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Disruption of footshock-induced theta rhythms by stimulating median raphe nucleus reduces anxiety in rats
Behavioural Brain Research 247 (2013) 193–200
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Disruption of footshock-induced theta rhythms by stimulating median raphe nucleus reduces anxiety in rats Yi-Tse Hsiao a , Pei-Lu Yi a,b,∗∗ , Chiung-Hsiang Cheng a , Fang-Chia Chang a,c,d,∗ a
Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan Department of Sports, Health & Leisure, College of Sports Knowledge, Aletheia University, Tainan Campus, Taiwan Graduate Institute of Brain & Mind Sciences, College of Medicine, National Taiwan University, Taipei, Taiwan d Graduate Institute of Acupuncture Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan b c
h i g h l i g h t s • • • •
Inescapable footshock stimulation increases LF theta rhythms and anxiety. High frequency (100 Hz) stimulation of MRN desynchronizes LF theta power. Administration of bicuculline into MRN decreases LF theta power. Desynchronization of LF oscillations reduces footshock-induced anxiety.
a r t i c l e
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Article history: Received 10 September 2012 Received in revised form 8 March 2013 Accepted 16 March 2013 Available online 28 March 2013 Keywords: Anxiety Footshock ␥-Aminobutyric acid (GABA) Median raphe nucleus Theta waves
a b s t r a c t Theta rhythms generated in the hippocampus are controlled by the pacemaker in the medial septumdiagonal band of Broca (MS-DBB). The median raphe nucleus (MRN) transmits serotonergic signals to the MS-DBB, which suppresses the septo-hippocampus-produced theta waves, whereas GABAergic interneurons in the MRN facilitate the generation of theta oscillations. Animal studies have indicated that fear increases theta oscillations. Moreover, anxiolytics reduce reticular formation-elicited theta rhythms and theta blockade decreases anxiety. In this study, we hypothesized that the MRN mediates anxiety reduction caused by the theta blockade. Our results demonstrated that inescapable-footshock stimulation significantly increased the power of low-frequency theta oscillations (4–7 Hz) in rats. Both the electrical stimulation of MRN and administration of bicuculline into the MRN successfully desynchronized footshock-induced theta oscillations. Compared to the naïve rats, inescapable-footshock stimulation diminished the entry percentage and time spent in the open arms of the elevated plus maze (EPM), behavioral indicators of anxiety. Rats treated with either MRN stimulation or bicuculline administration to desynchronize theta oscillations reduced anxiety caused by the inescapable-footshock stimulation. Our results demonstrated that the electrical stimulation of MRN or blockade of the GABAergic pathways in the MRN interferes with theta oscillations and reduces anxiety, implicating the role of MRN. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Theta waves are sinusoidal oscillations which are generated by the hippocampus and several subcortical nuclei (e.g., medial septum-diagonal band of Broca (MS-DBB), dorsal raphe nucleus,
∗ Corresponding author at: School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4., Roosevelt Road, Taipei, Taiwan. Tel.: +886 2 3366 3883. ∗∗ Corresponding author at: Department of Sports, Health & Leisure, College of Sports Knowledge, Aletheia University, Tainan Campus, Taiwan. Tel: +886 6 570 3100x7716. E-mail addresses: [email protected] (P.-L. Yi), [email protected] (F.-C. Chang). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.03.032
ventral tegmental nucleus of Gudden, and anterior thalamic nuclei) [1]. The frequency ranges of theta rhythms differ among species. In rats, the acceptable spectrum of theta rhythms is between 4 and 10 Hz [1]. Although theta oscillation is commonly observed in the electroencephalogram (EEG) spectra, its function is still unclear. In general, theta waves occur during stress [2,3], memory processing [1,4,5], orienting, exploratory [1,3], or rapid eye movement (REM) sleep [1,6]. In fact, theta rhythms can be divided into two types: type-1 and type-2 theta rhythms [1]. These two types of theta waves are determined by pharmacological manipulations [7–10]. Administration of atropine inhibits type-2 oscillations, but type-1 theta waves are resistant to anticholinergics [7–10]. Under normal condition, frequencies of type-2 theta waves (around 4–9 Hz) are lower than type-1 theta oscillations (around 6–12 Hz) [2,9,11,12].
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According to behavioral properties, type-1 theta is predominant during waking periods in rats, especially during voluntary movement [8–10]. In contrast, type-2 theta dominates EEGs during REM sleep [6] and arousal immobility in rats [8–10,12]. Type-2 theta is also observed when predators are nearby [2,9]. Frequencies of type1 and type-2 theta waves may be overlapped, and the predominant frequency may change slightly in different situations. For example, type-2 theta waves are detected at a frequency higher than 9 Hz in the presence of predators [9]. After treatment with atropine to suppress type-2 theta, dominant theta waves with frequencies lower than 7 Hz are still observed when animals elicit low-speed motor behaviors, such as face washing and teeth chattering in rats [10], as well as rearing and hopping in rabbits [9]. Thus, there is no exact frequency range that distinguishes between type-1 and type-2 waves. Particularly with electrically elicited theta, it can be shown that frequency and type are independent of each other – with movement or non-movement being the key determinant of cholinergic sensitivity. In the present study we simply defined theta waves, with frequencies between 4 and 7 Hz defined as lowfrequency (LF) theta waves, and frequencies between 7 and 10 Hz as high-frequency (HF) theta waves [13]. The relationship between theta waves and animal behaviors (e.g., learning, memory, fear and anxiety) has been demonstrated. For example, the blockade of theta waves impairs initial learning in water mazes, and the restoration of theta rhythms recovers the ability to learn [14]. Both theta waves and context-dependent fear are disrupted by blocking neuronal gap junctions within the dorsal hippocampus in freely moving rats [15]. Studies have shown that the oscillation of theta waves is correlated to stress and anxiety. Anxiolytic drugs inhibit reticularly-elicited theta powers in rats, therefore the strength of theta waves could be used as a neurophysiological index for evaluating the efficacy of anxiolytics [16,17]. To further investigate the role of theta oscillations in anxiety, our current study was designed to manipulate the generation of theta waves and subsequently try to influence the anxiety level. Several brain regions contribute to the production of theta waves [1], and these brain structures could be potential candidates for the disruption of theta wave generation. The MS-DBB is the primary structure controlling theta rhythms. Inactivation or lesion of MS-DBB neurons completely abolishes theta oscillations [18]. The median raphe nucleus (MRN) could further modulate theta rhythms. Studies have reported that GABAergic interneurons of MS-DBB, which suppress theta processing, receive serotoninergic afferents from the MRN [19–21]. Jackson et al. have demonstrated that directly applying 100 Hz of electrical stimulation to the MRN desynchronizes hippocampal theta waves [22]. In addition to its role in the regulation of theta waves, the MRN modulates behaviors in response to the stressors. Our previous study revealed that inescapable footshock stimulation increases the power of LF (4–7 Hz) theta oscillations through the activation of GABAergic pathways in the MRN, which blocks the ability of MRN to desynchronize theta waves [13]. Sainsbury et al. have also demonstrated that low-frequency theta waves increase after inescapable footshock stimuli and that the increase is not due to conditioning [12]. The aforementioned evidence suggests that anxiety and theta oscillations may influence each other via MRN activities. In the present study, we hypothesized that high frequency (100 Hz) electrical stimuli of MRN or blockade of the GABAergic pathways in the MRN interferes with theta functions and reduces anxiety. We herein employed inescapable footshock stimulation as an acute stressor in rats to trigger the enhancement of theta waves. A high frequency electrical stimulus (100 Hz) of MRN, or an administration of bicuculline into the MRN, was applied to desynchronize theta waves, and then the responses of the elevated plus maze (EPM) were employed to measure the footshock-induced anxiety levels.
2. Material and methods Stock solution of bicuculline (Sigma–Aldrich, St. Louis, MO, USA) was dissolved in pyrogen-free saline (PFS). The stock solutions were stored at −20 ◦ C until used. The dose of bicuculline employed in these experiments was 0.025 g based upon our previous results [13]. The total volume for each injection was 1 l and the duration of injection was 3–5 min. Our previous study has indicated that microinjection of 1 l solution into the MRN did not cause the MRN lesion [13]. Male Wistar rats (250–300 g; National Laboratory Animal Breeding and Research Center, Taiwan) were used in the present study. Animals were anesthetized by 50 mg/kg Zoletil® (Tiletamine:Zolazepam = 1:1; Virbac, Carros, France). Three screw EEG electrodes were surgically implanted on the right frontal and parietal lobes and the left occipital lobe as previously described [6]. The occipital electrode was used as a reference. Rats that received electrical stimulation to desynchronize theta oscillations were implanted with a concentric bipolar electrode (model: CBBPE10, 33 gauge, pencil point tip, I.D. 50 m, FHC, Bowdoinham, ME, USA) into the midline area of both sides of the MRNs (AP, −7.9 mm; ML, 0 mm; DV 7.5 mm relative to bregma). This electrode could stimulate both right and left sides of the MRNs. A microinjection guide cannula (26 gauge, O.D. 0.46 mm, I.D. 0.24 mm, Plastics One, Roanoke, VA, USA) was slowly implanted into the MRN (AP, −7.9 mm; ML, 0 mm; DV 7.5 mm relative to bregma) in rats which received bicuculline administration. Coordinates were adopted from the Paxino and Waton rat atlas [23]. The insulated leads from EEG screw electrodes were routed to a Teflon pedestal (Plastic One). The Teflon pedestal was then cemented to the skull with dental acrylic (Tempron, GC Co., Tokyo, Japan). The incision was treated topically with polysporin (polymixin B sulfate–bacitracin zinc) and the animals were allowed to recover for seven days prior to the initiation of experiments. These rats were housed separately in individual recording cages (home cages) in an isolated room, in which the temperature was maintained at 23 ± 1 ◦ C and the light:dark (L:D) rhythm was controlled in a 12:12 h L:D cycle (40 W × 4 tubes illumination). Food and water were available ad libitum. Ibuprofen was dissolved in drinking water and applied for three days after surgery. Experiments were performed one week after recovery from surgery. All procedures performed in this study were approved by the National Taiwan University Animal Care and Use Committee. On the second postsurgical day, rats were connected to the recording apparatus via a flexible tether. Experimental protocols were executed after one week of recovery. The location of the microinjection cannula was confirmed by injecting 0.5% trypan blue dye at the end of the experiment. The recording data were included for subsequent analyses only when the injection target had been confirmed inside the rats’ MRN. Animals were habituated by daily handling and injections of PFS timed to coincide with scheduled experimental administrations. Signals from the EEG electrodes were fed into an amplifier (model V75-01, Coulbourn Instruments, Lehigh Valley, PA, USA). The EEG was amplified (factor of 10,000) and analog bandpass was filtered between 0.1 and 40 Hz (frequency response: ±3 dB; filter frequency roll off: 12 dB/octave). The amplified EEG signals were then input to the iWorx system, an analog-digital converter (model: IX-214, iWorx Systems, Inc., Dover. NH, USA). The EEG signals were recorded by LabScribe 2.0 Software (iWorx Systems, Inc.) with a sampling rate of 100 Hz. EEGs were analyzed with LabScribe 2.0 and the open-source Chronux algorithms (http://chronux.org/) run by the Matlab Signal Processing Toolkit for the fast Fourier transform (FFT) and multi-taper time-frequency spectrum. The concentric bipolar electrode in the MRN was connected to a stimulus isolator (model: A360, World Precision Instruments, Sarasota,
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FL, USA) that was linked to an accupulser (model: A310, World Precision Instruments). A custom-made electrical foot stimulation box (40 cm × 22 cm × 29 cm) was used to initiate acute stress. PT-104 pulse plethysmographs (iWorx Systems, Inc.) were placed under the floors of home cages or footshock boxes to detect locomotion. The vibration signals caused by rat’s movements were converted into the electrical signals by the iWorx system (model: IX-214, iWorx Systems, Inc.). The intensity of vibration is represented by the absolute integral area, which uses zero volts as the zero reference for the integral. Values above and below zero were added to the integral. 2.1. Behavioral tests The EPM consisted of two open (50 cm long × 10 cm wide) and two enclosed arms (50 cm long × 10 cm wide × 40 cm height), and was elevated 60 cm above the floor. Behavioral activities and movements were recorded by a digital camera; this video file was imported to and analyzed by the custom software of Ethovision® (Version XT; Noldus, Wageninmgen, Netherlands). Rats were placed in the cross area of the four arms and faced toward an open arm. The time duration spent in the open arms and the ratio of open arm entries (open arm entries/(open + enclosed arm entries)) was determined as the major index of anxiety [24], and more ambulation toward the open arms indicates less anxiety. The performance of EPM lasted for 5 min. The EPM task was performed in an isolated room under 40 W × 4 tubes illumination. 2.2. Experimental protocols The experimental data were acquired during the first 25 min of the light period. A total of 35 Wistar rats were used and divided into five groups. Group 1 (n = 7) was the naïve control group without manipulation. The EPM task was performed on the naïve group at 20 min after the light onset to match the timing of EPM task in the rest of groups. In group 2 (n = 7), 10-min undisturbed (baseline) EEGs were acquired when rats were in their home cages. Then rats were placed in the footshock box for 10 min without being given electrical stimuli to determine the influence of the novel environment. Rats in group 3 (n = 7) and group 4 (n = 7) had similar protocols as those in group 2, except that rats in group 3 received a 10-min footshock manipulation in the footshock box, and rats in group 4 received a series of electrical MRN stimuli (100 Hz, 40 A, event interval: 15 s, pulse width: 0.1 ms, duration: 2 s) within the 10-min footshock manipulation. After this 10-min footshock manipulation, a 5-min EPM task was performed. The 10min footshock manipulation consists of twelve electrical stimuli, which were randomly given within this 10-min period. The current for each footshock stimulus was 5.0 mA and the stimulation duration was 50 ms. The role of GABAergic effect in the MRN was evaluated in group 5 (n = 7). A dose of 0.025 g bicuculline was microinjected into the MRN prior to the 10-min footshock manipulation, and then the EPM task was performed after the 10-min footshock manipulation. A diagram of the experimental protocol is shown in Fig. 1. 2.3. Statistical analysis All values of spectral powers were presented as mean ± SEM. The values of EEG powers in five distinct spectra (delta (0.5–4 Hz), LF theta (4–7 Hz), HF theta (7–10 Hz), alpha (10–13 Hz), and beta (13–30 Hz)) were calculated by FFT within a 30-s epoch. As aforementioned, type-1 and type-2 theta waves [9,10] differ primarily in terms of whether movement occurs or not and fundamentally in their sensitivity to anticholinergics [1]. Since our current recording for movements could not provide enough sensitivity to
Fig. 1. A diagram of experimental protocol. The thick line depicts the timing for EEG recordings in rat’s home box, dash line represents the timing for EEG recordings in the footshock box, and thin line indicates the timing for the EPM task. Black bar: dark period; white bar: light period; arrow head: administration of 0.025 g bicuculline; bicu: bicuculline; IS: inescapable footshock stimuli.
determine the low speed movement (e.g., head movement), we could not demonstrate here that the LF theta is or is not type2 theta by providing the matching movement data. Therefore, in the present study we simply defined the theta waves with frequencies between 4 and 7 Hz as LF theta and frequencies between 7 and 10 Hz as HF theta waves [13]. The 30-s EEG epoch baseline was acquired before the manipulations. The 30-s EEG result was an average of the 30-s EEGs measured for each individual’s footshock stimulus without artifact. The 30-s EEG obtained immediately after bicuculline administration was used to demonstrate the effect of GABAergic neurons in the MRN. Paired Student’s t-test was used to compare the change () between baseline EEGs and EEGs recorded after different manipulations. One-way analyses of variance (ANOVA) were calculated for analyzing the difference of spectral power between groups. An ˛ level of p < 0.05 was taken as indicating a statistically significant difference. If statistically significant differences were detected, a Fisher’s multiple comparison was made to determine which values during experimental conditions deviated from those obtained from the control conditions. Since FFT power of EEGs is not normally distributed, we applied a log transformation to the raw EEG FFT powers to correct this problem. Symbol depicted the difference of the power values between the power obtained after manipulation and that acquired from baseline EEGs ( = manipulated EEG power − baseline EEG power).
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3.2. Inescapable footshock stimuli enhanced LF theta power Our spectrogram results have demonstrated that inescapable footshocks dramatically raised the LF theta power. The LF theta power was enhanced during inescapable footshock stimuli when compared to the values obtained before footshocks (Fig. 3A). The enhancement of LF theta power mainly occurred during 30 s after the inescapable footshock stimulation (Fig. 3B and C). Thus, we analyzed the powers of different EEG spectra within a 30-s epoch after the footshock stimulus (Fig. 2). The power of LF theta waves increased from 0.51 ± 0.05 log V2 obtained from baseline to 0.68 ± 0.08 log V2 (t = −4.23, df = 6, p < 0.01) after the inescapable footshock stimulation was administered (Fig. 2A, the closed bar). When compared with results acquired from group 2 (the open bars), the inescapable footshock stimulation significantly enhanced the LF theta power by +0.17 ± 0.04 log V2 (F(3,24) = 3.82, p = 0.02; Fisher’s comparison: p < 0.05 vs. the open bar). 3.3. The effects of MRN stimulation and bicuculline administration
Fig. 2. Summary for the alterations of spectral powers after manipulations of MRN activity. The white bars represent the powers acquired from rats when they were in their home cage (baseline) and in the footshock box, the black bars depict the powers obtained from rats when they were in their home cage and in the footshock box with inescapable footshock stimuli, the gray bars represent the powers obtained from rats when they were in their home cage and in the footshock box with footshock + 100 Hz MRN stimulation, and the hatched bars (adopted from [13]) indicate powers acquired from rats when they were in their home cage and in the footshock box with footshock + bicuculline. *p < 0.05 vs. baseline; # p < 0.05 vs. the white bar; § p < 0.05 vs. the black bar.
3. Results
The enhancement of LF theta powers induced after the inescapable footshock stimulation was diminished by the 100 Hz electrical stimulation of the MRN (Fig. 4). There was no significant change of LF theta power in the 30-s epoch between the spectrograms obtained from the baseline and those acquired immediately after the inescapable footshock stimulus along with 100 Hz MRN stimuli (Fig. 4B and C). Stimulation of the MRN reduced the footshock-induced increase of LF theta power to −0.005 ± 0.02 log V2 (the gray bar, Fig. 2A; Fisher’s comparison: p < 0.05 vs. the closed bar). Our results also demonstrated that administration of bicuculline into the MRN significantly blocked the footshock-induced increase of LF theta power (the hatched bar, Fig. 2A; Fisher’s comparison: p < 0.01 vs. the closed bar). No alteration of power in any EEG spectrum was revealed when rats only received the 100 Hz MRN stimulation or was administered with 0.025 g bicuculline into the MRN (data not shown). Our results further indicated that the enhancement of HF theta induced by novel environments was diminished when rats additionally received inescapable footshocks stimuli with/without the 100 Hz electrical stimulation of MRN and bicuculline administration (Fig. 2A).
3.1. HF theta power increased when rats explored in the footshock box
3.4. The results of EPM task
The spectral powers of the delta, LF theta, alpha, and beta waves showed no statistical difference when comparing the values obtained while rats were in the footshock box to those acquired when rats were in their home cage. In Fig. 2A and B, the open bars represent the difference of power values () obtained when rats were in the footshock box and in the home cage. However, the HF theta power was significantly increased when rats encountered the novel environment (Fig. 2A, the open bar). HF theta waves became predominant in the EEG spectra while rats were in the novel footshock box, and the HF theta power increased from 0.21 ± 0.11 log V2 obtained when rats were in their home cages to 0.71 ± 0.09 log V2 (t = −4.86, df = 6, p < 0.01). Since HF theta waves are highly correlated with the locomotion activity [8], we further analyzed the alteration of locomotion activity. The absolute integral area of floor vibration intensity during the locomotion was 1.14 ± 0.64 (arbitrary unit, a.u.) when rats were in their home cages, and the value significantly increased to 4.00 ± 1.34 a.u. (t = −2.97, df = 6, p < 0.05) while rats were in the novel footshock box. These data suggest that the novel environment triggered the rats’ curiosity to explore around the footshock box and resulted in the increase of HF theta power.
The time spent in the open arms of the EPM was significantly decreased from 69.54 ± 23.99 s obtained from the naïve rats to 16.95 ± 7.91 s after receiving inescapable footshock stimuli (F(3,24) = 2.43, p = 0.09, Fisher’s comparison: p < 0.05 vs. the open bar; Fig. 5A). Inescapable footshock stimuli also decreased the proportion of open arm entries when compared to values obtained from naïve rats (F(3,12) = 2.81, p = 0.06, Fisher’s comparison: p < 0.05 vs. the open bar; Fig. 5B). Both the decreased time and entry frequency into the open arms of EPM induced by inescapable footshock stimuli were attenuated by the 100 Hz electrical stimulation of MRN or administration of bicuculline into the MRN. The time spent in the open arms of the EPM appeared to be slightly increased to 36.02 ± 9.61 s when rats received inescapable footshocks with the MRN stimulation, although the change did not reach statistical significance (Fisher’s comparison: p = 0.151 vs. the closed bar; Fig. 5A). The inescapable footshock stimuli-induced decrease of open arm entries was also slightly increased to 31.14 ± 2.22% by the MRN stimulation (Fisher’s comparison: p = 0.29 vs. the closed bar; Fig. 5B), however it did not approach statistical significance. Administration of bicuculline dramatically increased the duration spent in the open arms to 59.63 ± 13.85 s (Fisher’s comparison:
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Fig. 3. The intensity of EEG spectra obtained from undisturbed baseline and inescapable footshock. (A) The changes of spectral powers before and after inescapable footshock stimuli. Red: high intensity; blue: low intensity; arrow heads: inescapable footshock stimuli; arrow: switch rat from home cage to footshock box. (B) A 30-s spectrogram obtained when a rat was in its home cage. (C) A 30-s spectrogram acquired immediately after the inescapable footshock stimulus.
p < 0.05 vs. the closed bar; Fig. 5A) and enhanced the proportion of open arms entries to 39.87 ± 2.94% (Fisher’s comparison: p < 0.05 vs. the closed bar; Fig. 5B). Trace analyses of rats’ movements in the EPM indicated that the inescapable footshock stimulation decreased the time spent in open arms and the proportion of open arm entries. Both 100 Hz MRN stimulation and bicuculline administration into the MRN blocked the avoidance of open arms induced by inescapable footshock stimuli (Fig. 6). 4. Discussion The present study has demonstrated that high frequency (100 Hz) MRN stimulation to desynchronize theta waves diminished inescapable footshock-induced anxiety. Studies have suggested that the theta power is one of neurophysiological indices for measuring anxiety in experimental animals. Nearly all classes of anxiolytics possess the ability to reduce the reticular-elicited theta oscillations; therefore desynchronization of theta waves has been employed as a feasible model to determine the efficacy of anxiolytics [1,17]. Desynchronization of theta waves has been hypothesized
and confirmed as a quickly screening method for searching effective anxiolytics by recent studies. The bulk of the evidence has demonstrated that anxiolytics (both traditional [benzodiazepine] and novel anxiolytics [buspirone]) impair theta activities [25–27]. Gray and McNaughton hypothesized that behavioral influences produced by anxiolytics in animals are similar to those of lesions in the septo-hippocampal system [25,26,28]. They proposed that the septo-hippocampal system acts as a comparator to receive many signal inputs (e.g. punishment, fear, or etc.) and to modulate downstream brain areas to execute conflict resolutions (e.g. increasing arousal, attention, or etc.). This hypothesis illustrates the function of septo-hippocampal system. Anxiolytic drugs reduce anxiety by impairing the subcortical control of hippocampal theta activity, which subsequently suppresses the function of septo-hippocampal system [25,26,28]. Our results demonstrated clear evidence of a relationship between theta waves and anxiety. We have considered that pharmacological methods may simultaneously influence both anxiety levels and theta waves, which makes it difficult to determine whether desynchronization of theta waves is a cause and reduction of anxiety is a consequence, or vice versa. Therefore,
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Fig. 4. The intensity of EEG spectra obtained from the baseline when rats were in their home cage and during footshock + 100 Hz MRN stimulation. (A) The changes of spectral powers before and after footshock + 100 Hz MRN stimulation. (B) A 30-s spectrogram obtained when a rat was in its home cage. (C) A 30-s spectrogram acquired immediately after footshock + 100 Hz MRN stimulation. Red: high intensity; blue: low intensity; arrow heads: inescapable footshock stimuli; arrow: switch rat from home cage to footshock box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
our current study was designed to elucidate the role of MRN in the generation of theta oscillations, the effect of MRN GABAergic neurons on theta waves, and the consequent changes in anxiety levels after manipulating MRN in the unanesthetized, free-moving rats. Bissiere et al. have shown that theta waves were disrupted by blocking neuronal gap junctions in freely moving rats. The neuronal gap junction is located in the amaydala-hippocampus-cortical circuits [29,30] and is correlated with hippocampal and cortical oscillations [31]. They demonstrated that intracerebroventricular administration of gap junction inhibitors disrupted the generation of theta waves during the non-movement and movement state in rats [15]. The gap junction inhibitors simultaneously impair context fear retrieval and speed up fear extinction. McNaughton et al. also published several data regarding that microinjection or systemic injection of anxiolytics impairs theta functions and some kinds of behaviors [25,32,33]. These results suggest that disruption
of theta generation affected some behavioral functions. Our current results further elucidated that interruption of the generation of theta oscillations by a high-frequency MRN stimulation reduced anxiety. The MRN is one of the major brain areas containing enriched serotonergic neurons [34], which transmit serotonergic efferents to GABAergic cells of MS-DBB to suppress the generation of hippocampal theta oscillations [19–21]. Indeed, the septo-hippocampus is believed to be an essential brain region that controls memory processing and anxiety [25]. Serotonergic neurons in the MRN are modulated by the GABA neurotransmitter, which is released by local interneurons [35–37] and the limbic forebrain [38,39]. Administration of GABAA agonist, muscimol, into the MRN produces theta waves in anesthetized rats [40]. Jackson et al. have demonstrated that 100 Hz of electrical stimulation of the MRN inhibits the hippocampal phasic theta-on and theta-off cells, in turn leading to the
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Fig. 5. Desynchronization of footshock-induced theta waves reduced anxiety in the EPM task. The white bars: naïve group, the black bars: footshock group, the gray bars: footshock + 100 Hz MRN stimulation, the hatched bars: footshock + bicuculline. (A) The time spent in the open arms of EPM. (B) The proportion of open arm entries. *p < 0.05 vs. the naïve group. # p < 0.05 vs. the footshock group.
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desynchronization of theta rhythms in urethane-anesthetized rats [22]. Our current results have shown that administration of bicuculline into the MRN suppressed the footshock-induced increase of theta powers, which further confirmed the role of GABAergic interneurons of MRN in the generation of theta oscillations. Our results have also demonstrated that the inescapable footshock stimulus-induced enhancement of LF theta waves was also reduced by the 100 Hz electrical stimulation of MRN. The mechanism of anxiety reduction caused by desynchronizing theta oscillations remains unclear. The EEG-defined theta oscillation reflects a consequence of the phase-locked neuronal firing pattern in a large number of hippocampal cells [25]. These phasic bursting cells may indicate that the hippocampal neurons in response to the stressor are synchronizing and firing in a phase-lock manner to generate the theta oscillation. It may be the reason why desynchronization of theta oscillations impairs hippocampal functions. Furthermore, the power of theta oscillations in the hippocampus is related to the generation of anxiety [17]. We hypothesized that disruption of theta oscillations will reduce anxiety levels, and our results indicated that desynchronizing LF theta oscillations by high frequency (100 Hz) MRN stimulation, or by administration of bicuculline into the MRN, reduced inescapable footshock-induced anxiety. One concern is that the reduction of anxiety may be caused directly by the MRN electrical stimulation, but not as a consequence of disruption in the theta waves. Graeff et al. have determined that electrical stimulation of the MRN with frequency of 60 Hz and intensities ranged from 40 to 200 A causes seemingly stressful behaviors, such as crouching, micturition, piloerection, and teeth chattering, in rats [41]. However, none of these behaviors were observed in rats receiving the MRN electrical stimulation in our current study. Furthermore, our results indicated that anxiety levels were reduced instead of increasing after rats received the MRN electrical stimulation, suggesting that the effect of MRN electrical stimulation on anxiety reduction is a consequence of desynchronizing theta oscillations, but not mediated by the direct MRN stimulation. However, this needs to be confirmed in future studies. We defined LF theta waves as frequencies in the range of 4–7 Hz, which are also in the range of type-2 theta waves. Since type2 theta waves and stress are closely related [2,3], there may be some neurotransmitters that modulate both LF theta oscillations
Fig. 6. Examples of tracing for the rat’s movement in the EPM. The vertical arms are the open arms of the EPMs and horizontal arms are the closed arms. (A) naïve group; (B) footshock group; (C) footshock + 100 Hz MRN stimulation; (D) footshock + bicuculline.
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and stress responses. Our previous study indicates that high levels of hypocretin activated GABAergic interneurons in the MRN and subsequently disinhibited the generation of theta oscillations [13]. We have demonstrated that administration of a hypocretin receptor antagonist, TCS1102, directly into the MRN significantly blocked the footshock-induced enhancement of LF theta power. This observation suggests the involvement of MRN hypocretin and their receptors in the generation of theta waves [13]. Indeed, the role of hypocretin in the generation of theta oscillation [42,43] or its functions in response to the stressors has been elucidated [44,45]. Based on our previous and current findings, we concluded that hypocretin is a critical factor in the MRN in modulating both theta wave generation and anxiety. In summary, this study suggests that manipulation of GABA interneurons in the MRN or change of MRN neuronal activities may provide an efficient way to control anxiety levels. Disclosure statement This is not an industry supported study. Authors have indicated no financial conflicts of interest. Acknowledgements This work was supported by National Science Council grant NSC99-2320-B-002-026-MY3. We thank Mr. Brian Chang for reading and revising this manuscript. We also thank Mr. Yi-Fong Tsai’s technical assistance in this project. References [1] Buzsaki G. Theta oscillations in the hippocampus. Neuron 2002;33:325–40. [2] Sainsbury RS, Heynen A, Montoya CP. Behavioral correlates of hippocampal type 2 theta in the rat. Physiology & Behavior 1987;39:513–9. [3] Vanderwolf CH. Hippocampal electrical activity and voluntary movement in the rat. Electroencephalography and Clinical Neurophysiology 1969;26:407–18. [4] Raghavachari S, Kahana MJ, Rizzuto DS, Caplan JB, Kirschen MP, Bourgeois B, et al. Gating of human theta oscillations by a working memory task. Journal of Neuroscience 2001;21:3175–83. [5] Lisman JE, Idiart MA. Storage of 7+/− 2 short-term memories in oscillatory subcycles. Science 1995;267:1512–5. [6] Chang FC, Opp MR. Blockade of corticotropin-releasing hormone receptors reduces spontaneous waking in the rat. American Journal of Physiology 1998;275:R793–802. [7] Bland BH. The physiology and pharmacology of hippocampal formation theta rhythms. Progress in Neurobiology 1986;26:1–54. [8] Kramis R, Vanderwolf CH, Bland BH. Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane, and pentobarbital. Experimental Neurology 1975;49:58–85. [9] Buzsáki G, Vanderwolf CH, Grastyán E. Electrical activity of the archicortex. Budapest: Akadémiai Kiadó; 1985. [10] Vanderwolf CH. Neocortical and hippocampal activation relation to behavior: effects of atropine, eserine, phenothiazines, and amphetamine. Journal of Comparative & Physiological Psychology 1975;88:300–23. [11] Sainsbury RS, Montoya CP. The relationship between type 2 theta and behavior. Physiology & Behavior 1984;33:621–6. [12] Sainsbury RS, Harris JL, Rowland GL. Sensitization and hippocampal type 2 theta in the rat. Physiology & Behavior 1987;41:489–93. [13] Hsiao YT, Jou SB, Yi PL, Chang FC. Activation of GABAergic pathway by hypocretin in the median raphe nucleus (MRN) mediates stress-induced theta rhythm in rats. Behavioural Brain Researc 2012;233:224–31. [14] McNaughton N, Ruan M, Woodnorth MA. Restoring theta-like rhythmicity in rats restores initial learning in the Morris water maze. Hippocampus 2006;16:1102–10. [15] Bissiere S, Zelikowsky M, Ponnusamy R, Jacobs NS, Blair HT, Fanselow MS. Electrical synapses control hippocampal contributions to fear learning and memory. Science 2011;331:87–91. [16] Engin E, Stellbrink J, Treit D, Dickson CT. Anxiolytic and antidepressant effects of intracerebroventricularly administered somatostatin: behavioral and neurophysiological evidence. Neuroscience 2008;157:666–76. [17] McNaughton N, Kocsis B, Hajos M. Elicited hippocampal theta rhythm: a screen for anxiolytic and procognitive drugs through changes in hippocampal function? Behavioural Pharmacology 2007;18:329–46.
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Research report
Disruption of footshock-induced theta rhythms by stimulating median raphe nucleus reduces anxiety in rats Yi-Tse Hsiao a , Pei-Lu Yi a,b,∗∗ , Chiung-Hsiang Cheng a , Fang-Chia Chang a,c,d,∗ a
Department of Veterinary Medicine, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan Department of Sports, Health & Leisure, College of Sports Knowledge, Aletheia University, Tainan Campus, Taiwan Graduate Institute of Brain & Mind Sciences, College of Medicine, National Taiwan University, Taipei, Taiwan d Graduate Institute of Acupuncture Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan b c
h i g h l i g h t s • • • •
Inescapable footshock stimulation increases LF theta rhythms and anxiety. High frequency (100 Hz) stimulation of MRN desynchronizes LF theta power. Administration of bicuculline into MRN decreases LF theta power. Desynchronization of LF oscillations reduces footshock-induced anxiety.
a r t i c l e
i n f o
Article history: Received 10 September 2012 Received in revised form 8 March 2013 Accepted 16 March 2013 Available online 28 March 2013 Keywords: Anxiety Footshock ␥-Aminobutyric acid (GABA) Median raphe nucleus Theta waves
a b s t r a c t Theta rhythms generated in the hippocampus are controlled by the pacemaker in the medial septumdiagonal band of Broca (MS-DBB). The median raphe nucleus (MRN) transmits serotonergic signals to the MS-DBB, which suppresses the septo-hippocampus-produced theta waves, whereas GABAergic interneurons in the MRN facilitate the generation of theta oscillations. Animal studies have indicated that fear increases theta oscillations. Moreover, anxiolytics reduce reticular formation-elicited theta rhythms and theta blockade decreases anxiety. In this study, we hypothesized that the MRN mediates anxiety reduction caused by the theta blockade. Our results demonstrated that inescapable-footshock stimulation significantly increased the power of low-frequency theta oscillations (4–7 Hz) in rats. Both the electrical stimulation of MRN and administration of bicuculline into the MRN successfully desynchronized footshock-induced theta oscillations. Compared to the naïve rats, inescapable-footshock stimulation diminished the entry percentage and time spent in the open arms of the elevated plus maze (EPM), behavioral indicators of anxiety. Rats treated with either MRN stimulation or bicuculline administration to desynchronize theta oscillations reduced anxiety caused by the inescapable-footshock stimulation. Our results demonstrated that the electrical stimulation of MRN or blockade of the GABAergic pathways in the MRN interferes with theta oscillations and reduces anxiety, implicating the role of MRN. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Theta waves are sinusoidal oscillations which are generated by the hippocampus and several subcortical nuclei (e.g., medial septum-diagonal band of Broca (MS-DBB), dorsal raphe nucleus,
∗ Corresponding author at: School of Veterinary Medicine, National Taiwan University, No. 1, Sec. 4., Roosevelt Road, Taipei, Taiwan. Tel.: +886 2 3366 3883. ∗∗ Corresponding author at: Department of Sports, Health & Leisure, College of Sports Knowledge, Aletheia University, Tainan Campus, Taiwan. Tel: +886 6 570 3100x7716. E-mail addresses: [email protected] (P.-L. Yi), [email protected] (F.-C. Chang). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.03.032
ventral tegmental nucleus of Gudden, and anterior thalamic nuclei) [1]. The frequency ranges of theta rhythms differ among species. In rats, the acceptable spectrum of theta rhythms is between 4 and 10 Hz [1]. Although theta oscillation is commonly observed in the electroencephalogram (EEG) spectra, its function is still unclear. In general, theta waves occur during stress [2,3], memory processing [1,4,5], orienting, exploratory [1,3], or rapid eye movement (REM) sleep [1,6]. In fact, theta rhythms can be divided into two types: type-1 and type-2 theta rhythms [1]. These two types of theta waves are determined by pharmacological manipulations [7–10]. Administration of atropine inhibits type-2 oscillations, but type-1 theta waves are resistant to anticholinergics [7–10]. Under normal condition, frequencies of type-2 theta waves (around 4–9 Hz) are lower than type-1 theta oscillations (around 6–12 Hz) [2,9,11,12].
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According to behavioral properties, type-1 theta is predominant during waking periods in rats, especially during voluntary movement [8–10]. In contrast, type-2 theta dominates EEGs during REM sleep [6] and arousal immobility in rats [8–10,12]. Type-2 theta is also observed when predators are nearby [2,9]. Frequencies of type1 and type-2 theta waves may be overlapped, and the predominant frequency may change slightly in different situations. For example, type-2 theta waves are detected at a frequency higher than 9 Hz in the presence of predators [9]. After treatment with atropine to suppress type-2 theta, dominant theta waves with frequencies lower than 7 Hz are still observed when animals elicit low-speed motor behaviors, such as face washing and teeth chattering in rats [10], as well as rearing and hopping in rabbits [9]. Thus, there is no exact frequency range that distinguishes between type-1 and type-2 waves. Particularly with electrically elicited theta, it can be shown that frequency and type are independent of each other – with movement or non-movement being the key determinant of cholinergic sensitivity. In the present study we simply defined theta waves, with frequencies between 4 and 7 Hz defined as lowfrequency (LF) theta waves, and frequencies between 7 and 10 Hz as high-frequency (HF) theta waves [13]. The relationship between theta waves and animal behaviors (e.g., learning, memory, fear and anxiety) has been demonstrated. For example, the blockade of theta waves impairs initial learning in water mazes, and the restoration of theta rhythms recovers the ability to learn [14]. Both theta waves and context-dependent fear are disrupted by blocking neuronal gap junctions within the dorsal hippocampus in freely moving rats [15]. Studies have shown that the oscillation of theta waves is correlated to stress and anxiety. Anxiolytic drugs inhibit reticularly-elicited theta powers in rats, therefore the strength of theta waves could be used as a neurophysiological index for evaluating the efficacy of anxiolytics [16,17]. To further investigate the role of theta oscillations in anxiety, our current study was designed to manipulate the generation of theta waves and subsequently try to influence the anxiety level. Several brain regions contribute to the production of theta waves [1], and these brain structures could be potential candidates for the disruption of theta wave generation. The MS-DBB is the primary structure controlling theta rhythms. Inactivation or lesion of MS-DBB neurons completely abolishes theta oscillations [18]. The median raphe nucleus (MRN) could further modulate theta rhythms. Studies have reported that GABAergic interneurons of MS-DBB, which suppress theta processing, receive serotoninergic afferents from the MRN [19–21]. Jackson et al. have demonstrated that directly applying 100 Hz of electrical stimulation to the MRN desynchronizes hippocampal theta waves [22]. In addition to its role in the regulation of theta waves, the MRN modulates behaviors in response to the stressors. Our previous study revealed that inescapable footshock stimulation increases the power of LF (4–7 Hz) theta oscillations through the activation of GABAergic pathways in the MRN, which blocks the ability of MRN to desynchronize theta waves [13]. Sainsbury et al. have also demonstrated that low-frequency theta waves increase after inescapable footshock stimuli and that the increase is not due to conditioning [12]. The aforementioned evidence suggests that anxiety and theta oscillations may influence each other via MRN activities. In the present study, we hypothesized that high frequency (100 Hz) electrical stimuli of MRN or blockade of the GABAergic pathways in the MRN interferes with theta functions and reduces anxiety. We herein employed inescapable footshock stimulation as an acute stressor in rats to trigger the enhancement of theta waves. A high frequency electrical stimulus (100 Hz) of MRN, or an administration of bicuculline into the MRN, was applied to desynchronize theta waves, and then the responses of the elevated plus maze (EPM) were employed to measure the footshock-induced anxiety levels.
2. Material and methods Stock solution of bicuculline (Sigma–Aldrich, St. Louis, MO, USA) was dissolved in pyrogen-free saline (PFS). The stock solutions were stored at −20 ◦ C until used. The dose of bicuculline employed in these experiments was 0.025 g based upon our previous results [13]. The total volume for each injection was 1 l and the duration of injection was 3–5 min. Our previous study has indicated that microinjection of 1 l solution into the MRN did not cause the MRN lesion [13]. Male Wistar rats (250–300 g; National Laboratory Animal Breeding and Research Center, Taiwan) were used in the present study. Animals were anesthetized by 50 mg/kg Zoletil® (Tiletamine:Zolazepam = 1:1; Virbac, Carros, France). Three screw EEG electrodes were surgically implanted on the right frontal and parietal lobes and the left occipital lobe as previously described [6]. The occipital electrode was used as a reference. Rats that received electrical stimulation to desynchronize theta oscillations were implanted with a concentric bipolar electrode (model: CBBPE10, 33 gauge, pencil point tip, I.D. 50 m, FHC, Bowdoinham, ME, USA) into the midline area of both sides of the MRNs (AP, −7.9 mm; ML, 0 mm; DV 7.5 mm relative to bregma). This electrode could stimulate both right and left sides of the MRNs. A microinjection guide cannula (26 gauge, O.D. 0.46 mm, I.D. 0.24 mm, Plastics One, Roanoke, VA, USA) was slowly implanted into the MRN (AP, −7.9 mm; ML, 0 mm; DV 7.5 mm relative to bregma) in rats which received bicuculline administration. Coordinates were adopted from the Paxino and Waton rat atlas [23]. The insulated leads from EEG screw electrodes were routed to a Teflon pedestal (Plastic One). The Teflon pedestal was then cemented to the skull with dental acrylic (Tempron, GC Co., Tokyo, Japan). The incision was treated topically with polysporin (polymixin B sulfate–bacitracin zinc) and the animals were allowed to recover for seven days prior to the initiation of experiments. These rats were housed separately in individual recording cages (home cages) in an isolated room, in which the temperature was maintained at 23 ± 1 ◦ C and the light:dark (L:D) rhythm was controlled in a 12:12 h L:D cycle (40 W × 4 tubes illumination). Food and water were available ad libitum. Ibuprofen was dissolved in drinking water and applied for three days after surgery. Experiments were performed one week after recovery from surgery. All procedures performed in this study were approved by the National Taiwan University Animal Care and Use Committee. On the second postsurgical day, rats were connected to the recording apparatus via a flexible tether. Experimental protocols were executed after one week of recovery. The location of the microinjection cannula was confirmed by injecting 0.5% trypan blue dye at the end of the experiment. The recording data were included for subsequent analyses only when the injection target had been confirmed inside the rats’ MRN. Animals were habituated by daily handling and injections of PFS timed to coincide with scheduled experimental administrations. Signals from the EEG electrodes were fed into an amplifier (model V75-01, Coulbourn Instruments, Lehigh Valley, PA, USA). The EEG was amplified (factor of 10,000) and analog bandpass was filtered between 0.1 and 40 Hz (frequency response: ±3 dB; filter frequency roll off: 12 dB/octave). The amplified EEG signals were then input to the iWorx system, an analog-digital converter (model: IX-214, iWorx Systems, Inc., Dover. NH, USA). The EEG signals were recorded by LabScribe 2.0 Software (iWorx Systems, Inc.) with a sampling rate of 100 Hz. EEGs were analyzed with LabScribe 2.0 and the open-source Chronux algorithms (http://chronux.org/) run by the Matlab Signal Processing Toolkit for the fast Fourier transform (FFT) and multi-taper time-frequency spectrum. The concentric bipolar electrode in the MRN was connected to a stimulus isolator (model: A360, World Precision Instruments, Sarasota,
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FL, USA) that was linked to an accupulser (model: A310, World Precision Instruments). A custom-made electrical foot stimulation box (40 cm × 22 cm × 29 cm) was used to initiate acute stress. PT-104 pulse plethysmographs (iWorx Systems, Inc.) were placed under the floors of home cages or footshock boxes to detect locomotion. The vibration signals caused by rat’s movements were converted into the electrical signals by the iWorx system (model: IX-214, iWorx Systems, Inc.). The intensity of vibration is represented by the absolute integral area, which uses zero volts as the zero reference for the integral. Values above and below zero were added to the integral. 2.1. Behavioral tests The EPM consisted of two open (50 cm long × 10 cm wide) and two enclosed arms (50 cm long × 10 cm wide × 40 cm height), and was elevated 60 cm above the floor. Behavioral activities and movements were recorded by a digital camera; this video file was imported to and analyzed by the custom software of Ethovision® (Version XT; Noldus, Wageninmgen, Netherlands). Rats were placed in the cross area of the four arms and faced toward an open arm. The time duration spent in the open arms and the ratio of open arm entries (open arm entries/(open + enclosed arm entries)) was determined as the major index of anxiety [24], and more ambulation toward the open arms indicates less anxiety. The performance of EPM lasted for 5 min. The EPM task was performed in an isolated room under 40 W × 4 tubes illumination. 2.2. Experimental protocols The experimental data were acquired during the first 25 min of the light period. A total of 35 Wistar rats were used and divided into five groups. Group 1 (n = 7) was the naïve control group without manipulation. The EPM task was performed on the naïve group at 20 min after the light onset to match the timing of EPM task in the rest of groups. In group 2 (n = 7), 10-min undisturbed (baseline) EEGs were acquired when rats were in their home cages. Then rats were placed in the footshock box for 10 min without being given electrical stimuli to determine the influence of the novel environment. Rats in group 3 (n = 7) and group 4 (n = 7) had similar protocols as those in group 2, except that rats in group 3 received a 10-min footshock manipulation in the footshock box, and rats in group 4 received a series of electrical MRN stimuli (100 Hz, 40 A, event interval: 15 s, pulse width: 0.1 ms, duration: 2 s) within the 10-min footshock manipulation. After this 10-min footshock manipulation, a 5-min EPM task was performed. The 10min footshock manipulation consists of twelve electrical stimuli, which were randomly given within this 10-min period. The current for each footshock stimulus was 5.0 mA and the stimulation duration was 50 ms. The role of GABAergic effect in the MRN was evaluated in group 5 (n = 7). A dose of 0.025 g bicuculline was microinjected into the MRN prior to the 10-min footshock manipulation, and then the EPM task was performed after the 10-min footshock manipulation. A diagram of the experimental protocol is shown in Fig. 1. 2.3. Statistical analysis All values of spectral powers were presented as mean ± SEM. The values of EEG powers in five distinct spectra (delta (0.5–4 Hz), LF theta (4–7 Hz), HF theta (7–10 Hz), alpha (10–13 Hz), and beta (13–30 Hz)) were calculated by FFT within a 30-s epoch. As aforementioned, type-1 and type-2 theta waves [9,10] differ primarily in terms of whether movement occurs or not and fundamentally in their sensitivity to anticholinergics [1]. Since our current recording for movements could not provide enough sensitivity to
Fig. 1. A diagram of experimental protocol. The thick line depicts the timing for EEG recordings in rat’s home box, dash line represents the timing for EEG recordings in the footshock box, and thin line indicates the timing for the EPM task. Black bar: dark period; white bar: light period; arrow head: administration of 0.025 g bicuculline; bicu: bicuculline; IS: inescapable footshock stimuli.
determine the low speed movement (e.g., head movement), we could not demonstrate here that the LF theta is or is not type2 theta by providing the matching movement data. Therefore, in the present study we simply defined the theta waves with frequencies between 4 and 7 Hz as LF theta and frequencies between 7 and 10 Hz as HF theta waves [13]. The 30-s EEG epoch baseline was acquired before the manipulations. The 30-s EEG result was an average of the 30-s EEGs measured for each individual’s footshock stimulus without artifact. The 30-s EEG obtained immediately after bicuculline administration was used to demonstrate the effect of GABAergic neurons in the MRN. Paired Student’s t-test was used to compare the change () between baseline EEGs and EEGs recorded after different manipulations. One-way analyses of variance (ANOVA) were calculated for analyzing the difference of spectral power between groups. An ˛ level of p < 0.05 was taken as indicating a statistically significant difference. If statistically significant differences were detected, a Fisher’s multiple comparison was made to determine which values during experimental conditions deviated from those obtained from the control conditions. Since FFT power of EEGs is not normally distributed, we applied a log transformation to the raw EEG FFT powers to correct this problem. Symbol depicted the difference of the power values between the power obtained after manipulation and that acquired from baseline EEGs ( = manipulated EEG power − baseline EEG power).
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3.2. Inescapable footshock stimuli enhanced LF theta power Our spectrogram results have demonstrated that inescapable footshocks dramatically raised the LF theta power. The LF theta power was enhanced during inescapable footshock stimuli when compared to the values obtained before footshocks (Fig. 3A). The enhancement of LF theta power mainly occurred during 30 s after the inescapable footshock stimulation (Fig. 3B and C). Thus, we analyzed the powers of different EEG spectra within a 30-s epoch after the footshock stimulus (Fig. 2). The power of LF theta waves increased from 0.51 ± 0.05 log V2 obtained from baseline to 0.68 ± 0.08 log V2 (t = −4.23, df = 6, p < 0.01) after the inescapable footshock stimulation was administered (Fig. 2A, the closed bar). When compared with results acquired from group 2 (the open bars), the inescapable footshock stimulation significantly enhanced the LF theta power by +0.17 ± 0.04 log V2 (F(3,24) = 3.82, p = 0.02; Fisher’s comparison: p < 0.05 vs. the open bar). 3.3. The effects of MRN stimulation and bicuculline administration
Fig. 2. Summary for the alterations of spectral powers after manipulations of MRN activity. The white bars represent the powers acquired from rats when they were in their home cage (baseline) and in the footshock box, the black bars depict the powers obtained from rats when they were in their home cage and in the footshock box with inescapable footshock stimuli, the gray bars represent the powers obtained from rats when they were in their home cage and in the footshock box with footshock + 100 Hz MRN stimulation, and the hatched bars (adopted from [13]) indicate powers acquired from rats when they were in their home cage and in the footshock box with footshock + bicuculline. *p < 0.05 vs. baseline; # p < 0.05 vs. the white bar; § p < 0.05 vs. the black bar.
3. Results
The enhancement of LF theta powers induced after the inescapable footshock stimulation was diminished by the 100 Hz electrical stimulation of the MRN (Fig. 4). There was no significant change of LF theta power in the 30-s epoch between the spectrograms obtained from the baseline and those acquired immediately after the inescapable footshock stimulus along with 100 Hz MRN stimuli (Fig. 4B and C). Stimulation of the MRN reduced the footshock-induced increase of LF theta power to −0.005 ± 0.02 log V2 (the gray bar, Fig. 2A; Fisher’s comparison: p < 0.05 vs. the closed bar). Our results also demonstrated that administration of bicuculline into the MRN significantly blocked the footshock-induced increase of LF theta power (the hatched bar, Fig. 2A; Fisher’s comparison: p < 0.01 vs. the closed bar). No alteration of power in any EEG spectrum was revealed when rats only received the 100 Hz MRN stimulation or was administered with 0.025 g bicuculline into the MRN (data not shown). Our results further indicated that the enhancement of HF theta induced by novel environments was diminished when rats additionally received inescapable footshocks stimuli with/without the 100 Hz electrical stimulation of MRN and bicuculline administration (Fig. 2A).
3.1. HF theta power increased when rats explored in the footshock box
3.4. The results of EPM task
The spectral powers of the delta, LF theta, alpha, and beta waves showed no statistical difference when comparing the values obtained while rats were in the footshock box to those acquired when rats were in their home cage. In Fig. 2A and B, the open bars represent the difference of power values () obtained when rats were in the footshock box and in the home cage. However, the HF theta power was significantly increased when rats encountered the novel environment (Fig. 2A, the open bar). HF theta waves became predominant in the EEG spectra while rats were in the novel footshock box, and the HF theta power increased from 0.21 ± 0.11 log V2 obtained when rats were in their home cages to 0.71 ± 0.09 log V2 (t = −4.86, df = 6, p < 0.01). Since HF theta waves are highly correlated with the locomotion activity [8], we further analyzed the alteration of locomotion activity. The absolute integral area of floor vibration intensity during the locomotion was 1.14 ± 0.64 (arbitrary unit, a.u.) when rats were in their home cages, and the value significantly increased to 4.00 ± 1.34 a.u. (t = −2.97, df = 6, p < 0.05) while rats were in the novel footshock box. These data suggest that the novel environment triggered the rats’ curiosity to explore around the footshock box and resulted in the increase of HF theta power.
The time spent in the open arms of the EPM was significantly decreased from 69.54 ± 23.99 s obtained from the naïve rats to 16.95 ± 7.91 s after receiving inescapable footshock stimuli (F(3,24) = 2.43, p = 0.09, Fisher’s comparison: p < 0.05 vs. the open bar; Fig. 5A). Inescapable footshock stimuli also decreased the proportion of open arm entries when compared to values obtained from naïve rats (F(3,12) = 2.81, p = 0.06, Fisher’s comparison: p < 0.05 vs. the open bar; Fig. 5B). Both the decreased time and entry frequency into the open arms of EPM induced by inescapable footshock stimuli were attenuated by the 100 Hz electrical stimulation of MRN or administration of bicuculline into the MRN. The time spent in the open arms of the EPM appeared to be slightly increased to 36.02 ± 9.61 s when rats received inescapable footshocks with the MRN stimulation, although the change did not reach statistical significance (Fisher’s comparison: p = 0.151 vs. the closed bar; Fig. 5A). The inescapable footshock stimuli-induced decrease of open arm entries was also slightly increased to 31.14 ± 2.22% by the MRN stimulation (Fisher’s comparison: p = 0.29 vs. the closed bar; Fig. 5B), however it did not approach statistical significance. Administration of bicuculline dramatically increased the duration spent in the open arms to 59.63 ± 13.85 s (Fisher’s comparison:
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Fig. 3. The intensity of EEG spectra obtained from undisturbed baseline and inescapable footshock. (A) The changes of spectral powers before and after inescapable footshock stimuli. Red: high intensity; blue: low intensity; arrow heads: inescapable footshock stimuli; arrow: switch rat from home cage to footshock box. (B) A 30-s spectrogram obtained when a rat was in its home cage. (C) A 30-s spectrogram acquired immediately after the inescapable footshock stimulus.
p < 0.05 vs. the closed bar; Fig. 5A) and enhanced the proportion of open arms entries to 39.87 ± 2.94% (Fisher’s comparison: p < 0.05 vs. the closed bar; Fig. 5B). Trace analyses of rats’ movements in the EPM indicated that the inescapable footshock stimulation decreased the time spent in open arms and the proportion of open arm entries. Both 100 Hz MRN stimulation and bicuculline administration into the MRN blocked the avoidance of open arms induced by inescapable footshock stimuli (Fig. 6). 4. Discussion The present study has demonstrated that high frequency (100 Hz) MRN stimulation to desynchronize theta waves diminished inescapable footshock-induced anxiety. Studies have suggested that the theta power is one of neurophysiological indices for measuring anxiety in experimental animals. Nearly all classes of anxiolytics possess the ability to reduce the reticular-elicited theta oscillations; therefore desynchronization of theta waves has been employed as a feasible model to determine the efficacy of anxiolytics [1,17]. Desynchronization of theta waves has been hypothesized
and confirmed as a quickly screening method for searching effective anxiolytics by recent studies. The bulk of the evidence has demonstrated that anxiolytics (both traditional [benzodiazepine] and novel anxiolytics [buspirone]) impair theta activities [25–27]. Gray and McNaughton hypothesized that behavioral influences produced by anxiolytics in animals are similar to those of lesions in the septo-hippocampal system [25,26,28]. They proposed that the septo-hippocampal system acts as a comparator to receive many signal inputs (e.g. punishment, fear, or etc.) and to modulate downstream brain areas to execute conflict resolutions (e.g. increasing arousal, attention, or etc.). This hypothesis illustrates the function of septo-hippocampal system. Anxiolytic drugs reduce anxiety by impairing the subcortical control of hippocampal theta activity, which subsequently suppresses the function of septo-hippocampal system [25,26,28]. Our results demonstrated clear evidence of a relationship between theta waves and anxiety. We have considered that pharmacological methods may simultaneously influence both anxiety levels and theta waves, which makes it difficult to determine whether desynchronization of theta waves is a cause and reduction of anxiety is a consequence, or vice versa. Therefore,
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Fig. 4. The intensity of EEG spectra obtained from the baseline when rats were in their home cage and during footshock + 100 Hz MRN stimulation. (A) The changes of spectral powers before and after footshock + 100 Hz MRN stimulation. (B) A 30-s spectrogram obtained when a rat was in its home cage. (C) A 30-s spectrogram acquired immediately after footshock + 100 Hz MRN stimulation. Red: high intensity; blue: low intensity; arrow heads: inescapable footshock stimuli; arrow: switch rat from home cage to footshock box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
our current study was designed to elucidate the role of MRN in the generation of theta oscillations, the effect of MRN GABAergic neurons on theta waves, and the consequent changes in anxiety levels after manipulating MRN in the unanesthetized, free-moving rats. Bissiere et al. have shown that theta waves were disrupted by blocking neuronal gap junctions in freely moving rats. The neuronal gap junction is located in the amaydala-hippocampus-cortical circuits [29,30] and is correlated with hippocampal and cortical oscillations [31]. They demonstrated that intracerebroventricular administration of gap junction inhibitors disrupted the generation of theta waves during the non-movement and movement state in rats [15]. The gap junction inhibitors simultaneously impair context fear retrieval and speed up fear extinction. McNaughton et al. also published several data regarding that microinjection or systemic injection of anxiolytics impairs theta functions and some kinds of behaviors [25,32,33]. These results suggest that disruption
of theta generation affected some behavioral functions. Our current results further elucidated that interruption of the generation of theta oscillations by a high-frequency MRN stimulation reduced anxiety. The MRN is one of the major brain areas containing enriched serotonergic neurons [34], which transmit serotonergic efferents to GABAergic cells of MS-DBB to suppress the generation of hippocampal theta oscillations [19–21]. Indeed, the septo-hippocampus is believed to be an essential brain region that controls memory processing and anxiety [25]. Serotonergic neurons in the MRN are modulated by the GABA neurotransmitter, which is released by local interneurons [35–37] and the limbic forebrain [38,39]. Administration of GABAA agonist, muscimol, into the MRN produces theta waves in anesthetized rats [40]. Jackson et al. have demonstrated that 100 Hz of electrical stimulation of the MRN inhibits the hippocampal phasic theta-on and theta-off cells, in turn leading to the
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Fig. 5. Desynchronization of footshock-induced theta waves reduced anxiety in the EPM task. The white bars: naïve group, the black bars: footshock group, the gray bars: footshock + 100 Hz MRN stimulation, the hatched bars: footshock + bicuculline. (A) The time spent in the open arms of EPM. (B) The proportion of open arm entries. *p < 0.05 vs. the naïve group. # p < 0.05 vs. the footshock group.
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desynchronization of theta rhythms in urethane-anesthetized rats [22]. Our current results have shown that administration of bicuculline into the MRN suppressed the footshock-induced increase of theta powers, which further confirmed the role of GABAergic interneurons of MRN in the generation of theta oscillations. Our results have also demonstrated that the inescapable footshock stimulus-induced enhancement of LF theta waves was also reduced by the 100 Hz electrical stimulation of MRN. The mechanism of anxiety reduction caused by desynchronizing theta oscillations remains unclear. The EEG-defined theta oscillation reflects a consequence of the phase-locked neuronal firing pattern in a large number of hippocampal cells [25]. These phasic bursting cells may indicate that the hippocampal neurons in response to the stressor are synchronizing and firing in a phase-lock manner to generate the theta oscillation. It may be the reason why desynchronization of theta oscillations impairs hippocampal functions. Furthermore, the power of theta oscillations in the hippocampus is related to the generation of anxiety [17]. We hypothesized that disruption of theta oscillations will reduce anxiety levels, and our results indicated that desynchronizing LF theta oscillations by high frequency (100 Hz) MRN stimulation, or by administration of bicuculline into the MRN, reduced inescapable footshock-induced anxiety. One concern is that the reduction of anxiety may be caused directly by the MRN electrical stimulation, but not as a consequence of disruption in the theta waves. Graeff et al. have determined that electrical stimulation of the MRN with frequency of 60 Hz and intensities ranged from 40 to 200 A causes seemingly stressful behaviors, such as crouching, micturition, piloerection, and teeth chattering, in rats [41]. However, none of these behaviors were observed in rats receiving the MRN electrical stimulation in our current study. Furthermore, our results indicated that anxiety levels were reduced instead of increasing after rats received the MRN electrical stimulation, suggesting that the effect of MRN electrical stimulation on anxiety reduction is a consequence of desynchronizing theta oscillations, but not mediated by the direct MRN stimulation. However, this needs to be confirmed in future studies. We defined LF theta waves as frequencies in the range of 4–7 Hz, which are also in the range of type-2 theta waves. Since type2 theta waves and stress are closely related [2,3], there may be some neurotransmitters that modulate both LF theta oscillations
Fig. 6. Examples of tracing for the rat’s movement in the EPM. The vertical arms are the open arms of the EPMs and horizontal arms are the closed arms. (A) naïve group; (B) footshock group; (C) footshock + 100 Hz MRN stimulation; (D) footshock + bicuculline.
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and stress responses. Our previous study indicates that high levels of hypocretin activated GABAergic interneurons in the MRN and subsequently disinhibited the generation of theta oscillations [13]. We have demonstrated that administration of a hypocretin receptor antagonist, TCS1102, directly into the MRN significantly blocked the footshock-induced enhancement of LF theta power. This observation suggests the involvement of MRN hypocretin and their receptors in the generation of theta waves [13]. Indeed, the role of hypocretin in the generation of theta oscillation [42,43] or its functions in response to the stressors has been elucidated [44,45]. Based on our previous and current findings, we concluded that hypocretin is a critical factor in the MRN in modulating both theta wave generation and anxiety. In summary, this study suggests that manipulation of GABA interneurons in the MRN or change of MRN neuronal activities may provide an efficient way to control anxiety levels. Disclosure statement This is not an industry supported study. Authors have indicated no financial conflicts of interest. Acknowledgements This work was supported by National Science Council grant NSC99-2320-B-002-026-MY3. We thank Mr. Brian Chang for reading and revising this manuscript. We also thank Mr. Yi-Fong Tsai’s technical assistance in this project. References [1] Buzsaki G. Theta oscillations in the hippocampus. Neuron 2002;33:325–40. [2] Sainsbury RS, Heynen A, Montoya CP. Behavioral correlates of hippocampal type 2 theta in the rat. Physiology & Behavior 1987;39:513–9. [3] Vanderwolf CH. Hippocampal electrical activity and voluntary movement in the rat. Electroencephalography and Clinical Neurophysiology 1969;26:407–18. [4] Raghavachari S, Kahana MJ, Rizzuto DS, Caplan JB, Kirschen MP, Bourgeois B, et al. Gating of human theta oscillations by a working memory task. Journal of Neuroscience 2001;21:3175–83. [5] Lisman JE, Idiart MA. Storage of 7+/− 2 short-term memories in oscillatory subcycles. Science 1995;267:1512–5. [6] Chang FC, Opp MR. Blockade of corticotropin-releasing hormone receptors reduces spontaneous waking in the rat. American Journal of Physiology 1998;275:R793–802. [7] Bland BH. The physiology and pharmacology of hippocampal formation theta rhythms. Progress in Neurobiology 1986;26:1–54. [8] Kramis R, Vanderwolf CH, Bland BH. Two types of hippocampal rhythmical slow activity in both the rabbit and the rat: relations to behavior and effects of atropine, diethyl ether, urethane, and pentobarbital. Experimental Neurology 1975;49:58–85. [9] Buzsáki G, Vanderwolf CH, Grastyán E. Electrical activity of the archicortex. Budapest: Akadémiai Kiadó; 1985. [10] Vanderwolf CH. Neocortical and hippocampal activation relation to behavior: effects of atropine, eserine, phenothiazines, and amphetamine. Journal of Comparative & Physiological Psychology 1975;88:300–23. [11] Sainsbury RS, Montoya CP. The relationship between type 2 theta and behavior. Physiology & Behavior 1984;33:621–6. [12] Sainsbury RS, Harris JL, Rowland GL. Sensitization and hippocampal type 2 theta in the rat. Physiology & Behavior 1987;41:489–93. [13] Hsiao YT, Jou SB, Yi PL, Chang FC. Activation of GABAergic pathway by hypocretin in the median raphe nucleus (MRN) mediates stress-induced theta rhythm in rats. Behavioural Brain Researc 2012;233:224–31. [14] McNaughton N, Ruan M, Woodnorth MA. Restoring theta-like rhythmicity in rats restores initial learning in the Morris water maze. Hippocampus 2006;16:1102–10. [15] Bissiere S, Zelikowsky M, Ponnusamy R, Jacobs NS, Blair HT, Fanselow MS. Electrical synapses control hippocampal contributions to fear learning and memory. Science 2011;331:87–91. [16] Engin E, Stellbrink J, Treit D, Dickson CT. Anxiolytic and antidepressant effects of intracerebroventricularly administered somatostatin: behavioral and neurophysiological evidence. Neuroscience 2008;157:666–76. [17] McNaughton N, Kocsis B, Hajos M. Elicited hippocampal theta rhythm: a screen for anxiolytic and procognitive drugs through changes in hippocampal function? Behavioural Pharmacology 2007;18:329–46.
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