

Data analysis Data are presented as mean⫾standard error. For extracellular recordings, cells were considered responders if the mean firing rate in the 5–10 min epoch following application of the drug changed by ⬎20% relative to the 5 min period before drug addition. Responses were excluded from further analysis when the baseline firing data exhibited a pre-existing trend of the same polarity as the response. To validate this approach, previously established using single unit recording techniques (e.g. Reed et al., 2002), we analyzed 20 cells from our database, which were recorded for 5– 6 h and did not receive any drug treatments. Using a moving window advancing through the data with 1 min increments we analyzed between 210 and 265 time points per cell (total 4983 from 20 cells) for spontaneous activations or suppressions. On average 1.7⫾0.3% of time points analyzed were interpreted as suppressions and 1.6⫾0.2% as activations. Thus, our criteria are robust enough to reliably detect drug-induced changes in firing rate and do not interpret circadian or spontaneous transient fluctuations in firing rate as responses. For voltage clamp recordings the frequency of postsynaptic currents in the 2– 4 min epoch after drug addition was compared

T. M. Brown et al. / Neuroscience 154 (2008) 839 – 847 with that in the 2 min period before drug addition. The peak, area, rise and decay slopes of currents evoked by GABA pulses were compared in a similar manner. Proportions of responding cells were compared by ␹2 test and other parameters by one-way ANOVA or t-tests as appropriate. Significance was set at P⫽0.05. All statistical tests were carried out using GraphPad Prism 3.0 (San Diego, USA).

Drugs ANG II was obtained from Bachem (St. Helens, UK), N-methyl-Daspartate (NMDA) and (⫺)-bicuculline methiodide were purchased from Sigma (Dorset, UK), all other reagents were supplied by Tocris (Bristol, UK).

RESULTS We examined the effects of 5 min application of ANGII (1 nM–10 ␮M) on a total of 166 SCN neurons, discriminated from dual-electrode MUA electrophysiological recordings from 34 mouse brain slices. Most of these recordings (32 slices, 148 cells) were performed during the projected day (ZT 3–9). During this time epoch, we observed dose-dependent effects of ANGII (Fig. 1), with higher concentrations typically inducing increases in fir-

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ing rate and lower concentrations causing suppressions. Activations were variable in magnitude (113⫾47% control at 10 ␮M; n⫽9) and decayed to baseline values within 5–20 min of washout. Inhibitions induced by ANGII were less variable in magnitude (40 –50% of baseline firing at ANGII concentrations from 0.001– 0.03 ␮M) and were typically biphasic in nature, with the initial suppression followed by a transient recovery and then a second decrease in firing rate (e.g. 2B–D). Both types of effect were observed whether the recording electrode was placed in dorsal or ventral portions of the SCN. Further, in most cases these biphasic effects were apparent in the responses of individual SCN neurons to varying doses of ANGII (Fig. 1C, D). As a positive control, we tested 10 SCN neurons with NMDA (20 ␮M, 5 min), an excitatory agent known to alter the firing rate of most SCN neurons (Reed et al., 2002; Cutler et al., 2003). We found responses consistent with these previous studies which used different methodology to monitor SCN neuronal firing; NMDA induced transient (5–10 min) increases in firing rate in 7/10 cells (magnitude: 208⫾67% predrug values; data

Fig. 1. ANGII dose-dependently alters SCN neuronal firing rates. High concentrations of ANGII tended to increase the spontaneous discharge of SCN neurons (A, F, G) while lower doses tended to decrease spiking (B, F, G). These biphasic effects were apparent in the responses of individual cells to different concentrations of ANGII (C, D). The effects of 10 ␮M and 0.01 ␮M ANGII were blocked by the AT1 receptor antagonist ZD7155 (E, F). Firing rate traces (A–E) represent the mean firing rate (in Hz) every 10 s, bars above traces indicate timing and duration of drug addition. Bar chart in F shows the percentage of cells exhibiting increases/decreases in firing rate considered significant (see Experimental Procedures) at indicated concentrations of ANGII. Numbers above the bars indicate the number of cells tested at each concentration. Bar chart in G shows the % change in firing rate of SCN neurons considered responders at each concentration of ANGII. Data were omitted from G where the number of cells activated/suppressed was ⬍4. * And *** indicate significant reductions in the proportion of responding cells in the presence on ZD7155 compared ANGII alone (P⬍0.05 and 0.001 respectively; ␹2 test).

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not shown), while one cell exhibited a brief reduction in firing rate (47% predrug values). To investigate the receptor dependency of these actions of ANGII on SCN neurons we applied doses that primarily cause activations (10 ␮M) or inhibitions (0.01 ␮M) of neuronal discharge in the presence of 0.03 ␮M ZD7155, an AT1 receptor antagonist (Thomas et al., 1992; Fig. 1E). Pre-treatment with this AT1 receptor antagonist significantly reduced the proportion of cells showing changes in firing rate following application of either 10 ␮M (␹2 test, P⬍0.05; Fig. 1F) or 0.01 ␮M ANGII (␹2 test, P⬍0.001). Drug-induced alterations in SCN neuronal discharge such as those observed in this study might result from direct actions on the recorded neuron, actions on other cells that input to the recorded neuron or a combination of both. Since most SCN neurons are GABAergic (Moore and Speh, 1993), we investigated whether any of the observed effects of ANGII resulted from a change in GABAergic signaling within the SCN. When applied in the presence of the GABAA antagonist bicuculline (20 ␮M), we observed a reduction in the proportion of cells showing decreases in firing rate in response to ANGII with significantly fewer cells deceasing their firing rate in response to ANGII concentrations between 0.1 and 0.01 ␮M (Fig. 2C, ␹2 tests, P⬍0.05). Interestingly, under these conditions of GABAA receptor blockade, the overall proportion of cells responding (activation or suppression) did not change (␹2 tests, P⬍0.05), and a clear dose-dependent activation in neuronal discharge was still apparent with increasing concentrations of ANGII (Fig. 2). Further, we no longer observed

neurons suppressed at low doses of ANGII but activated at higher doses, indicating that the complex dose effects induced by this peptide under control conditions involve a GABAergic mechanism. In a small group of slices, we also examined the actions of ANGII during the projected night (ZT14 –19: four slices). When given during this early-mid projected night phase, 5 min application of 1 ␮M ANGII increased neuronal discharge in 4/12 cells and decreased firing in 3/12. When co-applied with bicuculline, 6/12 cells increased in firing rate and no cells exhibited suppressions. These proportions were not significantly different to those observed during the projected day (␹2 tests, P⬎0.05), nor were the magnitudes of the responses (data not shown). Based on these initial findings we did not investigate day–night variation in SCN neuronal responses to ANGII further. In our previous experiments using the AT1 receptor antagonist ZD7155, slices were treated with the antagonist for prolonged periods (2–3 h, starting at least 40 min before ANGII treatments). To determine whether endogenous AT1 receptor signaling regulated SCN neuronal discharge, we examined the effects of brief (5 min) applications of 0.03 ␮M ZD7155 alone. These treatments altered action potential discharge in a subset of SCN neurons, primarily suppressing firing (Fig. 3). The proportion of cells responding to ZD7155 and the magnitude of responses did not vary significantly between day and night (␹2 test and t-test respectively, both P⬍0.05). Similar results were obtained when ZD7155 was applied in the presence of 20 ␮M bicuculline (␹2 test and t-test respectively, both P⬍0.05),

Fig. 2. The suppressive effects of ANGII on SCN neuronal discharge involve enhanced GABAA signaling. SCN neurons showed dose dependent increases in firing rate when ANGII was applied in the presence of 20 ␮M bicuculline (BIC: A–D). The proportion of cells exhibiting suppressions in firing rate at ANGII concentrations between 0.1 and 0.01 ␮M was significantly reduced compare with ANGII alone (C). Firing rate traces (A, B) represent the mean firing rate (in Hz) every 10 s, bars above traces indicate timing and duration of drug addition. Bar chart in C shows the percentage of cells exhibiting increases/decreases in firing rate considered significant (see Experimental Procedures) at indicated concentrations of ANGII. Numbers above the bars indicate the number of cells tested at each concentration. Bar chart in D shows the % change in firing rate of SCN neurons that significantly increased their firing rate at each concentration of ANGII. Data were omitted from D where the number of cells activated or suppressed was ⬍4. *, ** And *** indicate significant reductions in the proportion of suppressed cells in the presence on BIC compared ANGII alone (P⬍0.05, 0.01 and 0.001 respectively; ␹2 test).

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Fig. 3. Tonic AT1 receptor signaling regulates the firing rate of SCN neurons. The AT1 receptor antagonist ZD7155 suppressed the firing rate of SCN neurons during the projected day (A, C) and night (B, D) in the presence (A, B) and absence (C, D) of bicuculline (BIC). The proportion of cells responding to ANGII (E) between day and night, in the presence or absence of BIC, did not differ significantly (␹2 tests, P⬎0.05). The magnitudes of ZD7155 induced suppressions in neuronal discharge (F), in the presence and absence of BIC were also not significantly different from those observed during the projected day (t-tests, P⬎0.05). Firing rate traces (A–D) represent the mean firing rate (in Hz) every 10 s, bars above traces indicate timing and duration of drug addition. Bar chart in E shows the percentage of cells exhibiting increases/decreases in firing rate considered significant (see Experimental Procedures). Numbers above the bars indicate the number of cells tested for each condition. Bar chart in F shows the % change in firing rate of SCN neurons that significantly decreased their firing rate in the presence and absence of BIC.

thus, unlike ANGII-induced suppressions, these actions of ZD7155 did not involve changes in GABAergic signaling. Our extracellular recording studies established that applying ANGII to SCN slices enhanced GABAA receptor mediated signaling. In order to evaluate the mechanism behind these effects, we used whole-cell patch clamp techniques, voltage-clamping SCN neurons at ⫺70 mV and recording spontaneous inhibitory postsynaptic currents (IPSCs). IPSCs recorded in this manner appear as inward currents, since the holding potential was more negative than the chloride equilibrium potential (Fig. 4; Ecl⫽⫺43.6 mV at 25 °C for our ionic conditions) and exhibited similar characteristics to those previously reported (Itri and Colwell, 2003; Itri et al., 2004; frequency: 3.8⫾0.6 Hz; amplitude: 15⫾1 pA; time to rise: 2.5⫾0.3 ms; time to decay 13.9⫾0.6 ms). These IPSCs were completely blocked by 20 ␮M bicuculline (n⫽8; Fig. 4A) but unaffected by a cocktail of ionotropic glutamate receptor antagonists (20 ␮M CNQX and 50 ␮M D-AP5; n⫽5; data not shown), indicating they were mediated by GABAA receptors. Brief applications of ANGII (2 min) dose-dependently enhanced the frequency of IPSCs (Fig. 4B), but had little effect on their amplitude (Fig. 4C), indicating that ANGII enhanced presynaptic GABA release. In the presence of 0.03 ␮M ZD7155, ANGII (10 ␮M) did not increase IPSC frequency (Fig. 4D; Tukey test P⬎0.01). The antagonist alone had no effects on IPSC frequency (frequency 2– 4 min after antagonist addition⫽98⫾4% predrug values;

n⫽6). Further, 10 ␮M ANGII did not enhance the frequency of miniature IPSCs (Fig. 4D; Tukey test P⬎0.001) when applied in the presence of 1 ␮M TTX. To further investigate whether ANGII altered GABAA receptor signaling in a manner other than via enhancing GABA release, we next examined postsynaptic responses to exogenously applied GABA (100 ␮M, 1 s each min; Fig. 4E). Following (2 min) treatments with 10 ␮M ANGII, peak GABA currents were not significantly different of those observed before application of the peptide (% predrug values: peak⫽96⫾3; area⫽99⫾11; rise slope⫽99⫾5; decay slope⫽99⫾4; paired t-tests all P⬎0.05; n⫽6). These voltage clamp experiments indicated that ANGII-induced increases in presynaptic GABA release required action potential firing. To determine whether this effect arose through a local stimulatory action on GABAergic cells present within the SCN, we performed current clamp recordings from SCN neurons in the presence of TTX. In four of eight SCN neurons, 10 ␮M ANGII caused a membrane depolarization (Fig. 5; 5.3⫾1.2 mV). These membrane depolarizations were associated with a decrease in input resistance (⫺228⫾64 M⍀). Smaller, but still significant, membrane depolarization and decreases in input resistance were also observed in four of seven cells treated with 0.03 ␮M ANGII (3.3⫾0.7 mV, ⫺154⫾54 M⍀ respectively). These data demonstrate that ANGII directly stimulates SCN neurons.

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Fig. 4. ANGII increases GABA release in the SCN in an action potential-dependent manner. (A) A representative example of an SCN neuron in which 10 ␮M ANGII increased the frequency of GABAA receptor mediated IPSCs. (B, C) Cumulative interevent and event amplitude plots for the neuron in A before and after ANGII application. ANGII caused a leftward shift in the interval but not amplitude distributions indicating an increase in GABA release. ANGII caused a dose dependent increase in IPSC frequency that was prevented by the AT1 receptor antagonist ZD7155 (D). ANG II did not enhance the frequency of miniature IPSCs recorded in the presence of TTX. ANG II also failed to alter the postsynaptic response of SCN neurons to exogenous applied GABA (E: 10 ␮M; 1 s each min, representative example of six cells tested). Numbers above bars in F shows the number of cells tested for each condition. ** And *** indicate significant differences form 10 ␮M ANGII alone (P⬍0.01 and 0.001 respectively; Tukey tests).

DISCUSSION These data constitute the first examination of the effects of ANGII on mouse SCN neurons. We show that ANGII depolarizes cells and enhances SCN neuronal discharge and that approximately half of SCN neurons tested respond to the peptide. These effects are mediated via AT1 receptors, since they are blocked by ZD7155, and they lead to a global enhancement of GABAergic signaling within the SCN, specifically, by increasing action potential-dependent GABA release. We also provide the first demonstration that endogenous AT1 receptor-mediated signaling tonically regulates the activities of some SCN neurons. Although we did not set out to examine the ionic mechanisms by which ANGII alters SCN neuronal activity, our current clamp studies indicate that ANGII activates a cation conductance in SCN cells, since ANGII depolarized neurons when spiking was blocked by TTX and concomitantly decreased their input resistance. Similar results have been observed in the hypothalamic paraventricular

nucleus (Latchford and Ferguson, 2005) and ascribed to a nonselective cation conductance. We believe that the increased action potential firing we observed in our extracellular recordings reflects a direct stimulatory effect on SCN neurons, at least partly, due to this inward cation current. Excitatory effects of ANGII are also frequently reported in other hypothalamic structures (Li et al., 2003; Cato and Toney, 2005; Spanswick and Renaud, 2005; Hagiwara and Kubo, 2007; Stocker and Toney, 2007). The results of our extracellular recordings are consistent with those of a previous investigation, which examined the effects of ANGII on SCN neurons in ovariectomized female rats (Tang and Pan, 1995). This earlier study also found that ANGII induced a mixture of activational and suppressive effects on SCN neuronal discharge, but did not examine whether such effects arose through altering the activities of other neurotransmitter/neuromodulator– receptor pathways or whether such actions exhibited circadian variation. We demonstrate that during both projected day and night phases, the proportion of SCN neu-

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Fig. 5. ANGII directly depolarizes SCN neurons. (A) A representative example on an SCN neuron exhibiting a membrane depolarization following application of 10 ␮M ANGII in the presence of TTX. (B) The response of the cell in A to negative current pulses (20 pA, 250 mS) before, during, and after application of ANGII. The decreased membrane deflection in response to current pulses, coupled with the depolarization in resting membrane potential, indicates an increase in cation conductance.

rons reducing their firing rate in response to ANGII is dramatically decreased when bicuculline is present in the perfusion media, indicating that many of these suppressions arise, indirectly, through enhanced GABAA receptor signaling. In agreement with this hypothesis, most studies examining the effects of GABA on rodent SCN neurons demonstrate that its actions are inhibitory and are predominantly mediated via GABAA receptors (Mason et al., 1991; Gribkoff et al., 1999, 2003; and see Brown and Piggins, 2007). Since some suppressive effects of ANGII remain during GABAA receptor blockade it is possible that ANGII directly reduces the firing rate of some SCN neurons, although, we did not observe inhibitory actions of ANGII on SCN neurons when spiking was blocked by TTX in our patch clamp studies. A more likely explanation is that these residual suppressions reflect enhanced activity at GABAB receptors (Liou et al., 1990) or ANGII evoked changes in the activities of other neurochemical signaling systems (for review see Brown and Piggins, 2007). We confirm that ANGII, acting via the AT1 receptor, enhances the release of GABA, rather than changing postsynaptic sensitivity of GABAA receptors by demonstrating that it increases the frequency but not amplitude of IPSCs and does not alter postsynaptic responses to exogenously applied GABA. These observations are surprising, since, in the rat hypothalamic paraventricular nuclei ANGII induces a presynaptic inhibition of GABA release (Li et al., 2003; Chen and Pan, 2007). Interestingly, ANGII does not increase the frequency of miniature IPSCs recorded in the presence of TTX, indicating that the enhanced GABA release observed following ANGII application is dependent upon action potential firing. In this respect, ANGII differs from VIP, another SCN neuropeptide that suppresses cellular discharge (Reed et al., 2002) but enhances GABA

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release in an action potential independent manner (Itri and Colwell, 2003). Since we provide evidence that ANGII directly activates SCN neurons, the enhanced GABA release observed in our study most likely results from increased firing of AT1 receptor expressing neurons located within the SCN. Consistent with this view, SCN neurons are known to receive GABAA-receptor-mediated synaptic input from other SCN cells (Strecker et al., 1997). However, we cannot rule out the possibility that ANGII also stimulates GABA release from terminals of neurons afferent to the SCN. Based on our findings we propose that the response of SCN neurons to ANGII reflects a mixture of its direct excitatory and indirect inhibitory effects (Fig. 6). In this model, we postulate that in addition to SCN neurons that are either directly activated or indirectly inhibited by ANGII, a population of SCN cells experiences both effects. Support for this view comes from our findings that: (1) many cells were activated at higher concentrations but suppressed at lower concentrations; (2) these suppressions were typically biphasic, with a transient recovery in firing which could arise through opposing direct and indirect effects; and (3) these complex effects were absent under GABAA blockade which reduced the proportion of cells suppressed but not the overall proportion of cells responding to ANGII. In agreement with the direct excitatory effects of ANGII discussed above, we observed that blockade of AT1 receptors with ZD7155 reduces the firing rate of ⬃30% cells during both day and night phases. These data indicate that some SCN neurons receive a tonic excitatory drive from

Fig. 6. Model of ANGII effects on SCN neurons. Acting via AT1 receptors, ANGII directly stimulates some SCN neurons (left). Increased GABA release from these cells inhibits postsynaptic SCN neurons which do not express AT1 receptors (top right), whereas, if the postsynaptic neuron also expresses AT1 receptors the two effects oppose each other (bottom right), with the net change in firing rate dependent on the concentration of ANGII and the extent of GABAergic input to the postsynaptic neuron from other AT1 expressing cells.

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endogenous AT1 receptor-mediated signaling that, in vitro, does not exhibit any dramatic day–night variation. Unlike our results with ANGII, these suppressive effects of ZD7155 cannot be attributed to alterations in GABAA signaling, since they are not blocked by bicuculline. This observation further supports the idea that the ZD7155 prevents a direct excitatory effect of endogenous AT1 receptor stimulation on SCN neurons. Immunohistochemical, receptor binding and in situ-hybridization studies indicate that the AT1A receptor is the most likely target to mediate any effects of ANGII on SCN neurons (Gehlert et al., 1986; Obermuller et al., 1991; Kakar et al., 1992; Sasamura et al., 1992; Burson et al., 1994; Johren et al., 1997). These studies are in accord with our data indicating that the actions of ANGII of SCN neuronal firing rate are blocked by an AT1 receptor antagonist. We did not observe any dramatic day–night difference in endogenous AT1 receptor tone or responsiveness (although we cannot rule out subtle circadian variation) consistent with the observation that core circadian functions in mice lacking AT1A receptors remain intact (Mistlberger et al., 2001). Indeed, circadian rhythms in such animals actually appear increased in amplitude due to higher levels of activity during the subjective night. Based on their analysis of the response of AT1A knockout mice to light pulses, Mistlberger et al. argue that the apparent increase in rhythm amplitude results from alterations outside the SCN rather than a direct effect on the clock. Our data suggest that SCN neurons in AT1A knockout mice would be more likely to show reduced rather than increased firing activity and would, therefore, support such a hypothesis. Since we demonstrate robust effects of ANGII, but the available evidence argues against a role of this peptide in modulating the amplitude or timing or circadian rhythms, the exact roles that this system plays within the SCN remain to be determined. We showed that ANGII enhances GABA release and that there is a fine balance between concentrations of ANGII that result in a net increase or decrease in the firing rate of a given cell. GABAA receptor-mediated IPSCs are involved in determining the regularity of spike firing by SCN neurons (Kononenko and Dudek, 2004), therefore, ANGII may play a more important physiological role in determining the patterns of spike firing by SCN neurons on millisecond, rather than circadian time scales.

CONCLUSION In conclusion we show that ANGII stimulates SCN neuronal discharge and that this leads to a widespread increase in GABA release within the SCN. Future studies will need to determine the exact ionic mechanisms underlying this effect of ANGII and how it impacts on information processing within the SCN. Acknowledgments—We thank Rayna Samuels for expert technical assistance and Dr. Mino Belle for helpful comments on a draft of this manuscript. This research was funded by a grant to H. D.

Piggins from the Biotechnology and Biological Sciences Research Council (UK).

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(Accepted 25 March 2008) (Available online 7 April 2008)