HIPPOCAMPUS 25:1393–1406 (2015)
Orexinergic Theta Rhythm in the Rat Hippocampal Formation: In Vitro and In Vivo Findings Renata Bocian, Paulina Kazmierska, Paulina Kłos-Wojtczak, Tomasz Kowalczyk, and Jan Konopacki*
ABSTRACT: Previous in vivo data suggested that orexin neuropeptides (ORXA and ORXB) synthetized in hypothalamic neurons were involved in the mechanism of generation of the hippocampal formation theta rhythm. Surprisingly, this suggestion has never been directly proved by experiments using intraseptal or intrahippocampal administration of orexins. In this study, involving the use of in vitro hippocampal formation slices and in vivo model of anesthetized rat, we provide the first convergent electropharmacological evidence that in the presence of both ORXA and ORXB the hippocampal formation neuronal network is capable of producing oscillations in the theta band. This effect of orexin peptides was antagonized by selective blockers of orexin receptors (OX1R and OX2R), SB 334867 and TCS OX2 29, respectively. These results provide evidence for a novel, orexinergic mechanism responsible for the production of theta rhythm in the hippocampal formation neuroC 2015 Wiley Periodicals, Inc. nal network. V KEY WORDS: orexin peptides; theta oscillations; hippocampal formation; in vitro; in vivo
INTRODUCTION The neuropeptides orexin-A (ORXA) and orexin-B (ORXB), also called hypocretin-1 and hypocretin-2, were originally identified in neurons of hypothalamus, including the perifornical area, lateral hypothalamus, and dorsomedial nucleus (de Lecea et al., 1998; Sakurai et al., 1998; Henny and Jones, 2006; Hahn, 2010; Mieda et al., 2013). The actions of orexin peptides are mediated by two receptors, OX1R and OX2R (Sakurai et al., 1998). OX1R has preferential affinity for ORXA, whereas OX2R exhibits similar affinities for ORXA/B (Sakurai et al., 1998). Both orexins receptors are widely distributed in a number of structures of the mammalian central nervous system (CNS), including the hippocampal formation, cerebral cortex, olfactory bulb, amygdala, diagonal band of Broca, thalamus, midbrain, brainstem, and spinal cord (Peyron et al., 1998; Sakurai et al., 1998; Nambu et al., 1999; van den
Department of Neurobiology, Faculty of Biology and Environmental Protection, University of Lodz, Poland Grant sponsor: National Science Centre Poland; Grant numbers: UMO2011/01/N/NZ4/01722 and UMO-2013/08/T/NZ3/00026. *Correspondence to: Jan Konopacki, Department of Neurobiology, Faculty of Biology and Environmental Protection, The University of Lodz, 90-236 Lodz, Pomorska str.141/143, Poland. E-mail: [email protected] Accepted for publication 17 March 2015. DOI 10.1002/hipo.22459 Published online 26 March 2015 in Wiley Online Library (wileyonlinelibrary.com). C 2015 WILEY PERIODICALS, INC. V
Pol, 1999; Smart and Jermann, 2002; Kukkonen, 2013; Mieda et al., 2013). This broad distribution of orexin receptors in CNS suggests the involvement of those peptides in many distinct physiological functions (Peyron et al., 1998; Smart and Jerman, 2002; Xu et al., 2013). Indeed, initially the pivotal role of orexins in short-term feeding was well documented (Sakurai et al., 1998; Dube et al., 1999; B€ackeberg et al., 2002; Thorpe and Kotz, 2005; Xu et al., 2013). Other evidence linked orexins to metabolic regulation and thermogenesis (Kukkonen et al., 2002; Monda et al., 2004; Funato et al., 2009; Kukkonen, 2013), stress response (Huang et al., 2010; Gerashchenko et al., 2011; Kukkonen, 2013), circadian rhythms (Deboer et al., 2004; Pekala et al., 2011), the regulation of sleep/wakefulness (Gerashchenko et al., 2001; Inutsuka and Yamanaka, 2013; Mieda et al., 2013; de Lecea and Huertra, 2014), memory processing (Akbari et al., 2008; Selbach et al., 2010), pathogenesis of Alzheimer disease (Kang et al., 2009), and epilepsy (Doreulee et al., 2010). It was also demonstrated that orexins modulate arousal: specifically, rodents treated with orexins spend more time awake (Hagan et al., 1999; Piper et al., 2000), and manifest increased locomotor activity (Alexandre et al., 2013). Electrophysiological studies conducted on cultured nucleus basalis neurons and on rat brain slices demonstrated that ORXA-induced depolarization and increased cell firing were accompanied by a decrease of membrane conductance in cholinergic neurons (Eggermann et al., 2001; Hoang et al., 2004; Mieda et al., 2013). The mechanisms of orexin-induced cell excitation were demonstrated to be linked to the inhibition of inward rectifier K1 current (Ivanov and Aston-Jones, 2000; Hoang et al., 2004), activation of electrogenic sodium-calcium exchanger and activation of non-selective cation channels (Liu et al., 2002; Wu et al., 2002). All tested cholinergic neurons were excited by ORXA, and even more potently by ORXB, suggesting mainly OX2R receptor involvement. ORXA has also been shown in vivo to elicit excitation in both cholinergic and non-cholinergic neurons of the laterodorsal tegmental nucleus (Takahashi et al., 2002). In contrast, it has been demonstrated that in vivo administration of ORXA inhibits the activation of the pedunculopontine tegmental nucleus (PPT) cholinergic neurons by excitation of GABAergic
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neurons of PPT itself (Takakusaki et al., 2005). PPT has been earlier demonstrated to be involved in the regulation of hippocampal formation theta rhythm (Vertes et al., 1993; Kinney et al., 1998; Matulewicz et al., 2010). It sends afferents to the medial septum the structure giving rise to cholinergic and GABAergic projections that are widely known to determine the hippocampal formation theta rhythm (Steward and Fox, 1990; Smythe et al., 1992; Vertes and Kocsis, 1997; Bland and Oddie, 1998; Buzsaki, 2002; Sotty et al., 2003; Ma et al., 2012; Pignatelli et al., 2012; Colgin, 2013).The medial septum/vertical limb of the diagonal band of Broca (MS/vDBB) also receives a dense innervation from hypothalamic orexinergic neurons, and some neurons in MS/vDBB express very high levels of OX2R (de Lecea et al., 1998; Wu et al., 2002, 2004; Mieda et al., 2013). Interestingly, Gerashchenko et al. (2001) demonstrated that injection of orexin-saporin toxin into the medial septal area completely eliminated theta rhythm in rats. Furthermore, it has just recently been demonstrated that intracerebroventricular administration of ORXA increased the power of theta rhythm in freely moving rats (Toth et al., 2012). This data suggests that orexin peptides are involved in the mechanism of generation of HPC theta (Geraschenko et al., 2001; Selbach et al., 2004; Wu et al., 2004; Toth et al., 2012). Surprisingly, this suggestion has never been directly proven by experiments with use of intraseptal or intrahippocampal administration of orexins. In the present study we address the issue of orexinergic control of theta rhythm at the level of hippocampal formation. Using in vitro hippocampal formation slices as a model of inducing theta oscillations (Konopacki, 1998; Kowalczyk et al., 2013a) and an in vivo model of anesthetized rat, we provide the first convergent electropharmacological evidence that in the presence of both ORXA and ORXB peptides the hippocampal formation neuronal network is capable of producing the oscillations in theta band. Preliminary reports of these findings appeared in an abstract form (Bocian et al., 2011; Kazmierska, 2012; Kazmierska et al., 2013).
MATERIALS AND METHODS All experiments described below were monitored by a Local Ethical Commission (permission no. 24/LB 547/2011 in accordance with the European Communities Council Directive of 24 November 1986).
In Vitro Experiments Subject and experimental procedure Primary experiments were performed on 401 hippocampal formation slices delivered from 74 male Wistar rats (120– 150 g). The brain was removed and placed in oxygenated (95% O2 1 5% CO2), cold (3–5 C) artificial cerebrospinal fluid (ACSF; composition in mM: NaCl 121; KCl 5; CaCl2 Hippocampus
2.5; KH2PO4 1.25; MgSO4 1.3; NaHCO3 26; glucose 10; Sigma Chemical). ACSF was made fresh before each experiment using prefiltrated and deionized water (Easy Pure RF, Barnstead). Transverse slices (500 mm) were made from both hippocampi using a tissue slicer (Stoelting). Slices were preincubated and submerged in oxygenated ACSF at a temperature of 20 C for 40 min after dissection. Next the slices were transferred into a gas-liquid interface-recording chamber and maintained on a nylon mesh. Slices were continuously perfused with oxygenated and prewarmed ACSF (always 35 C) at a flow rate of 1 ml/min.
Drugs In the separate control experiments performed on 24 slice preparations, field potentials were induced between 10 and 20 min after incubation with 50 mM carbachol (carbamylcholine chloride, Sigma Chemical, St. Louis) dissolved in ACSF. ORXA and ORXB (PolyPeptide Laboratoires, SAS, France) dissolved in ACSF were administered in doses ranging from 0.01 to 1.0 mM. TCS OX2 29, the selective antagonist of OX2R (Tocris Bioscience, UK), was dissolved in ACSF and administered in a concentration of 10 mM. SB 334867, the selective antagonist of OX1R (Tocris Bioscience, UK), was dissolved in DMSO and then in ACSF. The maximal concentration of DMSO in ACSF (vehicle group, data not shown) did not exceed 1%. The final concentration of SB 334867 was 10 mM.
Recording procedure and data analysis Glass recording electrodes (3–5 MX) filled with 2.0 M sodium acetate were made from Kwik-Fill capillaries (World Precision Instruments, Sarasota, FL). All recordings were performed in the CA3c region of HPC, which is considered to be the main generator of theta rhythm in HPC slices (Konopacki et al., 1988; Kowalczyk et al., 2009). The electrodes were positioned with the use of a micropositioner (IVM, Scientifica, UK). In all experiments field potentials were recorded with respect to the ground. Signals were filtered (0.001–0.3 kHz, band pass) and amplified (10003) using a P-511 preamplifier (Grass-Astromed, West Warwick). The HPC field activity was displayed on a digital storage oscilloscope (Tektronix TDS 3014) and stored on a computer hard drive using data acquisition interface CED-1401 (Cambridge Electronic Design, Cambridge, GB). The HPC field activity was recorded continuously and analysed in five-minute fragments after 10, 20, 30, and 40 min (i.e., 10–15 min fragment; 20–25 min fragment, etc.). Off-line spectral analysis of these fragments was conducted with the use of Spike 2.7 software (Cambridge Electronic Design, GB). The following parameters of the hippocampal field activity were analysed: frequency, amplitude and power. The detailed analysis of theta rhythm covered three samples of well-synchronized theta epochs, selected from each 5 min recording for the specified time period (20, 30, 40 min after compound administration). Additionally, a final recording was also performed after 30 min of washout. The frequency and
OREXINERGIC THETA RHYTHM IN THE HIPPOCAMPAL FORMATION power were determined using the Fast Fourier Transform (FFT) algorithm. Theta amplitude was determined directly from digital recordings. Mean values and standard errors of the mean (x 6 SEM) of three measured theta parameters (frequency, amplitude, and power), obtained from successive time periods after treatment with ORXA and ORXB, were computed and compared with carbachol (CCH) in vitro testing using the ANOVA test and Tukey’s post-hoc test (Statistica 10).
In Vivo Experiments Subjects and surgical procedure The data was obtained from 62 male Wistar rats (120– 150 g). The rats were initially anesthetized (Dragger vapor 19.3) with halothane (Sigma Chemical) while a jugular cannula was inserted. Halothane was then discontinued, and urethane (0.6 g/ml, Sigma Chemical) was administered via the jugular cannula to maintain anesthesia throughout the experiment. In separate experiments the second jugular cannula was used for iv administration of atropine sulfate (30 mg/kg). Anesthesia levels were maintained such that theta field potentials and the transition from theta to LIA could occur spontaneously. Body temperature was maintained at 37 C by a heating pad and heart rate was monitored constantly throughout the experiment. The rats were placed in a stereotaxic frame with the plane between the bregma and lambda levelled to horizontal. An uninsulated tungsten wire placed in the cortex, 2 mm anterior to the bregma, served as an indifferent electrode, and the stereotaxic frame was connected to a ground. A tungsten microelectrode (0.1 – 0.9 MX) for recording hippocampal field activity was placed in the right dorsal HPC, in stratum lacunosum-moleculare (3.2 mm posterior from bregma, 2.0 – 2.2 mm lateral from the midline, and 2.6 – 2.8 mm ventral to the dural surface; Paxinos and Watson, 1998). An AC amplifier (P-511, Grass-Astromed, West Warwick) was used for recording field potentials, with the low filter set at 1 Hz and the high filter set at 0.3 kHz. The field activity was displayed using a digital storage oscilloscope (Tektronix TDS 3014). EEG signals were digitized by interface (1410 plus, Cambridge Electronic Design, GB) and recorded onto computer hard disk for subsequent off-line analysis (Spike 2.29, Cambridge Electronic Design, GB).
Drug administration procedure In the first stage (preliminary experiments) doses of ORXA from 0.2 mg/0.5 ml to 1.0 mg/0.5 ml were tested (data not shown). ORXA was dissolved in physiological saline. The effective, threshold concentrations of ORXA were recognized as inducing hippocampal theta rhythm repeatedly. In further in vivo experiments, only intrahippocampal microinjections of ORXA in concentrations of 0.4 mg/0.5 ml were applied, as concentrations of ORXA below and above 0.4 mg/0.5 ml were found to be ineffective or to induce epileptiform
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discharges (concentration of 1.0 mg/0.5 ml, data not shown). ORXB was administrated in concentrations ranging from 0.1 mg/0.5 ml to 0.8 mg/0.5 ml. ORXB was dissolved in physiological saline. The effective, threshold concentrations of ORXB were recognized as inducing hippocampal theta rhythm repeatedly. In further in vivo experiments only intrahippocampal microinjections of ORXB in concentrations of 0.2 mg/0.5 ml were applied, as concentrations of ORXB below and above 0.2 mg/0.5 ml were found to be ineffective or to induce epileptiform discharges (concentration of 0.8 mg/0.5 ml, data not shown) TCS OX2 29: the selective antagonist of OX2R (Tocris Bioscience, UK) was dissolved in physiological saline and administered in a concentration of 10 mg/0.5 ml. SB 334867: the selective antagonist of OX1R (Tocris Bioscience, UK), was dissolved in DMSO and also administered in a concentration of 10 mg/0.5 ml. The doses of orexin agonists and antagonists were calculated accordingly from in vitro applied concentrations. Orexin antagonists were administrated only in the presence of orexins and dissolved in physiological saline. The maximal concentration of DMSO in physiological saline (vehicle group, data not shown) did not exceed 1%.
Intrahippocampal drugs injection We did not find evidence for lateralization of effects of intrahippocampal injection of drugs used on hippocampal theta (data not shown). Hence, in the primary experiments microinjection of DMSO or saline and all drugs used were always performed into the right hippocampal formation (26 gauge, 5 ml Hamilton 701N microsyringe). The coordinates of Hamilton canulae were as follows: 3.2 mm posterior from bregma, 2.2 mm lateral from the midline, and 2.6 mm ventral to the dural surface (Paxinos and Watson, 1998).
Experimental procedure To characterize the effect of orexin peptides on hippocampal spontaneous theta, 4 min continuous, control recordings of hippocampal field potentials were made prior to microinjection. Four min recordings were also continued in successive time periods: 15 min, 30 min, 45 and 90 min postinjection. Ten 2-s EEG samples containing theta rhythms were selected for analysis from successive time periods. To characterize the effect of orexins on hippocampal field potential in rats pretreated with iv atropine sulfate, 4 min continuous control recordings of hippocampal field potentials were made prior to microinjection.
Data analysis The EEG data obtained from in vivo experiments was analyzed off-line using the Spike 2.7 software computing system. Two-second samples of hippocampal EEG, registered during the spontaneous theta, were subjected to power/frequency (FFT) analysis. The mean peak-to-peak amplitude of theta was determined directly from epochs of theta. Hippocampus
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FIGURE 1. Field potentials of hippocampal formation slices recorded in three different experimental arrangements: A: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in the CA3c field of hippocampal formation slices before and at 10, 20, 30, and 40 min after administration of 50 lM carbachol (CCH). B: Analogue examples of EEG activity and FFT histograms related with indi-
vidual theta samples recorded in the CA3c field of hippocampal formation slices before and at 10, 20, 30, and 40 min after administration of 0.01 2 1.0 lM ORXA. C: Analogue examples of EEG activity recorded in the CA3c field of hippocampal formation slices before and 20 and 40 min after administration of orexins’ antagonists, SB 334867 and TCS OX2 29. Calibration 0.5 s, 500 lV.
Mean values and standard errors of the mean (x 6 SEM) of three measured theta parameters (frequency, amplitude, and power), obtained from control and 45 min after the injection of orexin peptides, were compared using the Mann-Whitney U-test (Statistica 10).
the sagital plane. Slices were then stained with cresyl violet for subsequent verification of electrode and cannulae placement.
RESULTS Histological procedure Following data acquisition, the rat was deeply anesthetized with urethane, perfused with saline, and then with 10% formalin. The brain was removed and stored in 10% formalin at a low temperature. Brains were frozen and sectioned at 20 mm slices in Hippocampus
In Vitro Experiments The effect of carbachol In the control experiments (Figs. 1A and 2A) the effect of CCH (50 mM) on field potentials was studied in the HPC slice preparations. Figures 1A and 2A demonstrates analog
OREXINERGIC THETA RHYTHM IN THE HIPPOCAMPAL FORMATION
FIGURE 2. Field potentials of hippocampal formation slices recorded in three different experimental arrangements: A: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in the CA3c field of hippocampal formation slices before and at 10, 20, 30, and 40 min after administration of 50 lM carbachol (CCH). B: Analogue examples of EEG activity and FFT histograms related with individual
samples of field activity recorded in HPC slices before, and in different time periods from the CCH perfusion onset, with one corresponding FFT histogram taken in 40 min. In the first 10 min after the CCH perfusion onset, all preparations (n 5 24 slices) typically generated epileptiform discharges. CCH-induced theta field potential developed gradually between 10 - 20 min from the perfusion onset. After 40 min of CCH perfusion, mean (6 SEM) theta frequency was 12.35 6 0.63 Hz, mean amplitude was 1339.28 6 274.02 mV, and power 73313.71 6 282.71 mV2. The effect of CCH was reversible 30 min after washout with ACSF.
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theta samples recorded in the CA3c field of hippocampal formation slices before and at 10, 20, 30, and 40 min after administration of 0.01 2 1.0 lM ORXB. C: Analogue examples of EEG activity recorded in the CA3c field of hippocampal formation slices before and 20 and 40 min after administration of orexins’ antagonists, SB 334867 and TCS OX2 29. Calibration 0.5 s, 500 lV.
The effect of ORXA In separate experiments the effect of different concentrations of ORXA on the HPC slice field potentials was evaluated (Fig. 1B). This figure demonstrates analog samples of field activity recorded in HPC slices before, and in different time period from the ORXA perfusion onset, with single corresponding FFT histogram taken at 40 min. When perfused with ORXA at a concentration of 0.01 mM, the HPC preparations (n 5 22 slices) responded only with epileptiform discharges. These hippocampal field potentials appeared usually in the first 10 min of a compound administration and lasted to the end of the experiment (at least 40 min). In the presence of ORXA in Hippocampus
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concentrations of 0.03 mM (Fig. 1B), HPC slice preparations responded with two different patterns of field potentials: epileptiform discharges and theta activity. In 12 out of 20 preparations (60%) mainly theta field potential was noted. The mean frequency of theta measured at 40 min from the perfusion onset was 11.14 6 0.92 Hz, mean amplitude was 833.53 6 246.63 mV and power 25220.92 6 11349.24 mV2. The application of 0.1 mM of ORXA resulted in the highest probability of theta appearance: this particular pattern of field potential appeared in 15 out of 23 slices (65.2%). In this case the mean dominant frequency noted at 40 min from the perfusion onset was lower (9.58 6 1.73 Hz; P < 0.05 vs. 40 min after the onset of perfusion with CCH) than the frequency of theta rhythm recorded at the same time after perfusion of CCH. The mean amplitude was higher (3065.75 6 779.95 mV; P < 0.05 vs. 40 min after the onset of perfusion with CCH), and mean power was higher (619057.81 6 199232.81 lV2, P < 0.05 vs. 40 min after the onset of perfusion with CCH) in comparison to those parameters of theta rhythm recorded at the same time after perfusion of CCH. The bath perfusion of HPC slices with 0.3 mM of ORXA resulted in the appearance of theta activity in 8 out of 21 preparations (38%). The mean dominant frequency registered 40 min from the perfusion onset was 9.31 6 1.86 Hz, mean amplitude was 1016.20 6 247.56 mV, and power 145840.73 6 77684.61 lV2. A further increase of ORXA concentration to 1.0 mM never produced theta field potential. All tested preparations (n 5 22 slices, 100%) responded only with epileptiform discharges. As was the case with CCH perfusion, the effect of ORXA was also reversible after 30 min washout with ACSF. Due to the fact that the highest probability of inducing theta field activity arose after administration of ORXA in the dose of 0.1 mM, this particular concentration was chosen for further pharmacological investigations with the use of orexins’ antagonists (n 5 57; Fig. 1C). These experiments were specifically designed to evaluate the capability of 0.1 mM ORXA to induce theta oscillations in the presence of OX1R and/or OX2R antagonists (SB 334867 and TCS OX2 29 respectively). Generally, in the in vitro condition ORXA never produced theta oscillations in the presence of OX1R and/or OX2R antagonists (Fig. 1C).
The effect of ORXB The analogue examples concerning the perfusion of CCH presented in Figure 2A are precisely the same as shown in a Figure 1A. This data was presented only for comparison of the effect of CCH with that of ORXB on HPC field potentials. In a separate series of experiments (Fig. 2B) the effect of different concentrations of ORXB on HPC slice field potentials was evaluated. Figure 2B demonstrates analogue samples of field activity recorded in HPC slices before and at different time periods from the ORXB perfusion onset with corresponding FFT histograms (taken after 40 min). When perfused with ORXB at a concentration of 0.01 – 0.03 mM, the HPC preparations (n 5 45 slices) responded only with epileptiform Hippocampus
discharges. These hippocampal field potentials appeared usually in the first 10 min of a compound administration and lasted to the end of the experiment (at least 40 min). In a presence of 0.1 mM ORXB (Fig. 2B), HPC slice preparations responded with two different patterns of field potentials: epileptiform discharges and theta activity. A perfusion of HPC slices with 0.1 mM ORXB resulted in the highest probability of theta appearance: in 13 out of 24 preparations (54.1%) mainly theta field potential was noted. In this case the mean frequency of theta measured 40 min from the perfusion onset was lower (10.14 6 0.81 Hz, P < 0.05 vs. 40 min after the onset of perfusion with CCH) than the frequency of theta rhythm recorded at the same time after perfusion of CCH. The mean amplitude was higher (2416.55 6 449.67 mV, P < 0.05 vs. 40 min after the onset of perfusion with CCH) and power was higher (553774.92 6 65093.75 mV2, P < 0.05 vs. 40 min after the onset of perfusion with CCH) in comparison to those parameters of theta rhythm recorded at the same time after perfusion of CCH. The application of 0.3 mM of ORXB resulted in the appearance of theta activity in 10 out of 20 slices (50%). The mean dominant frequency noted 40 min from the perfusion onset was 8.26 6 0.94 Hz, mean amplitude was 1011.48 6 271.03 mV, and power 117934.08 6 45462.86 lV2. The bath perfusion of HPC slices with 1.0 mM of ORXB resulted in the appearance of theta activity in 11 out of 23 preparations (47.8%). The mean dominant frequency registered 40 min from the perfusion onset was 9.21 6 1.04 Hz, mean amplitude was 817.88 6 134.40 mV, and power 43672.36 6 22526.89 lV2. The effect of ORXB was reversible 30 min after washout with ACSF. Due to the fact that the highest probability of inducing theta field activity arose after administration of ORXB in the dose of 0.1 mM, this particular concentration was chosen for further pharmacological investigation, with the use of orexins’ antagonists (n 5 67; Fig. 2C). These experiments were designed to evaluate the capability of 0.1 mM ORXB to induce theta oscillations in the presence of OX1R and/or OX2R antagonists (SB 334867 and TCS OX2 29 respectively). Generally, as it was in the case of ORXA, in the in vitro condition the ORXB never produced theta oscillations in the presence of OX1R and/or OX2R antagonists (Fig. 2C). Control application of the vehicle (DMSO, n 5 33) did not induce any apparent changes in the HPC field potential (data not shown).
In Vivo Experiments The effect of ORXA Three different concentrations of ORXA were tested in preliminary experiments: 0.2 mg/0.5 ml, 0.4 mg/0.5 ml, and 1.0 mg/0.5 ml. The concentration of 0.2 mg/0.5 ml of ORXA was found to be a subthreshold and did not induce any apparent effect on hippocampal field potential (data not shown). The concentration of 1.0 mg/0.5 ml in turn induced epileptiform discharges (data not shown). Hence, the concentration of 0.4 mg/0.5 ml of this agent was chosen for further pharmacological
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FIGURE 3. Field potentials of hippocampal formation recorded in anesthetized rats in three different experimental arrangements: A: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in in stratum lacunosum-moleculare of hippocampal formation before and at 15, 30, 45, and 90 min after administration of 0.4 lg/0.5 ll ORXA. B: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in in stratum lacunosum-moleculare of hippocampal formation before and after
iv administration of atropine sulphate (30 mg/kg) and at 15, 30, 45, and 90 min after the intrahippocampal administration of 0.4 lg/0.5 ll ORXA. C: Analogue examples of EEG activity recorded in the stratum lacunosum-moleculare of hippocampal formation before and after iv administration of atropine sulfate (30 mg/kg) and at 15, 30, 45, and 90 min after the intrahippocampal administration of 0.4 lg/0.5 ll ORXA and SB 334867, ORXA, and TCS OX2 29, ORXA, and SB 334867 and TCS OX2 29. Calibration 1.0 s, 200 lV.
investigation. The effect of ORXA in concentration of 0.4 mg/ 0.5 ml on HPC theta rhythm is shown in Figure 3. Intrahippocampal administration (n 5 7) of ORXA (0.4 mg/ 0.5 ml) in anesthetized rats induced well synchronized epochs of theta rhythm (Fig. 3A). In comparison with the preinjection basic parameters of spontaneous theta (frequency 5.74 6 0.23 Hz, amplitude 601.23 6 10.81 lV, and power 20262.42 6 951.13 lV2) ORXA theta recorded 45 min from the injection onset has a similar frequency (5.69 6 0.11 Hz), increased amplitude 661.29 6 13.15 lV (P < 0.001), and increased power 32990.75 6 753.68 lV2 (P < 0.001). This enhancement of the spontaneous theta amplitude and power was reversible 90 min after the injection. In the separate experiments (n 5 5) the effect of ORXA was tested in anesthetized rats pretreated iv by atropine sulphate (Fig. 3B). As Figure 3B demonstrates, atropine sulphate abolished spontaneous theta rhythm. Intrahippocampal administration of ORXA (0.4 mg/0.5 ml) induced well-synchronized epochs of theta rhythm, which were found to be reversible about 90 min after the injection. In the next experiments (n 5 17) the effect of ORXA on HPC field potential was tested in anesthetized and atropinepretreated rats in the presence of OX1R and/or OX2R
antagonists (SB 334867 – 10 mg/1.0 ml and TCS OX2 29 – 10 mg/1.0 ml, respectively, Fig. 3C). As is shown in Figure 3C, SB 334867 and TCS OX2 29 antagonists never blocked ORXA-induced theta when injected separately. The abolishing effect developed only when both antagonists were administrated simultaneously. The recovery of spontaneous theta rhythm was usually observed 80 – 90 min after the injection.
The effect of ORXB Three different concentrations of ORXB were tested in preliminary experiments: 0.1 mg/0.5 ml, 0.2 mg/0.5 ml, and 0.8 mg/0.5 ml. The concentration of 0.1 mg/0.5 ml of ORXB was found to be a subthreshold and did not induce any apparent effect on hippocampal field potential (data not shown). The concentration of 0.8 mg/0.5 ml in turn induced epileptiform discharges (data not shown). Hence, the concentration of 0.2 mg/0.5 ml of this agent was chosen for further pharmacological investigation. The effect of ORXB in a concentration of 0.2 mg/0.5 ml on HPC theta rhythm is shown in Figure 4. Intrahippocampal administration (n 5 10) of ORXB (0.2 mg/0.5 ml) in anesthetized rats induced well-synchronized epochs of theta rhythm (Fig. 4A). In comparison with preinjection basic Hippocampus
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FIGURE 4. Field potentials of hippocampal formation recorded in anesthetized rats in three different experimental arrangements: A: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in the stratum lacunosum-moleculare of hippocampal formation before and at 15, 30, 45, and 90 min after administration of 0.2 lg/0.5 ll ORXB. B: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in the stratum lacunosum-moleculare of hippocampal formation before and after
iv administration of atropine sulfate (30 mg/kg) and at 15, 30, 45, and 90 min after the intrahippocampal administration of 0.2 lg/0.5 ll ORXB. C: Analogue examples of EEG activity recorded in the stratum lacunosum-moleculare of hippocampal formation before and after iv administration of atropine sulfate (30 mg/kg) and at 15, 30, 45, and 90 min after the intrahippocampal administration of 0.2 lg/0.5 ll ORXB and SB 334867, ORXB and TCS OX2 29, ORXB and SB 334867 and TCS OX2 29. Calibration 1.0 s, 200 lV.
parameters of spontaneous theta (frequency 5.23 6 0.18 Hz, amplitude 620.07 6 35.25 lV, and power 23723.83 6 1703.36 lV2) ORXB-induced theta recorded 45 min after the injection has a similar frequency (5.12 6 0.21 Hz, P > 0.05), increased amplitude (691.42 6 7.88 lV, P < 0.001), and increased power (34494.75 6 944.49 lV2, P < 0.001). This enhancement of ORXB-induced theta amplitude and power was reversible 90 min after the injection. In a separate experiments (n 5 5) the effect of ORXB was tested in anesthetized and atropine-pretreated rats (Fig. 4B). As is shown in Figure 4B, atropine sulphate abolished spontaneous theta rhythm. Intrahippocampal administration of ORXB (0.2 mg/0.5 ml) induced well-synchronized epochs of theta rhythm, which was reversible about 90 min after the injection. In the next series of experiments (n 5 15), the effect of ORXB on HPC field potential was tested in anesthetized and atropine-pretreated rats in the presence of OX1R and/or OX2R antagonists (SB 334867 and TCS OX2 29, respectively, Fig. 4C). As is shown in Figure 4C, SB 334867 and TCS OX2 29 never blocked ORXB-induced theta when injected separately. The abolishing effect developed only in the presence of both orexin receptors antagonists, and was also reversible 80 – 90 min after the injection.
The control intrahippocampal injection of the vehicle (DMSO, n 5 3) did not induce any apparent changes in HPC field potential (data not shown).
Hippocampus
DISCUSSION In the present study we provided the first convergent in vitro and in vivo data concerning the modulation or the production of hippocampal formation theta rhythm by orexin peptides. Specifically, we demonstrated that both ORXA and ORXB induced theta in hippocampal formation slices delivered from adult (3 weeks old) rats. This orexin-induced in vitro theta rhythm was antagonized by selective blockers of OX1R and OX2R receptors, SB 334867 and TCS OX2 29 respectively. As a next step, this in vitro finding was verified in experiments conducted in vivo with the use of anesthetized rats of the same age. Overall, in vivo experiments supported our in vitro observations: intrahippocampal injection of both ORXA and ORXB augmented spontaneous HPC type II theta rhythm. In addition, intrahippocampal administration of ORXA and ORXB
OREXINERGIC THETA RHYTHM IN THE HIPPOCAMPAL FORMATION also induced theta oscillations in atropine-pretreated rats. In anesthetized and atropine-treated rats the abolishing effect of orexin receptors antagonists developed only when both agents, SB 334867 and TCS OX2 29, were simultaneously injected. The current results were obtained on HPC tissue maintained in two different experimental environments: in vivo and in vitro in completely deafferented hippocampal formation slices. Despite this fact, the resemblance of the electrophysiological response of the HPC neuronal network to the administration of ORXA and ORXB in the in vitro and in vivo conditions was similar. This finding provides further evidence for the validation of the earlier-developed in vitro model of HPC theta rhythm (Konopacki, 1998; Kowalczyk et al., 2013a) in the study of the neural mechanisms responsible for the generation of this hippocampal formation rhythmic field potential. The basic assumption of the current in vitro and in vivo experiments was the presence of OX1R and OX2R receptors in the HPC. This supposition was earlier supported by a number of reports demonstrating a high density of OX1R and OX2R in different regions of HPC, including the dentate gyrus and hippocampus proper (Trivedi et al., 1998; Kilduff and Payron, 2000; Marcus et al., 2001; Akbari et al., 2011). Soon after the identification of orexins’ neuropeptides, several studies have shown that orexin signalling is essential for the stability of the arousal state. For example, mice deficient in OX1R receptors have a normal amount of sleep and wakefulness across the light/dark cycle, but the stability of these behavioural states is considerably reduced (Mochizuki et al., 2011). In addition, dogs with mutations of OX2R exhibit narcolepsy and cataplexy (Lin et al., 1999). It was also demonstrated that patients that suffer from narcolepsy have also a very low level of ORXA in their cerebrospinal fluid (Thannickal et al., 2000). Furthermore, the intracerebrovetricular administration of ORXA was found to increase wakefulness and decrease both slow wave and REM sleep (Piper et al., 2000). Thus, orexins are generally considered to be excitatory neuropeptides in CNS (Leonard and Kukkonen, 2014). They excite a number of neurons in different regions of CNS, including the basal forebrain, raphe nuclei, locus ceruleus, tuberomammillary nucleus, ventral tegmental area (Hagan et al., 1999; Nakamura et al., 2000; Eggerman et al., 2001; Liu et al., 2002; Yamanaka et al., 2002). Both orexin peptides also have a strong direct excitatory effect on septohippocampal cholinergic (Wu et al., 2004) and GABAergic (Wu et al., 2002) neurons localized in the medial septum/vertical limb of the diagonal band of Broca. MS/vDBB also receives a dense innervation from the orexin neurons, and neurons in this region express a very high level of OX2R (Mieda et al., 2013). It was earlier suggested that ORXBinduced activation of the septohippocampal GABAergic neurons will, by engaging disinhibitory processes in the HPC, promote the production of the hippocampal theta rhythm (Wu et al., 2002). The results of the present in vitro and in vivo study provide the evidence that orexin peptides not only promote but just induce theta rhythm when acting directly at the level of the HPC neuronal network. Specifically, our in vivo experiments demonstrated that both ORXA and ORXB
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peptides augmented HPC spontaneous theta rhythm (type II theta) and also induced theta in atropine-pretreated rats. This is the first direct and unequivocal demonstration of orexininduced theta field potentials recorded in the hippocampal formation. Interestingly, in contrast to the in vitro findings, theta rhythms induced by ORXA and ORXB in the in vivo conditions were abolished only in the presence of both OX1R and OX2R antagonists, SB 334867 and TCS OX2 29.The administration of SB 334867 or TCS OX2 29 alone was completely ineffective in blocking the ORXA- or ORXB-induced theta. This would suggest that in the hippocampal neuronal network, which underlies the mechanisms of production of local theta field potential, each orexin peptide excites both OX1R and OX2R, and there is no noticeable preference in affinity of either ORXA or ORXB for these receptors. From this point of view, the results of the present in vivo experiments conducted on anesthetized rats do not support the earlier suggestion of Sakurai et al. (1998) that OX1R has a preferential affinity for ORXA, whereas OX2R exhibits similar affinities for ORXA and ORXB. On the other hand the effect observed in our study was similar to that previously observed in in vivo and in vitro preparations. Specifically, Jia et al. (2012) reported that the excitatory action of ORXA on behaviour, EEG and spontaneous activity of prefrontal cortex neurons was completely blocked only in the presence of the selective OX1R antagonist SB 334867 and OX2R antagonist TCS OX2 29. It was also observed in vitro that ORXA and ORXB depolarized rostral ventrolateral medulla neurons. Only the simultaneous application of OX1R and OX2R antagonists completely abolished ORXA-induced depolarization (Huang et al., 2010). In this in vitro study, we also compared analogue examples of theta rhythm induced by 50 mM carbachol with theta evoked by ORXA and ORXB, applied in the similar concentration range of 0.01 – 1.0 mM. This rather high concentration of carbachol was earlier tested many times in in vitro conditions (Bland et al., 1988; Konopacki et al., 1988; Konopacki and GołeR biewski, 1993; Konopacki, 1998). Specifically, we demonstrated that carbachol at concentration of 25 lM never induced in vitro theta. The overall level of activation of the hippocampal neuronal network was probably insufficient for theta to appear. However, when the same concentration of carbachol (25 lM) was perfused simultaneously with bicuculine (25 lM), well-synchronized theta was observed (Konopacki and GołeR biewski,1993). Typically, for recording HPC theta rhythm in the in vitro conditions carbachol in a concentration range from 13 to 60 lM was used (MacVicar and Tse, 1989; Williams and Kauer, 1997; Chapman and Lacaille, 1999; Fellous and Sejnowski, 2000). The average concentration of ORXA in the human cerebrospinal fluid ranges >200 pg/ml and the concentration
Orexinergic Theta Rhythm in the Rat Hippocampal Formation: In Vitro and In Vivo Findings Renata Bocian, Paulina Kazmierska, Paulina Kłos-Wojtczak, Tomasz Kowalczyk, and Jan Konopacki*
ABSTRACT: Previous in vivo data suggested that orexin neuropeptides (ORXA and ORXB) synthetized in hypothalamic neurons were involved in the mechanism of generation of the hippocampal formation theta rhythm. Surprisingly, this suggestion has never been directly proved by experiments using intraseptal or intrahippocampal administration of orexins. In this study, involving the use of in vitro hippocampal formation slices and in vivo model of anesthetized rat, we provide the first convergent electropharmacological evidence that in the presence of both ORXA and ORXB the hippocampal formation neuronal network is capable of producing oscillations in the theta band. This effect of orexin peptides was antagonized by selective blockers of orexin receptors (OX1R and OX2R), SB 334867 and TCS OX2 29, respectively. These results provide evidence for a novel, orexinergic mechanism responsible for the production of theta rhythm in the hippocampal formation neuroC 2015 Wiley Periodicals, Inc. nal network. V KEY WORDS: orexin peptides; theta oscillations; hippocampal formation; in vitro; in vivo
INTRODUCTION The neuropeptides orexin-A (ORXA) and orexin-B (ORXB), also called hypocretin-1 and hypocretin-2, were originally identified in neurons of hypothalamus, including the perifornical area, lateral hypothalamus, and dorsomedial nucleus (de Lecea et al., 1998; Sakurai et al., 1998; Henny and Jones, 2006; Hahn, 2010; Mieda et al., 2013). The actions of orexin peptides are mediated by two receptors, OX1R and OX2R (Sakurai et al., 1998). OX1R has preferential affinity for ORXA, whereas OX2R exhibits similar affinities for ORXA/B (Sakurai et al., 1998). Both orexins receptors are widely distributed in a number of structures of the mammalian central nervous system (CNS), including the hippocampal formation, cerebral cortex, olfactory bulb, amygdala, diagonal band of Broca, thalamus, midbrain, brainstem, and spinal cord (Peyron et al., 1998; Sakurai et al., 1998; Nambu et al., 1999; van den
Department of Neurobiology, Faculty of Biology and Environmental Protection, University of Lodz, Poland Grant sponsor: National Science Centre Poland; Grant numbers: UMO2011/01/N/NZ4/01722 and UMO-2013/08/T/NZ3/00026. *Correspondence to: Jan Konopacki, Department of Neurobiology, Faculty of Biology and Environmental Protection, The University of Lodz, 90-236 Lodz, Pomorska str.141/143, Poland. E-mail: [email protected] Accepted for publication 17 March 2015. DOI 10.1002/hipo.22459 Published online 26 March 2015 in Wiley Online Library (wileyonlinelibrary.com). C 2015 WILEY PERIODICALS, INC. V
Pol, 1999; Smart and Jermann, 2002; Kukkonen, 2013; Mieda et al., 2013). This broad distribution of orexin receptors in CNS suggests the involvement of those peptides in many distinct physiological functions (Peyron et al., 1998; Smart and Jerman, 2002; Xu et al., 2013). Indeed, initially the pivotal role of orexins in short-term feeding was well documented (Sakurai et al., 1998; Dube et al., 1999; B€ackeberg et al., 2002; Thorpe and Kotz, 2005; Xu et al., 2013). Other evidence linked orexins to metabolic regulation and thermogenesis (Kukkonen et al., 2002; Monda et al., 2004; Funato et al., 2009; Kukkonen, 2013), stress response (Huang et al., 2010; Gerashchenko et al., 2011; Kukkonen, 2013), circadian rhythms (Deboer et al., 2004; Pekala et al., 2011), the regulation of sleep/wakefulness (Gerashchenko et al., 2001; Inutsuka and Yamanaka, 2013; Mieda et al., 2013; de Lecea and Huertra, 2014), memory processing (Akbari et al., 2008; Selbach et al., 2010), pathogenesis of Alzheimer disease (Kang et al., 2009), and epilepsy (Doreulee et al., 2010). It was also demonstrated that orexins modulate arousal: specifically, rodents treated with orexins spend more time awake (Hagan et al., 1999; Piper et al., 2000), and manifest increased locomotor activity (Alexandre et al., 2013). Electrophysiological studies conducted on cultured nucleus basalis neurons and on rat brain slices demonstrated that ORXA-induced depolarization and increased cell firing were accompanied by a decrease of membrane conductance in cholinergic neurons (Eggermann et al., 2001; Hoang et al., 2004; Mieda et al., 2013). The mechanisms of orexin-induced cell excitation were demonstrated to be linked to the inhibition of inward rectifier K1 current (Ivanov and Aston-Jones, 2000; Hoang et al., 2004), activation of electrogenic sodium-calcium exchanger and activation of non-selective cation channels (Liu et al., 2002; Wu et al., 2002). All tested cholinergic neurons were excited by ORXA, and even more potently by ORXB, suggesting mainly OX2R receptor involvement. ORXA has also been shown in vivo to elicit excitation in both cholinergic and non-cholinergic neurons of the laterodorsal tegmental nucleus (Takahashi et al., 2002). In contrast, it has been demonstrated that in vivo administration of ORXA inhibits the activation of the pedunculopontine tegmental nucleus (PPT) cholinergic neurons by excitation of GABAergic
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neurons of PPT itself (Takakusaki et al., 2005). PPT has been earlier demonstrated to be involved in the regulation of hippocampal formation theta rhythm (Vertes et al., 1993; Kinney et al., 1998; Matulewicz et al., 2010). It sends afferents to the medial septum the structure giving rise to cholinergic and GABAergic projections that are widely known to determine the hippocampal formation theta rhythm (Steward and Fox, 1990; Smythe et al., 1992; Vertes and Kocsis, 1997; Bland and Oddie, 1998; Buzsaki, 2002; Sotty et al., 2003; Ma et al., 2012; Pignatelli et al., 2012; Colgin, 2013).The medial septum/vertical limb of the diagonal band of Broca (MS/vDBB) also receives a dense innervation from hypothalamic orexinergic neurons, and some neurons in MS/vDBB express very high levels of OX2R (de Lecea et al., 1998; Wu et al., 2002, 2004; Mieda et al., 2013). Interestingly, Gerashchenko et al. (2001) demonstrated that injection of orexin-saporin toxin into the medial septal area completely eliminated theta rhythm in rats. Furthermore, it has just recently been demonstrated that intracerebroventricular administration of ORXA increased the power of theta rhythm in freely moving rats (Toth et al., 2012). This data suggests that orexin peptides are involved in the mechanism of generation of HPC theta (Geraschenko et al., 2001; Selbach et al., 2004; Wu et al., 2004; Toth et al., 2012). Surprisingly, this suggestion has never been directly proven by experiments with use of intraseptal or intrahippocampal administration of orexins. In the present study we address the issue of orexinergic control of theta rhythm at the level of hippocampal formation. Using in vitro hippocampal formation slices as a model of inducing theta oscillations (Konopacki, 1998; Kowalczyk et al., 2013a) and an in vivo model of anesthetized rat, we provide the first convergent electropharmacological evidence that in the presence of both ORXA and ORXB peptides the hippocampal formation neuronal network is capable of producing the oscillations in theta band. Preliminary reports of these findings appeared in an abstract form (Bocian et al., 2011; Kazmierska, 2012; Kazmierska et al., 2013).
MATERIALS AND METHODS All experiments described below were monitored by a Local Ethical Commission (permission no. 24/LB 547/2011 in accordance with the European Communities Council Directive of 24 November 1986).
In Vitro Experiments Subject and experimental procedure Primary experiments were performed on 401 hippocampal formation slices delivered from 74 male Wistar rats (120– 150 g). The brain was removed and placed in oxygenated (95% O2 1 5% CO2), cold (3–5 C) artificial cerebrospinal fluid (ACSF; composition in mM: NaCl 121; KCl 5; CaCl2 Hippocampus
2.5; KH2PO4 1.25; MgSO4 1.3; NaHCO3 26; glucose 10; Sigma Chemical). ACSF was made fresh before each experiment using prefiltrated and deionized water (Easy Pure RF, Barnstead). Transverse slices (500 mm) were made from both hippocampi using a tissue slicer (Stoelting). Slices were preincubated and submerged in oxygenated ACSF at a temperature of 20 C for 40 min after dissection. Next the slices were transferred into a gas-liquid interface-recording chamber and maintained on a nylon mesh. Slices were continuously perfused with oxygenated and prewarmed ACSF (always 35 C) at a flow rate of 1 ml/min.
Drugs In the separate control experiments performed on 24 slice preparations, field potentials were induced between 10 and 20 min after incubation with 50 mM carbachol (carbamylcholine chloride, Sigma Chemical, St. Louis) dissolved in ACSF. ORXA and ORXB (PolyPeptide Laboratoires, SAS, France) dissolved in ACSF were administered in doses ranging from 0.01 to 1.0 mM. TCS OX2 29, the selective antagonist of OX2R (Tocris Bioscience, UK), was dissolved in ACSF and administered in a concentration of 10 mM. SB 334867, the selective antagonist of OX1R (Tocris Bioscience, UK), was dissolved in DMSO and then in ACSF. The maximal concentration of DMSO in ACSF (vehicle group, data not shown) did not exceed 1%. The final concentration of SB 334867 was 10 mM.
Recording procedure and data analysis Glass recording electrodes (3–5 MX) filled with 2.0 M sodium acetate were made from Kwik-Fill capillaries (World Precision Instruments, Sarasota, FL). All recordings were performed in the CA3c region of HPC, which is considered to be the main generator of theta rhythm in HPC slices (Konopacki et al., 1988; Kowalczyk et al., 2009). The electrodes were positioned with the use of a micropositioner (IVM, Scientifica, UK). In all experiments field potentials were recorded with respect to the ground. Signals were filtered (0.001–0.3 kHz, band pass) and amplified (10003) using a P-511 preamplifier (Grass-Astromed, West Warwick). The HPC field activity was displayed on a digital storage oscilloscope (Tektronix TDS 3014) and stored on a computer hard drive using data acquisition interface CED-1401 (Cambridge Electronic Design, Cambridge, GB). The HPC field activity was recorded continuously and analysed in five-minute fragments after 10, 20, 30, and 40 min (i.e., 10–15 min fragment; 20–25 min fragment, etc.). Off-line spectral analysis of these fragments was conducted with the use of Spike 2.7 software (Cambridge Electronic Design, GB). The following parameters of the hippocampal field activity were analysed: frequency, amplitude and power. The detailed analysis of theta rhythm covered three samples of well-synchronized theta epochs, selected from each 5 min recording for the specified time period (20, 30, 40 min after compound administration). Additionally, a final recording was also performed after 30 min of washout. The frequency and
OREXINERGIC THETA RHYTHM IN THE HIPPOCAMPAL FORMATION power were determined using the Fast Fourier Transform (FFT) algorithm. Theta amplitude was determined directly from digital recordings. Mean values and standard errors of the mean (x 6 SEM) of three measured theta parameters (frequency, amplitude, and power), obtained from successive time periods after treatment with ORXA and ORXB, were computed and compared with carbachol (CCH) in vitro testing using the ANOVA test and Tukey’s post-hoc test (Statistica 10).
In Vivo Experiments Subjects and surgical procedure The data was obtained from 62 male Wistar rats (120– 150 g). The rats were initially anesthetized (Dragger vapor 19.3) with halothane (Sigma Chemical) while a jugular cannula was inserted. Halothane was then discontinued, and urethane (0.6 g/ml, Sigma Chemical) was administered via the jugular cannula to maintain anesthesia throughout the experiment. In separate experiments the second jugular cannula was used for iv administration of atropine sulfate (30 mg/kg). Anesthesia levels were maintained such that theta field potentials and the transition from theta to LIA could occur spontaneously. Body temperature was maintained at 37 C by a heating pad and heart rate was monitored constantly throughout the experiment. The rats were placed in a stereotaxic frame with the plane between the bregma and lambda levelled to horizontal. An uninsulated tungsten wire placed in the cortex, 2 mm anterior to the bregma, served as an indifferent electrode, and the stereotaxic frame was connected to a ground. A tungsten microelectrode (0.1 – 0.9 MX) for recording hippocampal field activity was placed in the right dorsal HPC, in stratum lacunosum-moleculare (3.2 mm posterior from bregma, 2.0 – 2.2 mm lateral from the midline, and 2.6 – 2.8 mm ventral to the dural surface; Paxinos and Watson, 1998). An AC amplifier (P-511, Grass-Astromed, West Warwick) was used for recording field potentials, with the low filter set at 1 Hz and the high filter set at 0.3 kHz. The field activity was displayed using a digital storage oscilloscope (Tektronix TDS 3014). EEG signals were digitized by interface (1410 plus, Cambridge Electronic Design, GB) and recorded onto computer hard disk for subsequent off-line analysis (Spike 2.29, Cambridge Electronic Design, GB).
Drug administration procedure In the first stage (preliminary experiments) doses of ORXA from 0.2 mg/0.5 ml to 1.0 mg/0.5 ml were tested (data not shown). ORXA was dissolved in physiological saline. The effective, threshold concentrations of ORXA were recognized as inducing hippocampal theta rhythm repeatedly. In further in vivo experiments, only intrahippocampal microinjections of ORXA in concentrations of 0.4 mg/0.5 ml were applied, as concentrations of ORXA below and above 0.4 mg/0.5 ml were found to be ineffective or to induce epileptiform
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discharges (concentration of 1.0 mg/0.5 ml, data not shown). ORXB was administrated in concentrations ranging from 0.1 mg/0.5 ml to 0.8 mg/0.5 ml. ORXB was dissolved in physiological saline. The effective, threshold concentrations of ORXB were recognized as inducing hippocampal theta rhythm repeatedly. In further in vivo experiments only intrahippocampal microinjections of ORXB in concentrations of 0.2 mg/0.5 ml were applied, as concentrations of ORXB below and above 0.2 mg/0.5 ml were found to be ineffective or to induce epileptiform discharges (concentration of 0.8 mg/0.5 ml, data not shown) TCS OX2 29: the selective antagonist of OX2R (Tocris Bioscience, UK) was dissolved in physiological saline and administered in a concentration of 10 mg/0.5 ml. SB 334867: the selective antagonist of OX1R (Tocris Bioscience, UK), was dissolved in DMSO and also administered in a concentration of 10 mg/0.5 ml. The doses of orexin agonists and antagonists were calculated accordingly from in vitro applied concentrations. Orexin antagonists were administrated only in the presence of orexins and dissolved in physiological saline. The maximal concentration of DMSO in physiological saline (vehicle group, data not shown) did not exceed 1%.
Intrahippocampal drugs injection We did not find evidence for lateralization of effects of intrahippocampal injection of drugs used on hippocampal theta (data not shown). Hence, in the primary experiments microinjection of DMSO or saline and all drugs used were always performed into the right hippocampal formation (26 gauge, 5 ml Hamilton 701N microsyringe). The coordinates of Hamilton canulae were as follows: 3.2 mm posterior from bregma, 2.2 mm lateral from the midline, and 2.6 mm ventral to the dural surface (Paxinos and Watson, 1998).
Experimental procedure To characterize the effect of orexin peptides on hippocampal spontaneous theta, 4 min continuous, control recordings of hippocampal field potentials were made prior to microinjection. Four min recordings were also continued in successive time periods: 15 min, 30 min, 45 and 90 min postinjection. Ten 2-s EEG samples containing theta rhythms were selected for analysis from successive time periods. To characterize the effect of orexins on hippocampal field potential in rats pretreated with iv atropine sulfate, 4 min continuous control recordings of hippocampal field potentials were made prior to microinjection.
Data analysis The EEG data obtained from in vivo experiments was analyzed off-line using the Spike 2.7 software computing system. Two-second samples of hippocampal EEG, registered during the spontaneous theta, were subjected to power/frequency (FFT) analysis. The mean peak-to-peak amplitude of theta was determined directly from epochs of theta. Hippocampus
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FIGURE 1. Field potentials of hippocampal formation slices recorded in three different experimental arrangements: A: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in the CA3c field of hippocampal formation slices before and at 10, 20, 30, and 40 min after administration of 50 lM carbachol (CCH). B: Analogue examples of EEG activity and FFT histograms related with indi-
vidual theta samples recorded in the CA3c field of hippocampal formation slices before and at 10, 20, 30, and 40 min after administration of 0.01 2 1.0 lM ORXA. C: Analogue examples of EEG activity recorded in the CA3c field of hippocampal formation slices before and 20 and 40 min after administration of orexins’ antagonists, SB 334867 and TCS OX2 29. Calibration 0.5 s, 500 lV.
Mean values and standard errors of the mean (x 6 SEM) of three measured theta parameters (frequency, amplitude, and power), obtained from control and 45 min after the injection of orexin peptides, were compared using the Mann-Whitney U-test (Statistica 10).
the sagital plane. Slices were then stained with cresyl violet for subsequent verification of electrode and cannulae placement.
RESULTS Histological procedure Following data acquisition, the rat was deeply anesthetized with urethane, perfused with saline, and then with 10% formalin. The brain was removed and stored in 10% formalin at a low temperature. Brains were frozen and sectioned at 20 mm slices in Hippocampus
In Vitro Experiments The effect of carbachol In the control experiments (Figs. 1A and 2A) the effect of CCH (50 mM) on field potentials was studied in the HPC slice preparations. Figures 1A and 2A demonstrates analog
OREXINERGIC THETA RHYTHM IN THE HIPPOCAMPAL FORMATION
FIGURE 2. Field potentials of hippocampal formation slices recorded in three different experimental arrangements: A: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in the CA3c field of hippocampal formation slices before and at 10, 20, 30, and 40 min after administration of 50 lM carbachol (CCH). B: Analogue examples of EEG activity and FFT histograms related with individual
samples of field activity recorded in HPC slices before, and in different time periods from the CCH perfusion onset, with one corresponding FFT histogram taken in 40 min. In the first 10 min after the CCH perfusion onset, all preparations (n 5 24 slices) typically generated epileptiform discharges. CCH-induced theta field potential developed gradually between 10 - 20 min from the perfusion onset. After 40 min of CCH perfusion, mean (6 SEM) theta frequency was 12.35 6 0.63 Hz, mean amplitude was 1339.28 6 274.02 mV, and power 73313.71 6 282.71 mV2. The effect of CCH was reversible 30 min after washout with ACSF.
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theta samples recorded in the CA3c field of hippocampal formation slices before and at 10, 20, 30, and 40 min after administration of 0.01 2 1.0 lM ORXB. C: Analogue examples of EEG activity recorded in the CA3c field of hippocampal formation slices before and 20 and 40 min after administration of orexins’ antagonists, SB 334867 and TCS OX2 29. Calibration 0.5 s, 500 lV.
The effect of ORXA In separate experiments the effect of different concentrations of ORXA on the HPC slice field potentials was evaluated (Fig. 1B). This figure demonstrates analog samples of field activity recorded in HPC slices before, and in different time period from the ORXA perfusion onset, with single corresponding FFT histogram taken at 40 min. When perfused with ORXA at a concentration of 0.01 mM, the HPC preparations (n 5 22 slices) responded only with epileptiform discharges. These hippocampal field potentials appeared usually in the first 10 min of a compound administration and lasted to the end of the experiment (at least 40 min). In the presence of ORXA in Hippocampus
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concentrations of 0.03 mM (Fig. 1B), HPC slice preparations responded with two different patterns of field potentials: epileptiform discharges and theta activity. In 12 out of 20 preparations (60%) mainly theta field potential was noted. The mean frequency of theta measured at 40 min from the perfusion onset was 11.14 6 0.92 Hz, mean amplitude was 833.53 6 246.63 mV and power 25220.92 6 11349.24 mV2. The application of 0.1 mM of ORXA resulted in the highest probability of theta appearance: this particular pattern of field potential appeared in 15 out of 23 slices (65.2%). In this case the mean dominant frequency noted at 40 min from the perfusion onset was lower (9.58 6 1.73 Hz; P < 0.05 vs. 40 min after the onset of perfusion with CCH) than the frequency of theta rhythm recorded at the same time after perfusion of CCH. The mean amplitude was higher (3065.75 6 779.95 mV; P < 0.05 vs. 40 min after the onset of perfusion with CCH), and mean power was higher (619057.81 6 199232.81 lV2, P < 0.05 vs. 40 min after the onset of perfusion with CCH) in comparison to those parameters of theta rhythm recorded at the same time after perfusion of CCH. The bath perfusion of HPC slices with 0.3 mM of ORXA resulted in the appearance of theta activity in 8 out of 21 preparations (38%). The mean dominant frequency registered 40 min from the perfusion onset was 9.31 6 1.86 Hz, mean amplitude was 1016.20 6 247.56 mV, and power 145840.73 6 77684.61 lV2. A further increase of ORXA concentration to 1.0 mM never produced theta field potential. All tested preparations (n 5 22 slices, 100%) responded only with epileptiform discharges. As was the case with CCH perfusion, the effect of ORXA was also reversible after 30 min washout with ACSF. Due to the fact that the highest probability of inducing theta field activity arose after administration of ORXA in the dose of 0.1 mM, this particular concentration was chosen for further pharmacological investigations with the use of orexins’ antagonists (n 5 57; Fig. 1C). These experiments were specifically designed to evaluate the capability of 0.1 mM ORXA to induce theta oscillations in the presence of OX1R and/or OX2R antagonists (SB 334867 and TCS OX2 29 respectively). Generally, in the in vitro condition ORXA never produced theta oscillations in the presence of OX1R and/or OX2R antagonists (Fig. 1C).
The effect of ORXB The analogue examples concerning the perfusion of CCH presented in Figure 2A are precisely the same as shown in a Figure 1A. This data was presented only for comparison of the effect of CCH with that of ORXB on HPC field potentials. In a separate series of experiments (Fig. 2B) the effect of different concentrations of ORXB on HPC slice field potentials was evaluated. Figure 2B demonstrates analogue samples of field activity recorded in HPC slices before and at different time periods from the ORXB perfusion onset with corresponding FFT histograms (taken after 40 min). When perfused with ORXB at a concentration of 0.01 – 0.03 mM, the HPC preparations (n 5 45 slices) responded only with epileptiform Hippocampus
discharges. These hippocampal field potentials appeared usually in the first 10 min of a compound administration and lasted to the end of the experiment (at least 40 min). In a presence of 0.1 mM ORXB (Fig. 2B), HPC slice preparations responded with two different patterns of field potentials: epileptiform discharges and theta activity. A perfusion of HPC slices with 0.1 mM ORXB resulted in the highest probability of theta appearance: in 13 out of 24 preparations (54.1%) mainly theta field potential was noted. In this case the mean frequency of theta measured 40 min from the perfusion onset was lower (10.14 6 0.81 Hz, P < 0.05 vs. 40 min after the onset of perfusion with CCH) than the frequency of theta rhythm recorded at the same time after perfusion of CCH. The mean amplitude was higher (2416.55 6 449.67 mV, P < 0.05 vs. 40 min after the onset of perfusion with CCH) and power was higher (553774.92 6 65093.75 mV2, P < 0.05 vs. 40 min after the onset of perfusion with CCH) in comparison to those parameters of theta rhythm recorded at the same time after perfusion of CCH. The application of 0.3 mM of ORXB resulted in the appearance of theta activity in 10 out of 20 slices (50%). The mean dominant frequency noted 40 min from the perfusion onset was 8.26 6 0.94 Hz, mean amplitude was 1011.48 6 271.03 mV, and power 117934.08 6 45462.86 lV2. The bath perfusion of HPC slices with 1.0 mM of ORXB resulted in the appearance of theta activity in 11 out of 23 preparations (47.8%). The mean dominant frequency registered 40 min from the perfusion onset was 9.21 6 1.04 Hz, mean amplitude was 817.88 6 134.40 mV, and power 43672.36 6 22526.89 lV2. The effect of ORXB was reversible 30 min after washout with ACSF. Due to the fact that the highest probability of inducing theta field activity arose after administration of ORXB in the dose of 0.1 mM, this particular concentration was chosen for further pharmacological investigation, with the use of orexins’ antagonists (n 5 67; Fig. 2C). These experiments were designed to evaluate the capability of 0.1 mM ORXB to induce theta oscillations in the presence of OX1R and/or OX2R antagonists (SB 334867 and TCS OX2 29 respectively). Generally, as it was in the case of ORXA, in the in vitro condition the ORXB never produced theta oscillations in the presence of OX1R and/or OX2R antagonists (Fig. 2C). Control application of the vehicle (DMSO, n 5 33) did not induce any apparent changes in the HPC field potential (data not shown).
In Vivo Experiments The effect of ORXA Three different concentrations of ORXA were tested in preliminary experiments: 0.2 mg/0.5 ml, 0.4 mg/0.5 ml, and 1.0 mg/0.5 ml. The concentration of 0.2 mg/0.5 ml of ORXA was found to be a subthreshold and did not induce any apparent effect on hippocampal field potential (data not shown). The concentration of 1.0 mg/0.5 ml in turn induced epileptiform discharges (data not shown). Hence, the concentration of 0.4 mg/0.5 ml of this agent was chosen for further pharmacological
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FIGURE 3. Field potentials of hippocampal formation recorded in anesthetized rats in three different experimental arrangements: A: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in in stratum lacunosum-moleculare of hippocampal formation before and at 15, 30, 45, and 90 min after administration of 0.4 lg/0.5 ll ORXA. B: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in in stratum lacunosum-moleculare of hippocampal formation before and after
iv administration of atropine sulphate (30 mg/kg) and at 15, 30, 45, and 90 min after the intrahippocampal administration of 0.4 lg/0.5 ll ORXA. C: Analogue examples of EEG activity recorded in the stratum lacunosum-moleculare of hippocampal formation before and after iv administration of atropine sulfate (30 mg/kg) and at 15, 30, 45, and 90 min after the intrahippocampal administration of 0.4 lg/0.5 ll ORXA and SB 334867, ORXA, and TCS OX2 29, ORXA, and SB 334867 and TCS OX2 29. Calibration 1.0 s, 200 lV.
investigation. The effect of ORXA in concentration of 0.4 mg/ 0.5 ml on HPC theta rhythm is shown in Figure 3. Intrahippocampal administration (n 5 7) of ORXA (0.4 mg/ 0.5 ml) in anesthetized rats induced well synchronized epochs of theta rhythm (Fig. 3A). In comparison with the preinjection basic parameters of spontaneous theta (frequency 5.74 6 0.23 Hz, amplitude 601.23 6 10.81 lV, and power 20262.42 6 951.13 lV2) ORXA theta recorded 45 min from the injection onset has a similar frequency (5.69 6 0.11 Hz), increased amplitude 661.29 6 13.15 lV (P < 0.001), and increased power 32990.75 6 753.68 lV2 (P < 0.001). This enhancement of the spontaneous theta amplitude and power was reversible 90 min after the injection. In the separate experiments (n 5 5) the effect of ORXA was tested in anesthetized rats pretreated iv by atropine sulphate (Fig. 3B). As Figure 3B demonstrates, atropine sulphate abolished spontaneous theta rhythm. Intrahippocampal administration of ORXA (0.4 mg/0.5 ml) induced well-synchronized epochs of theta rhythm, which were found to be reversible about 90 min after the injection. In the next experiments (n 5 17) the effect of ORXA on HPC field potential was tested in anesthetized and atropinepretreated rats in the presence of OX1R and/or OX2R
antagonists (SB 334867 – 10 mg/1.0 ml and TCS OX2 29 – 10 mg/1.0 ml, respectively, Fig. 3C). As is shown in Figure 3C, SB 334867 and TCS OX2 29 antagonists never blocked ORXA-induced theta when injected separately. The abolishing effect developed only when both antagonists were administrated simultaneously. The recovery of spontaneous theta rhythm was usually observed 80 – 90 min after the injection.
The effect of ORXB Three different concentrations of ORXB were tested in preliminary experiments: 0.1 mg/0.5 ml, 0.2 mg/0.5 ml, and 0.8 mg/0.5 ml. The concentration of 0.1 mg/0.5 ml of ORXB was found to be a subthreshold and did not induce any apparent effect on hippocampal field potential (data not shown). The concentration of 0.8 mg/0.5 ml in turn induced epileptiform discharges (data not shown). Hence, the concentration of 0.2 mg/0.5 ml of this agent was chosen for further pharmacological investigation. The effect of ORXB in a concentration of 0.2 mg/0.5 ml on HPC theta rhythm is shown in Figure 4. Intrahippocampal administration (n 5 10) of ORXB (0.2 mg/0.5 ml) in anesthetized rats induced well-synchronized epochs of theta rhythm (Fig. 4A). In comparison with preinjection basic Hippocampus
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FIGURE 4. Field potentials of hippocampal formation recorded in anesthetized rats in three different experimental arrangements: A: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in the stratum lacunosum-moleculare of hippocampal formation before and at 15, 30, 45, and 90 min after administration of 0.2 lg/0.5 ll ORXB. B: Analogue examples of EEG activity and FFT histograms related with individual theta samples recorded in the stratum lacunosum-moleculare of hippocampal formation before and after
iv administration of atropine sulfate (30 mg/kg) and at 15, 30, 45, and 90 min after the intrahippocampal administration of 0.2 lg/0.5 ll ORXB. C: Analogue examples of EEG activity recorded in the stratum lacunosum-moleculare of hippocampal formation before and after iv administration of atropine sulfate (30 mg/kg) and at 15, 30, 45, and 90 min after the intrahippocampal administration of 0.2 lg/0.5 ll ORXB and SB 334867, ORXB and TCS OX2 29, ORXB and SB 334867 and TCS OX2 29. Calibration 1.0 s, 200 lV.
parameters of spontaneous theta (frequency 5.23 6 0.18 Hz, amplitude 620.07 6 35.25 lV, and power 23723.83 6 1703.36 lV2) ORXB-induced theta recorded 45 min after the injection has a similar frequency (5.12 6 0.21 Hz, P > 0.05), increased amplitude (691.42 6 7.88 lV, P < 0.001), and increased power (34494.75 6 944.49 lV2, P < 0.001). This enhancement of ORXB-induced theta amplitude and power was reversible 90 min after the injection. In a separate experiments (n 5 5) the effect of ORXB was tested in anesthetized and atropine-pretreated rats (Fig. 4B). As is shown in Figure 4B, atropine sulphate abolished spontaneous theta rhythm. Intrahippocampal administration of ORXB (0.2 mg/0.5 ml) induced well-synchronized epochs of theta rhythm, which was reversible about 90 min after the injection. In the next series of experiments (n 5 15), the effect of ORXB on HPC field potential was tested in anesthetized and atropine-pretreated rats in the presence of OX1R and/or OX2R antagonists (SB 334867 and TCS OX2 29, respectively, Fig. 4C). As is shown in Figure 4C, SB 334867 and TCS OX2 29 never blocked ORXB-induced theta when injected separately. The abolishing effect developed only in the presence of both orexin receptors antagonists, and was also reversible 80 – 90 min after the injection.
The control intrahippocampal injection of the vehicle (DMSO, n 5 3) did not induce any apparent changes in HPC field potential (data not shown).
Hippocampus
DISCUSSION In the present study we provided the first convergent in vitro and in vivo data concerning the modulation or the production of hippocampal formation theta rhythm by orexin peptides. Specifically, we demonstrated that both ORXA and ORXB induced theta in hippocampal formation slices delivered from adult (3 weeks old) rats. This orexin-induced in vitro theta rhythm was antagonized by selective blockers of OX1R and OX2R receptors, SB 334867 and TCS OX2 29 respectively. As a next step, this in vitro finding was verified in experiments conducted in vivo with the use of anesthetized rats of the same age. Overall, in vivo experiments supported our in vitro observations: intrahippocampal injection of both ORXA and ORXB augmented spontaneous HPC type II theta rhythm. In addition, intrahippocampal administration of ORXA and ORXB
OREXINERGIC THETA RHYTHM IN THE HIPPOCAMPAL FORMATION also induced theta oscillations in atropine-pretreated rats. In anesthetized and atropine-treated rats the abolishing effect of orexin receptors antagonists developed only when both agents, SB 334867 and TCS OX2 29, were simultaneously injected. The current results were obtained on HPC tissue maintained in two different experimental environments: in vivo and in vitro in completely deafferented hippocampal formation slices. Despite this fact, the resemblance of the electrophysiological response of the HPC neuronal network to the administration of ORXA and ORXB in the in vitro and in vivo conditions was similar. This finding provides further evidence for the validation of the earlier-developed in vitro model of HPC theta rhythm (Konopacki, 1998; Kowalczyk et al., 2013a) in the study of the neural mechanisms responsible for the generation of this hippocampal formation rhythmic field potential. The basic assumption of the current in vitro and in vivo experiments was the presence of OX1R and OX2R receptors in the HPC. This supposition was earlier supported by a number of reports demonstrating a high density of OX1R and OX2R in different regions of HPC, including the dentate gyrus and hippocampus proper (Trivedi et al., 1998; Kilduff and Payron, 2000; Marcus et al., 2001; Akbari et al., 2011). Soon after the identification of orexins’ neuropeptides, several studies have shown that orexin signalling is essential for the stability of the arousal state. For example, mice deficient in OX1R receptors have a normal amount of sleep and wakefulness across the light/dark cycle, but the stability of these behavioural states is considerably reduced (Mochizuki et al., 2011). In addition, dogs with mutations of OX2R exhibit narcolepsy and cataplexy (Lin et al., 1999). It was also demonstrated that patients that suffer from narcolepsy have also a very low level of ORXA in their cerebrospinal fluid (Thannickal et al., 2000). Furthermore, the intracerebrovetricular administration of ORXA was found to increase wakefulness and decrease both slow wave and REM sleep (Piper et al., 2000). Thus, orexins are generally considered to be excitatory neuropeptides in CNS (Leonard and Kukkonen, 2014). They excite a number of neurons in different regions of CNS, including the basal forebrain, raphe nuclei, locus ceruleus, tuberomammillary nucleus, ventral tegmental area (Hagan et al., 1999; Nakamura et al., 2000; Eggerman et al., 2001; Liu et al., 2002; Yamanaka et al., 2002). Both orexin peptides also have a strong direct excitatory effect on septohippocampal cholinergic (Wu et al., 2004) and GABAergic (Wu et al., 2002) neurons localized in the medial septum/vertical limb of the diagonal band of Broca. MS/vDBB also receives a dense innervation from the orexin neurons, and neurons in this region express a very high level of OX2R (Mieda et al., 2013). It was earlier suggested that ORXBinduced activation of the septohippocampal GABAergic neurons will, by engaging disinhibitory processes in the HPC, promote the production of the hippocampal theta rhythm (Wu et al., 2002). The results of the present in vitro and in vivo study provide the evidence that orexin peptides not only promote but just induce theta rhythm when acting directly at the level of the HPC neuronal network. Specifically, our in vivo experiments demonstrated that both ORXA and ORXB
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peptides augmented HPC spontaneous theta rhythm (type II theta) and also induced theta in atropine-pretreated rats. This is the first direct and unequivocal demonstration of orexininduced theta field potentials recorded in the hippocampal formation. Interestingly, in contrast to the in vitro findings, theta rhythms induced by ORXA and ORXB in the in vivo conditions were abolished only in the presence of both OX1R and OX2R antagonists, SB 334867 and TCS OX2 29.The administration of SB 334867 or TCS OX2 29 alone was completely ineffective in blocking the ORXA- or ORXB-induced theta. This would suggest that in the hippocampal neuronal network, which underlies the mechanisms of production of local theta field potential, each orexin peptide excites both OX1R and OX2R, and there is no noticeable preference in affinity of either ORXA or ORXB for these receptors. From this point of view, the results of the present in vivo experiments conducted on anesthetized rats do not support the earlier suggestion of Sakurai et al. (1998) that OX1R has a preferential affinity for ORXA, whereas OX2R exhibits similar affinities for ORXA and ORXB. On the other hand the effect observed in our study was similar to that previously observed in in vivo and in vitro preparations. Specifically, Jia et al. (2012) reported that the excitatory action of ORXA on behaviour, EEG and spontaneous activity of prefrontal cortex neurons was completely blocked only in the presence of the selective OX1R antagonist SB 334867 and OX2R antagonist TCS OX2 29. It was also observed in vitro that ORXA and ORXB depolarized rostral ventrolateral medulla neurons. Only the simultaneous application of OX1R and OX2R antagonists completely abolished ORXA-induced depolarization (Huang et al., 2010). In this in vitro study, we also compared analogue examples of theta rhythm induced by 50 mM carbachol with theta evoked by ORXA and ORXB, applied in the similar concentration range of 0.01 – 1.0 mM. This rather high concentration of carbachol was earlier tested many times in in vitro conditions (Bland et al., 1988; Konopacki et al., 1988; Konopacki and GołeR biewski, 1993; Konopacki, 1998). Specifically, we demonstrated that carbachol at concentration of 25 lM never induced in vitro theta. The overall level of activation of the hippocampal neuronal network was probably insufficient for theta to appear. However, when the same concentration of carbachol (25 lM) was perfused simultaneously with bicuculine (25 lM), well-synchronized theta was observed (Konopacki and GołeR biewski,1993). Typically, for recording HPC theta rhythm in the in vitro conditions carbachol in a concentration range from 13 to 60 lM was used (MacVicar and Tse, 1989; Williams and Kauer, 1997; Chapman and Lacaille, 1999; Fellous and Sejnowski, 2000). The average concentration of ORXA in the human cerebrospinal fluid ranges >200 pg/ml and the concentration
