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Interaction of GABA and norepinephrine in the lateral division of the bed nucleus of the stria terminals in anesthetized rat, correlating single-unit and cardiovascular responses
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3 June 2017 Please cite this article in press as: Yeganeh F et al. Interaction of GABA and norepinephrine in the lateral division of the bed nucleus of the stria terminals in anesthetized rat, correlating single-unit and cardiovascular responses. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.05.044 1
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INTERACTION OF GABA AND NOREPINEPHRINE IN THE LATERAL DIVISION OF THE BED NUCLEUS OF THE STRIA TERMINALS IN ANESTHETIZED RAT, CORRELATING SINGLE-UNIT AND CARDIOVASCULAR RESPONSES
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FAHIMEH YEGANEH, a ALI NASIMI b AND MASOUMEH HATAM a*
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a Dept. of Physiology, Shiraz University of Medical Sciences, Shiraz, Iran
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Dept. of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract—The bed nucleus of the stria terminalis (BST) consists of multiple anatomically distinct nuclei. The lateral division, which receives dense noradrenergic innervation, has been implicated in cardiovascular regulation and modulation of responses to stress. This study is performed to identify the cardiovascular and single-unit responses of the lateral BST to norepinephrine (NE), involved adrenoceptors, and possible interaction with GABAergic system of the BST in urethane-anesthetized rats. NE, adrenoreceptor antagonists, and GABAA antagonist were microinjected into the lateral division of BST, while arterial pressure (AP), heart rate (HR), and single-unit responses were simultaneously recorded. NE microinjected into the lateral division of BST produced depressor and bradycardic responses. The decrease in AP and HR to NE was blocked by prazosin, an a1-adrenoreceptor antagonist, but not by yohimbine, an a2 antagonist. Furthermore, injections of the GABAA receptor antagonist, bicuculline methiodide (BMI), into the lateral BST abolished the NE-induced depressor and bradycardic responses. We also observed single-unit responses consisting of excitatory and inhibitory responses correlated with cardiovascular function to the microinjection of NE. In conclusion, these data provide the first evidence that microinjection of NE in the lateral division of BST produces depressor and bradycardic responses in urethaneanesthetized rat. The depressor and bradycardiac response are mediated by local a1- but not a2-adrenoceptors. a1-AR activates the GABAergic system within the BST, which in turn produces depressor and bradycardic responses. Ó 2017 Published by Elsevier Ltd on behalf of IBRO. Key words: BST, norepinephrine, GABA, cardiovascular regulation, single unit.
*Corresponding author. Address: Department of Physiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Fax: +98-71-32302026. E-mail address: [email protected] (M. Hatam). Abbreviations: AP, arterial pressure; BMI, bicuculline methiodide; BST, bed nucleus of the stria terminalis; DNB, dorsal noradrenergic bundle; HR, heart rate; MAP, mean arterial pressure; NE, norepinephrine; PSTH, peri-stimulus time histogram; VNB, ventral noradrenergic bundle. http://dx.doi.org/10.1016/j.neuroscience.2017.05.044 0306-4522/Ó 2017 Published by Elsevier Ltd on behalf of IBRO. 1
INTRODUCTION
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The bed nucleus of the stria terminalis (BST) is part of the limbic system (De Olmos et al., 1985; Ju and Swanson, 1989). The lateral division of the BST (BSTL) and the central nucleus of the amygdala are considered as components of the central extended amygdala, owing to their extensive bidirectional connection (Dong et al., 2001; Fudge and Haber, 2001; Sarhan et al., 2005; Gungor et al., 2015). The medial central nucleus of the amygdala projects to the medial part of the anterior division of BST (Dong et al., 2001). The BST is known for its role in response to stress and cardiovascular effect (Hazra et al., 2012; Ventura-Silva et al., 2012; Crestani et al., 2015; Crestani, 2016). The BST may adjust arterial pressure (AP) and heart rate (HR) during stress by activating or inhibiting the sympathetic nervous system (Alves et al., 2007; Zhang et al., 2009; Crestani et al., 2010; Nasimi and Hatam, 2011). Various neurotransmitters, such as acetylcholine (Alves et al., 2007; Nasimi and Hatam, 2011), Angiotensin II (Kafami and Nasimi, 2015), Glutamate (Ciriello and Janssen, 1993; Hatam and Nasimi, 2007), and GABA (Cullinan et al., 1993; Sun and Cassell, 1993; Hatam et al., 2009; Crestani et al., 2013) are involved in cardiovascular effect of the BST. The main source of GABAergic inputs to the BST is the central nucleus of the amygdala, to which it sends reciprocal projections (Dong et al., 2001). BST sends the GABAergic neurons to the region surrounding the hypothalamic paraventricular nucleus (Boudaba et al., 1996), influences the hypothala mic–pituitary–adrenal axis (Choi et al., 2007), and decreases the release of vasopressin (Hatam et al., 2009). GABA exerts its influence on the BST through the activation of GABAA, but not GABAB, receptors (Hatam et al., 2009). The BST is a major target for noradrenergic innervation (Swanson and Hartman, 1975; Moore and Bloom, 1979; Kilts and Anderson, 1986). Two important noradrenergic pathways in the brain are the dorsal noradrenergic bundle (DNB) and ventral noradrenergic bundle (VNB). The DNB originates in the locus coeruleus (Park et al., 2009), while VNB originates in the nucleus tractus solitarius and other noradrenergic cell groups (A1, A5, and A7) (Park et al., 2009; Crestani et al., 2013). The high density of noradrenergic fibers in the BST has been implicated in anxiety, the regulation of the hypothalamic–pitui
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tary–adrenal axis (Cecchi et al., 2002), and the cardiovascular response. The microinjection of NE into the BST of unanesthetized rats caused long-lasting dose-related pressor and bradycardic responses (Crestani et al., 2007). It has also been shown that both a1- and a2adrenoceptors mediate the pressor and bradycardiac responses (Crestani et al., 2008). In some preliminary observations, we found that NE may cause opposite cardiovascular effects depending on the stimulation site in the BST. This is one of the hypotheses which this report has addressed. Also, despite evidence showing the cardiovascular effects of NE and GABA in the BST, there is no study investigating possible interaction between them. This is another hypothesis this report has addressed. Therefore, this study was performed to find: – The cardiovascular effects of different doses of NE (3– 30 nmol/100 nl) in distinct BST regions in anesthetized rats and compare with those in conscious rats (Crestani et al., 2007). – The effect of blocking a1- and a2-adrenoceptors alone or in combination on cardiovascular responses to microinjection of NE into the BST. – The interaction between the GABAergic and noradrenergic systems of the BST. – The correlation between cardiovascular and single-unit responses.
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EXPERIMENTAL PROCEDURES
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General procedures
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The experiments were performed on 120 male Sprague– Dawley rats (250–300 g, 10–11 weeks old). They have been approved by the Animal Use and Care Committee of Shiraz University of Medical Sciences. The rats had free access to food and water under a 12-h light/dark cycle with a room temperature of 25 °C. Animals were anesthetized with urethane (1.4 g/kg, ip), and supplementary doses (0.7 g/kg) were given if needed. The trachea was cannulated to ease the ventilation. The body temperature was maintained at 37 ± 1 °C, using a controlled heat pad. For recording the blood pressure, the right femoral artery was cannulated with a polyethylene catheter (PE-50) filled with heparinized saline. Two holes were drilled above the BST at coordinates of ±1.6 to 2.0 mm mediolateral, 0.12 to 0.36 mm rostrocaudal, and 6.4–7.4 mm ventral to the cortical surface of bregma, according to a rat brain atlas (Paxinose and Watson, 2007).
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Drug microinjections
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A triple-barreled micropipette was made with the use of capillary tubing (Stoleting, USA). One of the barrels contained NE, the second barrel contained the antagonist, while the third barrel was used for recording extracellular action potentials. Drugs were microinjected using a pressurized air pulse applicator. The injection volume was measured by direct observation of the fluid meniscus in the micropipette using a microscope fitted
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with an ocular micrometer that allowed a 2-nl resolution (U.W.O, Canada). The injection volume for each drug was 100 nl. Blood pressure and heart rate were recorded using a ML T844 pressure transducer coupled to a pre-amplifier (FE221 Bridge amplifier, ADinstruments) connected to a power lab 4/35 data acquisition system (model PL3504 AD instruments).
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Single-unit recording
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Extracellular action potentials were recorded using a glass microelectrode pulled to a fine tip diameter (1–3 mm), filled with NaCl solution (2 M). Signals were amplified (10,000), filtered (0.3–3 kHz) by an amplifier (WPI, DAM 80, USA), and displayed on an oscilloscope (Tektronix 5103N, USA). Single-unit firings were digitized, saved in multiunit mode, and isolated using the WPI windows discriminator method with a program written by Nasimi et al. (2012) and Ranjbar et al. (2015). When blood pressure and firing were stable, both blood pressure and the spontaneous activity of the neurons were recorded simultaneously for five minutes; NE was then microinjected into the BST. If a change in blood pressure was observed, we waited for 30 min to an hour to make sure that the effect of NE disappeared before continuing with other interventions.
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Experimental groups
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The experiments consisted of the following groups:
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Fig. 1. Dose–response curve of NE microinjected into the lateral division of BST.
Please cite this article in press as: Yeganeh F et al. Interaction of GABA and norepinephrine in the lateral division of the bed nucleus of the stria terminals in anesthetized rat, correlating single-unit and cardiovascular responses. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.05.044
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The vehicle control group: 100 nl of the vehicle (saline or DMSO) was microinjected into the BSTL. Dose–response groups: To find the dose–response curve of the adrenergic system of the BST for the cardiovascular responses, four doses (3, 10, 20, and 30 nmol/100 nl) of NE (Sigma, USA) were microinjected into the BSTL. The NE control groups: In this group, two consecutive injections of NE were made into the BSTL, one hour apart. Since we aimed to compare the values before treatment with those after treatment, this group was designed to make sure that the effect of the second NE injection was comparable to that of the first. The alpha blocker groups: To find the role of different adrenoreceptors (AR), antagonists were microinjected into the BSTL in three separate groups. First, NE (20 nmol/100 nl) was microinjected Fig. 2. Microinjection of NE (20 nmol/100 nl) into the lateral part of BST caused a long depressor as the control. When blood pressure and bradycardic response (a–c) (paired t-test, MAP: P < 0.001, HR: P < 0. 01, n = 30 rats), with and heart rate returned to the either a long inhibitory (d) or excitatory single-unit responses (e). The arrow shows the injection baseline, a second injection of a time, a: arterial pressure change, b: MAP change, c: HR change, d and e: single-unit responses showed as PSTH. selective a1 antagonist, prazosin (10 nmol/100 nl Sigma), or an a2 antagonist, yohimbine (10 nmol/100 nl, Sigma), or a mixture of prazosin (10 nmol) and yohimbine (10 nmol), in a final volume of 100 nl was microinjected into the BST. Five to eight minutes later, the same site was retested by microinjection of NE (10 nmol/100 nl). The Bicuculline group: First, NE was injected into the BST. An hour later, a selective GABAA antagonist, bicuculline methiodide (BMI, 100 pmol/100 nl, Sigma) was microinjected into the BST. 5–8 min later, the same site was retested by microinjection of NE. Experiments were performed on one side of the brain, and if the condition of animal was stable and BP returned to baseline, another experiment was performed on the contralateral side.
Fig. 3. Time courses of changes in mean arterial pressure and heart rate in response to NE microinjection in the lateral division of BST. The asterisk shows significant changes compared to pre-injection value (paired t-test, MAP: P < 0.001, HR: P < 0. 01, n = 30 rats).
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Data analysis
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The mean arterial pressure (MAP) and heart rate (HR) values were expressed as mean ± SE. The time course of changes of MAP and HR was plotted and the maximum change was compared with that of the preinjection (paired t-test) and the control (independent ttest) values. A P < 0.05 was considered as the statistical significance. For neuronal activity, single-unit spikes were isolated and a peri-stimulus time histogram (PSTH) was plotted from the spike times. After this, the cardiovascular response patterns and the cell firing patterns for each injection were aligned and compared.
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Fig. 4. A sample of cardiovascular responses to NE (20 nmol/100 nl) injection into the lateral division of BST, before and after local injection of prazosin (10 nmol/100 nl, n = 12 rats), an a1adrenoreceptor antagonist. Prazosin caused a decrease in blood pressure and abolished the depressor and excitatory single-unit responses to the second injection of NE (paired t-test P < 0.05). The arrow shows the injection time, a: blood pressure change, b: MAP change, c: HR change, d: single-unit responses showed as PSTH.
Fig. 5. Microinjection of prazosin (10 nmol/100 nl) into the lateral division of the BST changed the depressor response to NE to pressor (20 nmol/100 nl). The arrow shows the injection time, a: blood pressure change, b: MAP change, c: HR change.
Histological verification of injection sites
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At the end of each experiment, the animal was sacrificed using a high dose of the anesthetic (urethane) and perfused transcardially with 100 ml of 0.9% saline followed by 100 ml of 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h. Frozen serial transverse sections (40 mm) of the forebrain were cut and stained with Cresyl Violet 1%. The injection sites were determined according to a rat brain atlas (Paxinose and Watson, 2007), using a light microscope.
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RESULTS
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Control group
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Microinjection of saline (100 nl, n = 5) into the lateral division of BST had no significant effect on mean arterial pressure (MAP), heart rate (HR) or firing rate of the neurons (DMAP = 0.2 ± 0.6 mmHg, DHR = 2 ± 0.9 beats/min, Dfiring rate = 0.2 ± 0.2).
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Dose–response curve of NE microinjected into the lateral division of BST
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To find the response to increasing doses of NE, four doses were injected into the BST. Microinjection of different doses (5, 10, 20, 35 nmol/100 nl, n = 6 for each dose) in the lateral part produced doserelated depressor and bradycardic responses (Fig. 1). The dose of 20 nmol of NE in 100 nl (ED50) was used for further experiments.
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Cardiovascular and single-unit responses to microinjection of NE into BSTL
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Microinjection of NE into the BSTL decreased both MAP and HR (DMAP = 15.5 ± 1 mmHg, DHR = 16 ± 2, n = 30 rats), which were significantly different from the preinjection values (paired t-test, P < 0.001). Samples of arterial pressure and heart rate tracings are shown in Fig. 2a–c. MAP and HR changes began shortly after the
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injection and reached a peak at two to four minutes and then returned to the baseline in about 60 min. The time courses of the changes are shown in Fig. 3. Action potentials of 147 mostly spontaneously active neurons were recorded from the lateral part of BST. In response to NE, 88 neurons (59.8%) showed no response, 37 neurons (25.2%) exhibited excitatory response (Fig. 2e), and 22 neurons (15%) exhibited
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inhibitory response (Fig. 2d). The response was concomitant with the change in MAP or HR. The duration of response was different in various neurons, spanning from 180 to more than 600 seconds.
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Consecutive NE injections
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In this control group, two injections of NE, one hour apart, were given in the lateral division of BST. NE injection (20 nmol/100 nl) produced depressor and bradycardic responses (first injection: DMAP = 12 ± 1.8 mmHg, and DHR = 14 ± 4.2 beats/min; second injection: DMAP = 13 ± 2.7 mmHg, and DHR = 9 ± 1.7 beats/min). There was no significant difference between the first and second injections (paired t-test, P > 0.05).
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Fig. 6. A sample of cardiovascular responses to NE (20 nmol/100 nl) injection into the lateral division of BST, before and after local injection of yohimbine, an a2-adrenoreceptor antagonist (10 nmol/100 nl). Yohimbine did not affect the depressor response to NE (n = 12 rats). The arrow shows the injection time, a: blood pressure change, b: MAP change, c: HR change.
Fig. 7. Microinjection of a combination of prazosin (10 nmol) and yohimbine (10 nmol) blocked the NE depressor responses into the lateral division of the BST (paired t-test, MAP: P < 0.001, HR: P < 0.01, n = 9 rats). The arrow shows the injection time, a Blood pressure change, b: MAP change, c: HR change.
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Cardiovascular and single-unit responses to microinjection of adrenoceptor (AR) blockers into the lateral part of BST
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To find the type of AR involved in mediating depressor response, first the NE was microinjected into the BST. Then, after an hour, 100 nl of the selective a1-AR antagonist, prazosin, or a2-AR antagonist, yohimbine, or a combination of the two was microinjected into the BST. Five to seven minutes later, NE was microinjected into the same site. Microinjection of prazosin (10 nmol/100 nl) into the lateral part of BST elicited a decrease in MAP ( 17.3 ± 3.4 mmHg, n = 11), with no significant change in HR ( 4.3 ± 2 bpm) (Fig. 4). Prazosin greatly attenuated the depressor response to NE by 68% compared to that of the control (DMAP = 15.45 ± 1.5 vs. 4.9 ± 2.8, paired t-test, P < 0.05), while the HR response was attenuated by 87% (DHR = 19 ± 5.1 vs. 2.45 ± 3.8, paired t-test, P < 0.05, n = 12 rats) (Figs. 4 and 9). In four rats, the NE depressor response changed to a pressor response without a significant change in HR (Fig. 5). Microinjection of yohimbine (10 nmol/100 nl) did not elicit any cardiovascular response (MAP = 78.7 ± 4.7 vs. 80.9 ± 5 mmHg and HR = 375.7 ± 15.2 vs. 357 ± 15 bpm, n = 12 rats). Yohimbine did not affect the depressor (before: DMAP = 13.1 ± 2.2, after: DMAP = 14.4 ± 3.5 mmHg) or HR
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response (before: DHR = 8.9 ± 2.4 beats/min, after: DHR = 5.7 ± 1.9 beats/min) to NE (Figs. 6 and 9). Injection of a combination of prazosin (10 nmol/100 nl) and yohimbine (10 nmol/100 nl) into the BSTL elicited a decrease in MAP (DMAP = 10.2 ± 3.5 mmHg, paired t-test P < 0.05) with no significant change in HR ( 6 ± 2.7 bpm), while greatly attenuating the depressor (DMAP = 18.5 1.9 vs. 2.5 ± 2.6, paired t-test, P < 0.05) and bradycardic (DHR 14.8 ± 3.1 vs 2.3 ± 1.8 paired t-test P < 0.05) responses to NE (Fig. 7). In most cases, the depressor response changed to a pressor response. Fig. 9 summarizes the results of these experiments. Excitatory (n = 11 neurons) and inhibitory (n = 12 neurons) singleunit responses to NE injection were abolished by prazosin (Fig. 4d). However, yohimbine had no significant effect on excitatory/ inhibitory effect of NE.
Cardiovascular and single-unit responses to microinjection of selective GABAA antagonist followed by NE into the BSTL
Fig. 8. A sample of cardiovascular responses to NE injection into the lateral division of BST, before and after local injection of BMI (100 pmol/100 nl, n = 7 rats), a GABAA receptor antagonist. BMI increased MAP, HR and firing rate. BMI abolished the depressor response to the second injection of NE. The arrow shows the injection time, a: blood pressure change, b: MAP change, c: HR change, d: single-unit responses showed as PSTH.
To investigate the synaptic mechanism involved in the depressor response of NE in the lateral part of BST, first NE was microinjected into the BST. Then, after one hour, the selective GABAA antagonist, bicuculline methiodide (BMI, 100 pmol/100 nl), was injected. Then, NE was injected into the same site again. As shown in Figs. 8 and 9, BMI elicited an increase in both MAP and HR (DMAP = 47.3 ± 10.8 mmHg, DHR = 45.6 ± 15.5 bpm, n = 7 rats, paired t-test P < 0.01). Pretreatment with BMI blocked the depressor response to NE injection (before: DMAP = 15 ± 2.8 mmHg, after: DMAP = 0.6 ± 0.7 mmHg, paired t-test P < 0.01; before: DHR = 12.4 ± 4.1 beats/min, after: DHR = 5 ± 5.3 beats/min, paired t-test P < 0.05). The extracellular action potentials of 22 neurons were recorded in these experiments. Injection of BMI caused excitation in 18 neurons (81.8%, Fig. 8d) and had no significant effect on the firing rate of four neurons (18.2%).
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Histology
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Distribution of the injection sites is shown in Fig. 10. Data of the injection sites outside the BSTL were not included in the analysis.
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DISCUSSION
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The BST is one of the major targets of noradrenergic innervation in the brain (Swanson and Hartman, 1975; Moore and Bloom, 1979; Kilts and Anderson, 1986) originating mainly from neurons located in the A1, A2, and A5 brainstem nuclei, and in a lesser degree from the A6 (Fallon and Moore, 1978; Byrum and Guyenet, 1987; Riche et al., 1990; Woulfe et al., 1990; Forray et al., 2000). Noradrenergic terminals are mainly located in the ventral portion (Phelix et al., 1992; Freedman and Cassell, 1994) of the anterior part of the lateral side of the BST (Fuentealba et al., 2000). These areas correspond to the regions in our experiments (Fig.10). The present study has been performed to find cardiovascular and electrophysiological effects of NE in the BSTL and to determine if its effects are mediated through GABAergic neuronal systems. To facilitate the discussion, a neural network is postulated based on the findings of the present and some previous papers (Fig.11). In response to stressful challenges, BST adjusts the levels of arterial pressure and heart rate, appropriate for the stressor, by either activating or inhibiting the sympathetic nervous system (Ciriello and Janssen, 1993; Roder and Ciriello, 1993; Dunn and Williams, 1995; Alves et al., 2007; Crestani et al., 2009; Zhang et al., 2009; Kuwaki, 2011). It has been shown that BST projects to CVLM, mediating sym-
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pathoinhibition, and to RVLM mediating sympathoexcitaon sympathoexcitatory neurons, as postulated in tion (Giancola et al., 1993; Dong et al., 2001; Dong and Fig. 11, provided that there is a tonic release of NE. Swanson, 2004; Hatam and Ganjkhani, 2012). As shown Another possible explanation of this result is based on in the postulated diagram (Fig. 11), the sympathoexcitaearlier observation that NE has an inhibitory action on tory neuron receives three inputs, one positive input (a1) local glutamatergic neurotransmission in vBST (Egli from noradrenergic terminals and one negative input from et al., 2005). This area corresponds to the regions where GABAergic neurons. GABA neuron receives one positive prazosin was injected in our experiments. Two studies noradrenergic input, a1. The network can produce either have demonstrated that stimulation of the lateral division depressor or pressor responses. It produces a depressor of BST with glutamate decreases BP and HR in response if activity of GABA overcomes the excitatory urethane-anesthetized rats (Ciriello and Janssen, 1993; effects of a1 on the sympathoexcitatory neuron. Hatam and Nasimi, 2007). Thus, blockade of a1-AR may We found that microinjection of NE into the lateral part disinhibit the glutamate release, consequently causing a of BST decreased MAP and HR in urethane-anesthetized depressor response; however, there is no tonic release rats (Fig. 2). It was shown that the injection of NE into the of glutamate in the lateral BST (Hatam and Nasimi, 2007). BST of unanesthetized rats caused long-lasting doseThe pretreatment of BST with yohimbine, an a2-AR related pressor and bradycardiac responses, which was antagonist, did not affect depressor or bradycardic greatly reduced in urethane-anesthetized rats but did responses to injection of NE (Fig. 6), indicating that a2not change to depressor response (Crestani et al., AR is not involved in the depressor response to NE. 2007). So, our finding for the lateral part is different from Similar to prazosin, the response to NE was blocked by the previous study. As seen in Fig. 2, two types of pretreatment with a combination of prazosin and single-unit responses were recorded along with the yohimbine (Fig. 7). depressor responses. The excitatory record could be from the GABAergic neuron stimulated by NE, and the long inhibitory response could be from the sympathoexcitatory neuron which was inhibited by the GABAergic inputs (Fig. 11). To find the possible involvement of a1 or a2 receptors in depressor response, we pretreated the BST with the selective a1-AR antagonist, prazosin, or the selective a2-AR antagonist, yohimbine. The depressor response to NE was blocked by the pretreatment of the BST with prazosin (Fig. 4), indicating that the pressor response is mediated by a1-AR. The singleunit response, shown in Fig.4, probably recorded from the GABA neuron which receives a1 input (Fig. 11). Therefore, by blocking this receptor, GABA neuron did not respond to NE and the depressor response disappeared. In some cases, the depressor response to NE altered to a pressor one by prazosin (Fig. 5), suggesting the possible involvement of b-AR in stimulating the sympathoexcitatory neuron. The presence of local b-AR in the BST and its effect on pressor response to NE microinjected into the BST has been previously demonstrated (Crestani et al., 2007). As seen in Figs. 4 and 5, prazosin caused a decrease in MAP and HR. The depressor effect of prazosin Fig. 9. Bar charts summarizing the effects of various drugs injected into the BSTL on MAP and could also be explained by the HR. Note that depressor and bradycardic responses were significantly attenuated after prazosin, whereas yohimbine alone did not significantly affect MAP possibility of direct positive a1 action or bicuculline or prazosin + yohimbine, * ** or HR (paired t-test, P < 0.05 , P < 0.01 ).
Please cite this article in press as: Yeganeh F et al. Interaction of GABA and norepinephrine in the lateral division of the bed nucleus of the stria terminals in anesthetized rat, correlating single-unit and cardiovascular responses. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.05.044
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Fig. 11. Schematic illustration of the proposed neuronal interaction pathways involved in mediating the depressor and bradycardia responses to NE inputs to the lateral BST neurons.
Fig. 10. Schematic coronal section of rat brain adopted from an atlas (Paxinose and Watson, 2007). The injection sites of NE were shown as star symbol, bicuculline as filled circles, prazosin as filled squares, yohimbine as open squares and prazosin + yohimbine as filled triangle ac: anterior commissure; BSTLD: BST, lateral division, dorsal part; BSTLI: BST, lateral division, intermediate part; BSTLP: BST, lateral division, posterior part; BSTLV: BST, lateral division, ventral part.
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As summarized in Fig. 9, the depressor and bradycardic effects of NE were mediated through a1-AR but not a2-AR in urethane-anesthetized rats. In another series of experiments, we explored whether the depressor and bradycardic responses to NE were mediated through activation of GABAA receptors. Injection of bicuculline methiodide (BMI, a GABAA receptor antagonist) into the lateral BST produced pressor response and tachycardia, which was consistent with our previous findings (Hatam et al., 2009). BMI blocked NE-induced depressor and bradycardic responses (Fig. 8). In other words, GABA mediates sympathoinhibitory and bradycardia effects of NE in the lateral
part of BST. Taken together, these data suggest that NE depressor effect in the lateral BST is mediated by a1-AR, which is probably located on the GABAergic neurons, as postulated in Fig. 11. In support of the postulated network (Fig. 11), it was reported that activation of a1- and b-adrenoceptors depolarizes the local GABAergic neurons in the BST (Dumont and Williams, 2004). In response to NE, this network could produce either pressor or depressor response, based on the strength of GABA transmission. The prolongation of synaptic inhibition by the positive modulation of GABAA receptor function is an important component of the central nervous system’s depressant effects of volatile anesthetics and several chemically distinct intravenous anesthetics (Tanelian et al., 1993). Therefore, anesthesia facilitates GABA release, resulting in a depressor effect of NE instead of pressor in the lateral BST. Also, when a1 is blocked, GABA release will decrease and the pressor effect of NE may appear, as shown in Fig. 5. GABAergic transmission, as shown in Fig. 11, may have the physiological benefit of modulating the pressor effects of NE by preventing over-stimulation of sympathoexcitatory neurons. In conclusion, these data provide the first evidence that microinjection of NE in the lateral division of BSTproduced depressor and bradycardia responses in urethane-anesthetized rat. The depressor and bradycardiac responses are mediated by local a1- but not a2-adrenoceptors. a1-AR activates GABAergic systems within the BST, which in turn produces a depressor and bradycardic response.
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Acknowledgment—This manuscript was extracted from a PhD thesis by Fahimeh Yeganeh sponsored by the Vice Chancellery of Research of Shiraz University of Medical Sciences (grant number: 7309). The authors would like to thank Shiraz University of Medical Sciences, Shiraz, Iran and also Center for Development of Clinical Research of Nemazee Hospital and Dr. Nasrin Shokrpour for editorial assistance.
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(Received 30 January 2017, Accepted 23 May 2017) (Available online xxxx)
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INTERACTION OF GABA AND NOREPINEPHRINE IN THE LATERAL DIVISION OF THE BED NUCLEUS OF THE STRIA TERMINALS IN ANESTHETIZED RAT, CORRELATING SINGLE-UNIT AND CARDIOVASCULAR RESPONSES
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FAHIMEH YEGANEH, a ALI NASIMI b AND MASOUMEH HATAM a*
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a Dept. of Physiology, Shiraz University of Medical Sciences, Shiraz, Iran
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b
Dept. of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract—The bed nucleus of the stria terminalis (BST) consists of multiple anatomically distinct nuclei. The lateral division, which receives dense noradrenergic innervation, has been implicated in cardiovascular regulation and modulation of responses to stress. This study is performed to identify the cardiovascular and single-unit responses of the lateral BST to norepinephrine (NE), involved adrenoceptors, and possible interaction with GABAergic system of the BST in urethane-anesthetized rats. NE, adrenoreceptor antagonists, and GABAA antagonist were microinjected into the lateral division of BST, while arterial pressure (AP), heart rate (HR), and single-unit responses were simultaneously recorded. NE microinjected into the lateral division of BST produced depressor and bradycardic responses. The decrease in AP and HR to NE was blocked by prazosin, an a1-adrenoreceptor antagonist, but not by yohimbine, an a2 antagonist. Furthermore, injections of the GABAA receptor antagonist, bicuculline methiodide (BMI), into the lateral BST abolished the NE-induced depressor and bradycardic responses. We also observed single-unit responses consisting of excitatory and inhibitory responses correlated with cardiovascular function to the microinjection of NE. In conclusion, these data provide the first evidence that microinjection of NE in the lateral division of BST produces depressor and bradycardic responses in urethaneanesthetized rat. The depressor and bradycardiac response are mediated by local a1- but not a2-adrenoceptors. a1-AR activates the GABAergic system within the BST, which in turn produces depressor and bradycardic responses. Ó 2017 Published by Elsevier Ltd on behalf of IBRO. Key words: BST, norepinephrine, GABA, cardiovascular regulation, single unit.
*Corresponding author. Address: Department of Physiology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. Fax: +98-71-32302026. E-mail address: [email protected] (M. Hatam). Abbreviations: AP, arterial pressure; BMI, bicuculline methiodide; BST, bed nucleus of the stria terminalis; DNB, dorsal noradrenergic bundle; HR, heart rate; MAP, mean arterial pressure; NE, norepinephrine; PSTH, peri-stimulus time histogram; VNB, ventral noradrenergic bundle. http://dx.doi.org/10.1016/j.neuroscience.2017.05.044 0306-4522/Ó 2017 Published by Elsevier Ltd on behalf of IBRO. 1
INTRODUCTION
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The bed nucleus of the stria terminalis (BST) is part of the limbic system (De Olmos et al., 1985; Ju and Swanson, 1989). The lateral division of the BST (BSTL) and the central nucleus of the amygdala are considered as components of the central extended amygdala, owing to their extensive bidirectional connection (Dong et al., 2001; Fudge and Haber, 2001; Sarhan et al., 2005; Gungor et al., 2015). The medial central nucleus of the amygdala projects to the medial part of the anterior division of BST (Dong et al., 2001). The BST is known for its role in response to stress and cardiovascular effect (Hazra et al., 2012; Ventura-Silva et al., 2012; Crestani et al., 2015; Crestani, 2016). The BST may adjust arterial pressure (AP) and heart rate (HR) during stress by activating or inhibiting the sympathetic nervous system (Alves et al., 2007; Zhang et al., 2009; Crestani et al., 2010; Nasimi and Hatam, 2011). Various neurotransmitters, such as acetylcholine (Alves et al., 2007; Nasimi and Hatam, 2011), Angiotensin II (Kafami and Nasimi, 2015), Glutamate (Ciriello and Janssen, 1993; Hatam and Nasimi, 2007), and GABA (Cullinan et al., 1993; Sun and Cassell, 1993; Hatam et al., 2009; Crestani et al., 2013) are involved in cardiovascular effect of the BST. The main source of GABAergic inputs to the BST is the central nucleus of the amygdala, to which it sends reciprocal projections (Dong et al., 2001). BST sends the GABAergic neurons to the region surrounding the hypothalamic paraventricular nucleus (Boudaba et al., 1996), influences the hypothala mic–pituitary–adrenal axis (Choi et al., 2007), and decreases the release of vasopressin (Hatam et al., 2009). GABA exerts its influence on the BST through the activation of GABAA, but not GABAB, receptors (Hatam et al., 2009). The BST is a major target for noradrenergic innervation (Swanson and Hartman, 1975; Moore and Bloom, 1979; Kilts and Anderson, 1986). Two important noradrenergic pathways in the brain are the dorsal noradrenergic bundle (DNB) and ventral noradrenergic bundle (VNB). The DNB originates in the locus coeruleus (Park et al., 2009), while VNB originates in the nucleus tractus solitarius and other noradrenergic cell groups (A1, A5, and A7) (Park et al., 2009; Crestani et al., 2013). The high density of noradrenergic fibers in the BST has been implicated in anxiety, the regulation of the hypothalamic–pitui
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tary–adrenal axis (Cecchi et al., 2002), and the cardiovascular response. The microinjection of NE into the BST of unanesthetized rats caused long-lasting dose-related pressor and bradycardic responses (Crestani et al., 2007). It has also been shown that both a1- and a2adrenoceptors mediate the pressor and bradycardiac responses (Crestani et al., 2008). In some preliminary observations, we found that NE may cause opposite cardiovascular effects depending on the stimulation site in the BST. This is one of the hypotheses which this report has addressed. Also, despite evidence showing the cardiovascular effects of NE and GABA in the BST, there is no study investigating possible interaction between them. This is another hypothesis this report has addressed. Therefore, this study was performed to find: – The cardiovascular effects of different doses of NE (3– 30 nmol/100 nl) in distinct BST regions in anesthetized rats and compare with those in conscious rats (Crestani et al., 2007). – The effect of blocking a1- and a2-adrenoceptors alone or in combination on cardiovascular responses to microinjection of NE into the BST. – The interaction between the GABAergic and noradrenergic systems of the BST. – The correlation between cardiovascular and single-unit responses.
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EXPERIMENTAL PROCEDURES
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General procedures
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The experiments were performed on 120 male Sprague– Dawley rats (250–300 g, 10–11 weeks old). They have been approved by the Animal Use and Care Committee of Shiraz University of Medical Sciences. The rats had free access to food and water under a 12-h light/dark cycle with a room temperature of 25 °C. Animals were anesthetized with urethane (1.4 g/kg, ip), and supplementary doses (0.7 g/kg) were given if needed. The trachea was cannulated to ease the ventilation. The body temperature was maintained at 37 ± 1 °C, using a controlled heat pad. For recording the blood pressure, the right femoral artery was cannulated with a polyethylene catheter (PE-50) filled with heparinized saline. Two holes were drilled above the BST at coordinates of ±1.6 to 2.0 mm mediolateral, 0.12 to 0.36 mm rostrocaudal, and 6.4–7.4 mm ventral to the cortical surface of bregma, according to a rat brain atlas (Paxinose and Watson, 2007).
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Drug microinjections
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A triple-barreled micropipette was made with the use of capillary tubing (Stoleting, USA). One of the barrels contained NE, the second barrel contained the antagonist, while the third barrel was used for recording extracellular action potentials. Drugs were microinjected using a pressurized air pulse applicator. The injection volume was measured by direct observation of the fluid meniscus in the micropipette using a microscope fitted
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with an ocular micrometer that allowed a 2-nl resolution (U.W.O, Canada). The injection volume for each drug was 100 nl. Blood pressure and heart rate were recorded using a ML T844 pressure transducer coupled to a pre-amplifier (FE221 Bridge amplifier, ADinstruments) connected to a power lab 4/35 data acquisition system (model PL3504 AD instruments).
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Single-unit recording
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Extracellular action potentials were recorded using a glass microelectrode pulled to a fine tip diameter (1–3 mm), filled with NaCl solution (2 M). Signals were amplified (10,000), filtered (0.3–3 kHz) by an amplifier (WPI, DAM 80, USA), and displayed on an oscilloscope (Tektronix 5103N, USA). Single-unit firings were digitized, saved in multiunit mode, and isolated using the WPI windows discriminator method with a program written by Nasimi et al. (2012) and Ranjbar et al. (2015). When blood pressure and firing were stable, both blood pressure and the spontaneous activity of the neurons were recorded simultaneously for five minutes; NE was then microinjected into the BST. If a change in blood pressure was observed, we waited for 30 min to an hour to make sure that the effect of NE disappeared before continuing with other interventions.
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Experimental groups
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The experiments consisted of the following groups:
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Fig. 1. Dose–response curve of NE microinjected into the lateral division of BST.
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The vehicle control group: 100 nl of the vehicle (saline or DMSO) was microinjected into the BSTL. Dose–response groups: To find the dose–response curve of the adrenergic system of the BST for the cardiovascular responses, four doses (3, 10, 20, and 30 nmol/100 nl) of NE (Sigma, USA) were microinjected into the BSTL. The NE control groups: In this group, two consecutive injections of NE were made into the BSTL, one hour apart. Since we aimed to compare the values before treatment with those after treatment, this group was designed to make sure that the effect of the second NE injection was comparable to that of the first. The alpha blocker groups: To find the role of different adrenoreceptors (AR), antagonists were microinjected into the BSTL in three separate groups. First, NE (20 nmol/100 nl) was microinjected Fig. 2. Microinjection of NE (20 nmol/100 nl) into the lateral part of BST caused a long depressor as the control. When blood pressure and bradycardic response (a–c) (paired t-test, MAP: P < 0.001, HR: P < 0. 01, n = 30 rats), with and heart rate returned to the either a long inhibitory (d) or excitatory single-unit responses (e). The arrow shows the injection baseline, a second injection of a time, a: arterial pressure change, b: MAP change, c: HR change, d and e: single-unit responses showed as PSTH. selective a1 antagonist, prazosin (10 nmol/100 nl Sigma), or an a2 antagonist, yohimbine (10 nmol/100 nl, Sigma), or a mixture of prazosin (10 nmol) and yohimbine (10 nmol), in a final volume of 100 nl was microinjected into the BST. Five to eight minutes later, the same site was retested by microinjection of NE (10 nmol/100 nl). The Bicuculline group: First, NE was injected into the BST. An hour later, a selective GABAA antagonist, bicuculline methiodide (BMI, 100 pmol/100 nl, Sigma) was microinjected into the BST. 5–8 min later, the same site was retested by microinjection of NE. Experiments were performed on one side of the brain, and if the condition of animal was stable and BP returned to baseline, another experiment was performed on the contralateral side.
Fig. 3. Time courses of changes in mean arterial pressure and heart rate in response to NE microinjection in the lateral division of BST. The asterisk shows significant changes compared to pre-injection value (paired t-test, MAP: P < 0.001, HR: P < 0. 01, n = 30 rats).
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Data analysis
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The mean arterial pressure (MAP) and heart rate (HR) values were expressed as mean ± SE. The time course of changes of MAP and HR was plotted and the maximum change was compared with that of the preinjection (paired t-test) and the control (independent ttest) values. A P < 0.05 was considered as the statistical significance. For neuronal activity, single-unit spikes were isolated and a peri-stimulus time histogram (PSTH) was plotted from the spike times. After this, the cardiovascular response patterns and the cell firing patterns for each injection were aligned and compared.
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Fig. 4. A sample of cardiovascular responses to NE (20 nmol/100 nl) injection into the lateral division of BST, before and after local injection of prazosin (10 nmol/100 nl, n = 12 rats), an a1adrenoreceptor antagonist. Prazosin caused a decrease in blood pressure and abolished the depressor and excitatory single-unit responses to the second injection of NE (paired t-test P < 0.05). The arrow shows the injection time, a: blood pressure change, b: MAP change, c: HR change, d: single-unit responses showed as PSTH.
Fig. 5. Microinjection of prazosin (10 nmol/100 nl) into the lateral division of the BST changed the depressor response to NE to pressor (20 nmol/100 nl). The arrow shows the injection time, a: blood pressure change, b: MAP change, c: HR change.
Histological verification of injection sites
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At the end of each experiment, the animal was sacrificed using a high dose of the anesthetic (urethane) and perfused transcardially with 100 ml of 0.9% saline followed by 100 ml of 10% formalin. The brain was removed and stored in 10% formalin for at least 24 h. Frozen serial transverse sections (40 mm) of the forebrain were cut and stained with Cresyl Violet 1%. The injection sites were determined according to a rat brain atlas (Paxinose and Watson, 2007), using a light microscope.
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Control group
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Microinjection of saline (100 nl, n = 5) into the lateral division of BST had no significant effect on mean arterial pressure (MAP), heart rate (HR) or firing rate of the neurons (DMAP = 0.2 ± 0.6 mmHg, DHR = 2 ± 0.9 beats/min, Dfiring rate = 0.2 ± 0.2).
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Dose–response curve of NE microinjected into the lateral division of BST
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To find the response to increasing doses of NE, four doses were injected into the BST. Microinjection of different doses (5, 10, 20, 35 nmol/100 nl, n = 6 for each dose) in the lateral part produced doserelated depressor and bradycardic responses (Fig. 1). The dose of 20 nmol of NE in 100 nl (ED50) was used for further experiments.
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Cardiovascular and single-unit responses to microinjection of NE into BSTL
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Microinjection of NE into the BSTL decreased both MAP and HR (DMAP = 15.5 ± 1 mmHg, DHR = 16 ± 2, n = 30 rats), which were significantly different from the preinjection values (paired t-test, P < 0.001). Samples of arterial pressure and heart rate tracings are shown in Fig. 2a–c. MAP and HR changes began shortly after the
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injection and reached a peak at two to four minutes and then returned to the baseline in about 60 min. The time courses of the changes are shown in Fig. 3. Action potentials of 147 mostly spontaneously active neurons were recorded from the lateral part of BST. In response to NE, 88 neurons (59.8%) showed no response, 37 neurons (25.2%) exhibited excitatory response (Fig. 2e), and 22 neurons (15%) exhibited
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inhibitory response (Fig. 2d). The response was concomitant with the change in MAP or HR. The duration of response was different in various neurons, spanning from 180 to more than 600 seconds.
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Consecutive NE injections
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In this control group, two injections of NE, one hour apart, were given in the lateral division of BST. NE injection (20 nmol/100 nl) produced depressor and bradycardic responses (first injection: DMAP = 12 ± 1.8 mmHg, and DHR = 14 ± 4.2 beats/min; second injection: DMAP = 13 ± 2.7 mmHg, and DHR = 9 ± 1.7 beats/min). There was no significant difference between the first and second injections (paired t-test, P > 0.05).
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Fig. 6. A sample of cardiovascular responses to NE (20 nmol/100 nl) injection into the lateral division of BST, before and after local injection of yohimbine, an a2-adrenoreceptor antagonist (10 nmol/100 nl). Yohimbine did not affect the depressor response to NE (n = 12 rats). The arrow shows the injection time, a: blood pressure change, b: MAP change, c: HR change.
Fig. 7. Microinjection of a combination of prazosin (10 nmol) and yohimbine (10 nmol) blocked the NE depressor responses into the lateral division of the BST (paired t-test, MAP: P < 0.001, HR: P < 0.01, n = 9 rats). The arrow shows the injection time, a Blood pressure change, b: MAP change, c: HR change.
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Cardiovascular and single-unit responses to microinjection of adrenoceptor (AR) blockers into the lateral part of BST
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To find the type of AR involved in mediating depressor response, first the NE was microinjected into the BST. Then, after an hour, 100 nl of the selective a1-AR antagonist, prazosin, or a2-AR antagonist, yohimbine, or a combination of the two was microinjected into the BST. Five to seven minutes later, NE was microinjected into the same site. Microinjection of prazosin (10 nmol/100 nl) into the lateral part of BST elicited a decrease in MAP ( 17.3 ± 3.4 mmHg, n = 11), with no significant change in HR ( 4.3 ± 2 bpm) (Fig. 4). Prazosin greatly attenuated the depressor response to NE by 68% compared to that of the control (DMAP = 15.45 ± 1.5 vs. 4.9 ± 2.8, paired t-test, P < 0.05), while the HR response was attenuated by 87% (DHR = 19 ± 5.1 vs. 2.45 ± 3.8, paired t-test, P < 0.05, n = 12 rats) (Figs. 4 and 9). In four rats, the NE depressor response changed to a pressor response without a significant change in HR (Fig. 5). Microinjection of yohimbine (10 nmol/100 nl) did not elicit any cardiovascular response (MAP = 78.7 ± 4.7 vs. 80.9 ± 5 mmHg and HR = 375.7 ± 15.2 vs. 357 ± 15 bpm, n = 12 rats). Yohimbine did not affect the depressor (before: DMAP = 13.1 ± 2.2, after: DMAP = 14.4 ± 3.5 mmHg) or HR
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response (before: DHR = 8.9 ± 2.4 beats/min, after: DHR = 5.7 ± 1.9 beats/min) to NE (Figs. 6 and 9). Injection of a combination of prazosin (10 nmol/100 nl) and yohimbine (10 nmol/100 nl) into the BSTL elicited a decrease in MAP (DMAP = 10.2 ± 3.5 mmHg, paired t-test P < 0.05) with no significant change in HR ( 6 ± 2.7 bpm), while greatly attenuating the depressor (DMAP = 18.5 1.9 vs. 2.5 ± 2.6, paired t-test, P < 0.05) and bradycardic (DHR 14.8 ± 3.1 vs 2.3 ± 1.8 paired t-test P < 0.05) responses to NE (Fig. 7). In most cases, the depressor response changed to a pressor response. Fig. 9 summarizes the results of these experiments. Excitatory (n = 11 neurons) and inhibitory (n = 12 neurons) singleunit responses to NE injection were abolished by prazosin (Fig. 4d). However, yohimbine had no significant effect on excitatory/ inhibitory effect of NE.
Cardiovascular and single-unit responses to microinjection of selective GABAA antagonist followed by NE into the BSTL
Fig. 8. A sample of cardiovascular responses to NE injection into the lateral division of BST, before and after local injection of BMI (100 pmol/100 nl, n = 7 rats), a GABAA receptor antagonist. BMI increased MAP, HR and firing rate. BMI abolished the depressor response to the second injection of NE. The arrow shows the injection time, a: blood pressure change, b: MAP change, c: HR change, d: single-unit responses showed as PSTH.
To investigate the synaptic mechanism involved in the depressor response of NE in the lateral part of BST, first NE was microinjected into the BST. Then, after one hour, the selective GABAA antagonist, bicuculline methiodide (BMI, 100 pmol/100 nl), was injected. Then, NE was injected into the same site again. As shown in Figs. 8 and 9, BMI elicited an increase in both MAP and HR (DMAP = 47.3 ± 10.8 mmHg, DHR = 45.6 ± 15.5 bpm, n = 7 rats, paired t-test P < 0.01). Pretreatment with BMI blocked the depressor response to NE injection (before: DMAP = 15 ± 2.8 mmHg, after: DMAP = 0.6 ± 0.7 mmHg, paired t-test P < 0.01; before: DHR = 12.4 ± 4.1 beats/min, after: DHR = 5 ± 5.3 beats/min, paired t-test P < 0.05). The extracellular action potentials of 22 neurons were recorded in these experiments. Injection of BMI caused excitation in 18 neurons (81.8%, Fig. 8d) and had no significant effect on the firing rate of four neurons (18.2%).
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Histology
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Distribution of the injection sites is shown in Fig. 10. Data of the injection sites outside the BSTL were not included in the analysis.
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DISCUSSION
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The BST is one of the major targets of noradrenergic innervation in the brain (Swanson and Hartman, 1975; Moore and Bloom, 1979; Kilts and Anderson, 1986) originating mainly from neurons located in the A1, A2, and A5 brainstem nuclei, and in a lesser degree from the A6 (Fallon and Moore, 1978; Byrum and Guyenet, 1987; Riche et al., 1990; Woulfe et al., 1990; Forray et al., 2000). Noradrenergic terminals are mainly located in the ventral portion (Phelix et al., 1992; Freedman and Cassell, 1994) of the anterior part of the lateral side of the BST (Fuentealba et al., 2000). These areas correspond to the regions in our experiments (Fig.10). The present study has been performed to find cardiovascular and electrophysiological effects of NE in the BSTL and to determine if its effects are mediated through GABAergic neuronal systems. To facilitate the discussion, a neural network is postulated based on the findings of the present and some previous papers (Fig.11). In response to stressful challenges, BST adjusts the levels of arterial pressure and heart rate, appropriate for the stressor, by either activating or inhibiting the sympathetic nervous system (Ciriello and Janssen, 1993; Roder and Ciriello, 1993; Dunn and Williams, 1995; Alves et al., 2007; Crestani et al., 2009; Zhang et al., 2009; Kuwaki, 2011). It has been shown that BST projects to CVLM, mediating sym-
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pathoinhibition, and to RVLM mediating sympathoexcitaon sympathoexcitatory neurons, as postulated in tion (Giancola et al., 1993; Dong et al., 2001; Dong and Fig. 11, provided that there is a tonic release of NE. Swanson, 2004; Hatam and Ganjkhani, 2012). As shown Another possible explanation of this result is based on in the postulated diagram (Fig. 11), the sympathoexcitaearlier observation that NE has an inhibitory action on tory neuron receives three inputs, one positive input (a1) local glutamatergic neurotransmission in vBST (Egli from noradrenergic terminals and one negative input from et al., 2005). This area corresponds to the regions where GABAergic neurons. GABA neuron receives one positive prazosin was injected in our experiments. Two studies noradrenergic input, a1. The network can produce either have demonstrated that stimulation of the lateral division depressor or pressor responses. It produces a depressor of BST with glutamate decreases BP and HR in response if activity of GABA overcomes the excitatory urethane-anesthetized rats (Ciriello and Janssen, 1993; effects of a1 on the sympathoexcitatory neuron. Hatam and Nasimi, 2007). Thus, blockade of a1-AR may We found that microinjection of NE into the lateral part disinhibit the glutamate release, consequently causing a of BST decreased MAP and HR in urethane-anesthetized depressor response; however, there is no tonic release rats (Fig. 2). It was shown that the injection of NE into the of glutamate in the lateral BST (Hatam and Nasimi, 2007). BST of unanesthetized rats caused long-lasting doseThe pretreatment of BST with yohimbine, an a2-AR related pressor and bradycardiac responses, which was antagonist, did not affect depressor or bradycardic greatly reduced in urethane-anesthetized rats but did responses to injection of NE (Fig. 6), indicating that a2not change to depressor response (Crestani et al., AR is not involved in the depressor response to NE. 2007). So, our finding for the lateral part is different from Similar to prazosin, the response to NE was blocked by the previous study. As seen in Fig. 2, two types of pretreatment with a combination of prazosin and single-unit responses were recorded along with the yohimbine (Fig. 7). depressor responses. The excitatory record could be from the GABAergic neuron stimulated by NE, and the long inhibitory response could be from the sympathoexcitatory neuron which was inhibited by the GABAergic inputs (Fig. 11). To find the possible involvement of a1 or a2 receptors in depressor response, we pretreated the BST with the selective a1-AR antagonist, prazosin, or the selective a2-AR antagonist, yohimbine. The depressor response to NE was blocked by the pretreatment of the BST with prazosin (Fig. 4), indicating that the pressor response is mediated by a1-AR. The singleunit response, shown in Fig.4, probably recorded from the GABA neuron which receives a1 input (Fig. 11). Therefore, by blocking this receptor, GABA neuron did not respond to NE and the depressor response disappeared. In some cases, the depressor response to NE altered to a pressor one by prazosin (Fig. 5), suggesting the possible involvement of b-AR in stimulating the sympathoexcitatory neuron. The presence of local b-AR in the BST and its effect on pressor response to NE microinjected into the BST has been previously demonstrated (Crestani et al., 2007). As seen in Figs. 4 and 5, prazosin caused a decrease in MAP and HR. The depressor effect of prazosin Fig. 9. Bar charts summarizing the effects of various drugs injected into the BSTL on MAP and could also be explained by the HR. Note that depressor and bradycardic responses were significantly attenuated after prazosin, whereas yohimbine alone did not significantly affect MAP possibility of direct positive a1 action or bicuculline or prazosin + yohimbine, * ** or HR (paired t-test, P < 0.05 , P < 0.01 ).
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Fig. 11. Schematic illustration of the proposed neuronal interaction pathways involved in mediating the depressor and bradycardia responses to NE inputs to the lateral BST neurons.
Fig. 10. Schematic coronal section of rat brain adopted from an atlas (Paxinose and Watson, 2007). The injection sites of NE were shown as star symbol, bicuculline as filled circles, prazosin as filled squares, yohimbine as open squares and prazosin + yohimbine as filled triangle ac: anterior commissure; BSTLD: BST, lateral division, dorsal part; BSTLI: BST, lateral division, intermediate part; BSTLP: BST, lateral division, posterior part; BSTLV: BST, lateral division, ventral part.
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As summarized in Fig. 9, the depressor and bradycardic effects of NE were mediated through a1-AR but not a2-AR in urethane-anesthetized rats. In another series of experiments, we explored whether the depressor and bradycardic responses to NE were mediated through activation of GABAA receptors. Injection of bicuculline methiodide (BMI, a GABAA receptor antagonist) into the lateral BST produced pressor response and tachycardia, which was consistent with our previous findings (Hatam et al., 2009). BMI blocked NE-induced depressor and bradycardic responses (Fig. 8). In other words, GABA mediates sympathoinhibitory and bradycardia effects of NE in the lateral
part of BST. Taken together, these data suggest that NE depressor effect in the lateral BST is mediated by a1-AR, which is probably located on the GABAergic neurons, as postulated in Fig. 11. In support of the postulated network (Fig. 11), it was reported that activation of a1- and b-adrenoceptors depolarizes the local GABAergic neurons in the BST (Dumont and Williams, 2004). In response to NE, this network could produce either pressor or depressor response, based on the strength of GABA transmission. The prolongation of synaptic inhibition by the positive modulation of GABAA receptor function is an important component of the central nervous system’s depressant effects of volatile anesthetics and several chemically distinct intravenous anesthetics (Tanelian et al., 1993). Therefore, anesthesia facilitates GABA release, resulting in a depressor effect of NE instead of pressor in the lateral BST. Also, when a1 is blocked, GABA release will decrease and the pressor effect of NE may appear, as shown in Fig. 5. GABAergic transmission, as shown in Fig. 11, may have the physiological benefit of modulating the pressor effects of NE by preventing over-stimulation of sympathoexcitatory neurons. In conclusion, these data provide the first evidence that microinjection of NE in the lateral division of BSTproduced depressor and bradycardia responses in urethane-anesthetized rat. The depressor and bradycardiac responses are mediated by local a1- but not a2-adrenoceptors. a1-AR activates GABAergic systems within the BST, which in turn produces a depressor and bradycardic response.
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Acknowledgment—This manuscript was extracted from a PhD thesis by Fahimeh Yeganeh sponsored by the Vice Chancellery of Research of Shiraz University of Medical Sciences (grant number: 7309). The authors would like to thank Shiraz University of Medical Sciences, Shiraz, Iran and also Center for Development of Clinical Research of Nemazee Hospital and Dr. Nasrin Shokrpour for editorial assistance.
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(Received 30 January 2017, Accepted 23 May 2017) (Available online xxxx)
Please cite this article in press as: Yeganeh F et al. Interaction of GABA and norepinephrine in the lateral division of the bed nucleus of the stria terminals in anesthetized rat, correlating single-unit and cardiovascular responses. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.05.044
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