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Cardiorespiratory and metabolic responses to injection of bicuculline into the hypothalamic paraventricular nucleus (PVN) of conscious rats
Brain Research 895 (2001) 33–40 www.elsevier.com / locate / bres
Research report
Cardiorespiratory and metabolic responses to injection of bicuculline into the hypothalamic paraventricular nucleus (PVN) of conscious rats Evelyn Schlenker*, Ludwig Barnes, Susan Hansen, Douglas Martin Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, 414 East Clark Street, Vermillion, SD 57069, USA Accepted 21 December 2000
Abstract Stimulation of the PVN increases mean arterial pressure (MAP) and heart rate (HR). However, little is known about its role in modulating ventilation. We tested the hypothesis that the stimulation of the PVN by microinjection of bicuculline methiodide (BMI), a g-aminobutyric acid (GABA)A receptor antagonist, increases ventilation in conscious rats. Oxygen consumption was also evaluated to determine if the ventilatory responses were associated with increases in metabolic rate. Male Sprague–Dawley rats were instrumented with femoral catheters to measure MAP and HR and cannulae were implanted 1 mm above the PVN. After 5 to 7 days of recovery, metabolic, ventilatory, and cardiovascular responses to artificial cerebrospinal fluid (aCSF) and BMI were evaluated. Rats were given a 50 nl unilateral microinjection of aCSF (the vehicle control) followed by 50 n1 of BMI (1 mM) into the other side. Microinjection of BMI significantly increased MAP compared to aCSF (14564 vs. 12465 mmHg, P,0.02), HR to 460617 from 362622 breaths / min (P,0.01). Ventilation increased by 300% (P50.01) by stimulating frequency of breathing (176614 compared to 79612 breaths / min, ] P,0.005) and increasing tidal volume. Concomitantly, O 2 consumption doubled (P,0.006). These data suggest that in the PVN GABA receptors may be important regulators of cardiopulmonary and metabolic function in conscious rats. 2001 Published by Elsevier Science B.V. Theme: Endocrine and autonomic regulation Topic: Respiratory regulation Keywords: Ventilation; Oxygen consumption; Blood pressure; Hypothalamus
1. Introduction Stimulation of paraventricular nucleus of the hypothalamus (PVN) by electrical or chemical methods regulates cardiovascular functions and modulates sympathetic outflow [26]. In conscious rats, previous work has shown that chemical stimulation of the PVN is associated with an increase in sympathoadrenal outflow [15] and increases in both mean arterial pressure (MAP) and heart rate (HR) [16]. In addition, stimulation of sympathetic and cardiovascular activity can also affect ventilation [26]. Moreover, direct anatomical connections have been described between the PVN and brain areas involved in regulation of *Corresponding author. Tel.: 11-605-677-5160; fax: 11-605-6776381. E-mail address: [email protected] (E. Schlenker).
ventilation, such as the nucleus tractus solitarius (NTS), nucleus ambiguus, phrenic motoneurons, and parabrachial nuclei [13,27,29]. Indeed, previous studies are consistent with a modulatory role of the PVN in the control of ventilation. Stimulation of the PVN in anesthetized rabbits was reported to increase respiratory rate [8]. Similarly, glutamate injection into the PVN increased diaphragm electromyographic activity in anesthetized rats [27]. Finally, neural activity in the PVN of conscious cats was altered during phasic respiratory events [13]. Thus, in addition to the appropriate anatomical substrates, the PVN also appears to modulate respiratory function. However, a limitation to previous work is that much of this work has been carried out under anesthesia that can markedly attenuate the responses to PVN stimulation [12]. Accordingly, this study tested the hypothesis that stimulation of the PVN would increase cardiorespiratory function. MAP, HR,
0006-8993 / 01 / $ – see front matter 2001 Published by Elsevier Science B.V. PII: S0006-8993( 01 )02011-X
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respiratory function and oxygen consumption were measured in conscious rats subjected to injection of bicuculline methiodide (BMI), a GABAA receptor antagonist, into the PVN.
2. Materials and methods
2.1. Surgical procedures Six adult male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing an average of 400 g were utilized in these studies. Rats were housed in a controlled environment and allowed free access to laboratory rat chow and water. Aseptic technique was used for all surgeries. On the day of the surgery the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg / kg) and atropine (0.4 mg / kg) and placed in a Stoelting stereotaxic apparatus. A 2 cm incision was made to expose the scalp and connective tissue was removed to expose the cranial sutures. The rats were instrumented bilaterally with 23-gauge stainless steel cannulae. The cannulae were directed bilaterally at the PVN at a 108 angle from the vertical using the following coordinates: 21.8 mm posterior from bregma, 1.6 mm lateral to the midline, and 26.3 mm ventral from dura. The animals were allowed 5 days to recover from the stereotaxic surgery. Two days prior to experimentation the rats were anesthetized with Enflurane (isoflurane). Lidocaine hydrochloride (2%; Schein) infiltration was used as a local anesthetic to supplement general anesthesia and provide postoperative analgesia at the incision sites. Cannulae were then inserted into the left femoral artery (Tygon 0.060 O.D. tipped with 28-gauge Teflon) for measurement of arterial blood pressure and into the femoral vein (PE50) for the administration of pentobarbital (150 mg / kg) to sacrifice the rats at the end of the experiments. The catheters were tunneled subcutaneously and exited at the nape of the neck. The incision was closed with surgical suture and the catheters were tied in place to prevent the rat from dislodging or pulling of the catheters. The catheters were filled with heparinized saline (25 ml / 1000 ml) to prevent clotting.
2.2. Measurement of ventilation and oxygen consumption Rats were acclimatized for 5 days in a Leucite restrainer prior to the experiment. Ventilation was evaluated using the plethysmographic technique as previously described [22]. Briefly, restrained conscious rats were placed into cylindrical Plexiglas chambers. Air entered and exited the chamber through ports. The flow rate of gases exiting the chamber was measured with a Gilmont rotameter. Chamber temperature and relative humidity were monitored with a Digetec thermometer and a Cole-Palmer hygrometer, respectively. Pressure changes related to ventilation were measured with a low-pressure Statham transducer coupled
to a Grass preamplifier and data acquisition system (Biopac Systems, Inc., Santa Barbara, CA). Ventilatory parameters included frequency of breathing, tidal volume, and it is product, ventilation. In addition, oxygen consumption (VO 2 ) was evaluated using open circuit method [22]. A Beckman OM-14 oxygen analyzer measured the fractional amount of oxygen that entered and exited the chamber. The difference in the fractional content of O 2 exiting and entering the chamber was multiplied by the flow rate to calculate VO 2 in ml / min.
2.3. Experimental protocols The rats were placed into loosely fitting Leucite restrainers to allow microinjection into the PVN and to record arterial pressure and heart rate. MAP and HR were monitored continuously by connecting the in-dwelling arterial catheter to a pressure transducer interfaced with the Biopac data acquisition unit. HR was calculated from the pulsatile blood pressure recording. The experiments were not started until the rat maintained stable (65%) blood pressure and heart rate measurements for at least 30 min. Injectors (30 gauge) extended 1.0 mm beyond the end of the guide cannulae. They were attached via PE20 polyethylene tubing to 1.0 ml Hamilton microsyringes (Hamilton Co., Reno, NV), and backfilled with artificial cerebrospinal fluid (aCSF) (vehicle) or a 1 mM solution of bicuculline methiodide (BMI) (Research Biochemicals Inc., Natick, MA) in aCSF. The volume of injectates was 50 nl. Thirty minutes prior to starting the protocol, the rat was placed in one of the Leucite restrainers with access ports for the arterial line and microinjection lines. The injectors were inserted into the PVN. Super glue was used to keep the injectors in place. A baseline period of 10 min was determined. Then aCSF was unilaterally microinjected into the PVN and responses were recorded for 20 min. Subsequently BMI was injected and metabolic and cardiopulmonary values were recorded beginning 2 min after injection and every 2 min for 10 min after which time the variables were recorded in 5 min intervals until 40 min post-injection. All procedures were approved by the Animal Care and Use Committee of the University of South Dakota and followed the NIH guidelines for the care and use of animals in laboratory research.
2.4. Data analysis Data were analyzed using a one-way analysis of variance with repeated measures to evaluate the effects of BMI administration over time. Post hoc tests consisted of the Dunnett test comparison of post-injection values with the pre-injection control. Significance was accepted as P, 0.05. Data are presented as means6S.E.M.
E. Schlenker et al. / Brain Research 895 (2001) 33 – 40
Fig. 1. Sites of injection for bicuculline (BMI) and artificial cerebrospinal fluid (aCSF) microinjections into the paraventricular nucleus of the hypothalamus (PVN). The figure is a schematic representation where BMI (stars) (n56) and aCSF (ovals) (n56) were microinjected. The black stars denote sites where BMI elicited the physiological responses described in the text. Open stars represent site at which BMI failed to elicit responses.
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150% (460620 bpm) after BMI injection, and displayed a similar pattern of response as MAP although HR only remained statistically different from baseline to the 8 min mark. BMI injection was also associated with marked changes in respiratory function. Frequency of ventilation increased from 7364 to 176614 b / min and remained significantly elevated for 15 min post-injection (Fig. 3). Tidal volume also showed an increase with BMI injection from 1.3660.21 to 2.0560.27 ml / b that plateaued between 2 and 6 min post-injection then returned to control values by 10 min post-injection (Fig. 3). Overall minute ventilation increased three-fold from 102619 to 305686 ml / min and remained significantly elevated for 8 min (Fig. 4). BMI stimulation of the PVN was also associated with an increase in oxygen consumption (maximum effect 10.1761.28 ml / min). However, the pattern of response of oxygen consumption to BMI administration was somewhat different than the other variables (Fig. 4). Oxygen consumption increased gradually reaching a peak at 6 min post-injection and then returned to baseline over the ensuing 4 min. Thus, BMI stimulated all variables, but not in the same manner nor for the same length of time.
2.5. Histology At the end of the experiments the animals were euthanized with sodium pentobarbital (150 mg / kg) and then perfused intracardially with a solution of 0.9% saline followed by 10% formaldehyde solution. The brains were then removed stored in a 10% formalin solution and cut with a sliding microtome at 60 mm coronal sections through the entire PVN region. The sections were then placed on gelatin-coated slides and allowed to air dry over night. Verification of the injection termination sites was performed under a low-power microscope using the atlas of Paxinos and Watson [19]. Injections were considered acceptable if they fell within a 0.6 mm radius of the PVN [23]. Several sites around the PVN served as anatomical controls for the BMI injections. No response was elicited from these microinjections (Fig. 1).
3. Results Baseline values for MAP, HR, oxygen consumption, ventilation, frequency and tidal volume were 12565.0 mm Hg, 352617 b.p.m., 5.5260.23 ml / min, 138.6622.8 ml / min, 8968 b / min, and 1.4760.23 ml / b, respectively. Injection of aCSF did not affect any variable relative to baseline measurements and all variables remained stable over the 20 min of measurements (see Figs. 2–4). By contrast, BMI microinjected into the PVN increased both HR and MAP within 2 min (Fig. 2). A significant elevation of MAP (maximum D51966 mmHg) was sustained for 15 min after injection and then gradually returned to baseline values by 20 min post-injection. Heart rate increased by
4. Discussion This study was undertaken to test the hypothesis that the PVN modulates respiratory function in conscious rats. The results indicate that the PVN is a pivotal region of the hypothalamus that influences not only oxygen consumption and cardiovascular parameters, but also control of breathing in conscious rats. The PVN is an important forebrain site involved in the integration of neurohumoral outflow to the cardiovascular system [25]. Previous studies have shown that stimulation of the PVN caused an increase in MAP, HR and sympathetic nervous system activity. In the present study we have confirmed the pressor and tachycardic responses to PVN stimulation in conscious rats. The responses we observed compare favorably with previous responses obtained in our hands [15,16] and those of others [10,12,20,28]. Thus, the cardiovascular outcome of the present studies is comparable previous work. In addition, we also observed that stimulation of the PVN was associated with a marked increase in respiratory function. Relatively few previous studies have addressed the role of the PVN in the control of ventilation. A separate study monitored neural activity in the PVN in freely-behaving cats and reported that PVN activity changed during phasic respiratory events suggesting a link between the PVN and respiratory function [13]. More directly, electrical stimulation the PVN in anesthetized rabbits caused a 44% increase in respiratory rate [8]. The study that most closely resembles the current work is that of Yeh and coworkers [27] who determined the effects of
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Fig. 2. Effects of bicuculline methiodide (BMI) (solid lines) and artificial cerebrospinal fluid (aCSF) (dashed lines) microinjected into the PVN on (A) mean arterial pressure and (B) heart rate (b.p.m.5beats per min). Pre-values represent mean baseline values immediately preceding the injection of either aCSF or BMI. The arrow and asterisks indicate significant (P,0.05) responses relative to the ‘pre’ values. Values are mean6S.E.M., n56.
bilateral microinjections of L-glutamate into the PVN respiratory function in anesthetized rats. They reported changes in inspiratory and expiratory times corresponding to an increase in frequency of approximately 25%. We also observed an increase in ventilatory frequency however in our study respiratory frequency doubled after BMI injection. We also recorded a 69% increase in tidal volume whereas Yeh et al. reported that peak diaphragmatic electromyographic activity (a surrogate of tidal volume) increased by approximately 100%. Thus, there are differences in the relative magnitudes of the responses of various respiratory functions to stimulation of the PVN between our study and that of Yeh et al. These could be the
result of differences between anesthetized and conscious preparations, since anesthesia may influence PVN-mediated responses [12]. On the other hand these differences may represent variations in the population of PVN neurons activated. Yeh et al. used glutamate as a stimulant, which may activate all neurons in the PVN whereas we used BMI, which should only activate those neurons tonically inhibited by GABA. Nevertheless, in the final analysis, we observed that PVN stimulation caused minute ventilation to increase to three times its baseline value. This agrees well with overall minute ventilation response (32.5) calculated from the study of Yeh et al. Thus, in addition to a clear effect on cardiovascular function, findings of the
E. Schlenker et al. / Brain Research 895 (2001) 33 – 40
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Fig. 3. Effects of bicuculline methiodide (BMI) (solid lines) and artificial cerebrospinal fluid (aCSF) (dashed lines) microinjected into the PVN on (A) frequency of breathing (b / m5breaths per min) and (B) tidal volume (ml / b5milliliters per breath). Pre-values represent mean baseline values immediately preceding the injection of either aCSF or BMI. Asterisks indicate significant (P,0.05) responses relative the ‘pre’ values. Values are mean6S.E.M., n56.
current study and those of others indicate that the PVN can also exert profound effects on respiratory function. Moreover, the cardiovascular and respiratory responses are interactive. We have previously shown that PVN stimulation increases MAP primarily by increasing cardiac output [14]. The increases in tidal volume and frequency recorded in the present study could facilitate venous return to support an increase in cardiac output. Thus, the PVN appears to elicit coordinated respiratory and cardiovascular responses. The pathways by which the PVN influences ventilation remain to be fully elucidated. It is known however that the PVN has extensive neural connections to other brain regions that are involved in control of respiration [26]. Zheng and coworkers [29] used anterograde tracing with Phaseolus vulgaris leucoagglutinin in rats to denote several pathways of efferent fibers from the PVN. One pathway projected to the central gray matter, Edinger–Westphal
nucleus, pedunculopontine tegmental nucleus, nucleus of the locus ceruleus and parabrachial nucleus. A second pathway had projections to the nucleus ambiguus, nucleus of the solitary tract and the dorsal motor nucleus of the vagus. The PVN also makes afferent connections with other areas such as the hippocampus and arcuate nucleus that have been shown to participate in control of breathing [6,22,25,26]. Thus the PVN may affect respiratory function via direct neural circuits to other respiratory control centers in the brain. Alternatively, the relationship between the PVN and respiration may be indirect. In view of evidence indicating that catecholamines influence ventilation [5] and that PVN stimulation increases sympathetic nerve activity [28] and circulation catecholamine concentrations [15,16], it is possible that increases in sympathetic nervous system outflow could account for the respiratory stimulation we observed. If this were the case, one would expect the
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Fig. 4. Effects of bicuculline methiodide (BMI) (solid lines) and artificial cerebrospinal fluid (aCSF) (dashed lines) microinjected into the PVN on (A) oxygen consumption and (B) ventilation. Pre-values represent mean baseline values immediately preceding the injection of either aCSF or BMI. Asterisks indicate significant (P,0.05) responses relative the ‘pre’ values. Values are mean6S.E.M., n56.
pattern of responses to be similar between the cardiovascular responses, which are mediated by the sympathetic nervous system [16], and the ventilatory responses. Indeed, the time-course of the cardiovascular and respiratory responses were quite similar suggesting that they might be mediated via similar pathways that include the sympathetic nervous system. The PVN is also well recognized as a brain site that participates in the control of metabolism [3]. Indeed we observed an increase in oxygen consumption of 200%. The PVN sends direct neural projection to brown adipose tissue (BAT), a major controller of metabolic rate in rodents [4]. Moreover, stimulation of the PVN has been observed to
increase BAT activity [2]. Similarly, increases in circulating catecholamine concentrations also stimulated BAT activity [9]. Thus, it seems reasonable that activation of BAT may have contributed significantly to the large increase in oxygen consumption observed in the present study. It could be argued that the respiratory responses we have reported were secondary to changes in metabolic rate and oxygen consumption, since the time-course of the oxygen consumption response lagged behind that for the ventilatory response. An alternative explanation is that an overshoot in the response of the respiratory system to oxygen demand, or inherent variability in the ventilatory data as reflected by the large S.E.M. contributed to these
E. Schlenker et al. / Brain Research 895 (2001) 33 – 40
apparent differences in time course. Thus, the ventilatory response could include both direct and indirect effects that have different time-courses with the initial ventilatory response (at 2 min) representing a direct effect and the later time-points driven by a metabolic increase. The neurochemical signals controlling the PVN outflow to respiratory circuits is not known. Previous studies have used glutamate as a chemical stimulant. While an effective neurophysiological tool, this approach yields little information concerning neurotransmitters involved in the control of respiration since almost all neurons are responsive to glutamate. Consequently, glutamate may simultaneously stimulate excitatory and inhibitory circuits. In the present study we used BMI to stimulate the PVN. BMI has been used as an antagonist of GABAA receptors, which are abundantly found in the PVN [7]. Recently, however it has been suggested that BMI may also block calcium-activated potassium channels in some [11,17], but not all brain nuclei [24]. The use of an antagonist is advantageous since a response will only be observed if significant endogenous inhibitory tone is present. Accordingly, one possible interpretation of our findings is that the activity of PVN neurons involved in the control of respiratory drive may be tonically suppressed by endogenous GABAergic systems. Consistent with a possible role for GABAergic systems in hypothalamic control of ventilation, other studies have shown that oxygen consumption was increased by baclofen (a GABA B receptor agonist) microinjected into the 3rd ventricle in rats [1] and we also noted that microinjection of baclofen directly into the arcuate nucleus of conscious rats stimulates ventilation [21], Alternatively, if BMI was acting primarily via inhibition of calcium-activated potassium channels, our data suggest that this neural control mechanism tonically inhibits PVN control of ventilatory function. Moreover, a recent study also suggests an interaction between PVN nitric oxide and GABAergic systems in the control of cardiovascular function [28]. Whether this interaction of signaling systems is involved in PVN modulation of respiratory function remains to be determined. In any case, the data from the present study do not allow discrimination between these interesting possibilities. In conclusion, the findings of the current study suggest that inhibitory systems in the PVN modulate oxygen consumption, MAP, HR and control of breathing in conscious rats. The PVN is an important integratory center for these parameters and is ideally suited to modulate cardiorespiratory coupling in pathological states, such as hypertension associated with sleep apnea [18].
Acknowledgements This work was supported by NIH HLBI [62167 (D.M.) and the USD Foundation (E.S.).
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[21] E.H. Schlenker, X.M. Liu, Muscimol and baclofen microinjected into the arcuate nucleus affect ventilation differently in male rats, Soc. Neurosci. Abstr. 22 (1996) 513.4. [22] E.H. Schlenker, D.S. Martin, X.M. Lin, M.C. Egland, Naloxone microinjected into the arcuate nucleus has differential effects on ventilation in male and female rats, Physiol. Behav. 62 (1997) 531–536. [23] T. Segura, D.S. Martin, P.J. Sheridan, J.R. Haywood, Measurement of the diffusion of 3 H-bicuculline microinjected into the rat hypothalamus, J. Neurosci. Methods 41 (1992) 175–186. [24] L.C. Solomon, Effects of blockade of GABAA receptors versus apamin sensitive calcium activated potassium channels in pre-botzinger complex on phrenic motor output in vivo, Soc. Neurosci. Abstr. 26 (2000) 928. [25] L. Swanson, P.E. Sawchenko, Hypothalamic integration: organization of the paraventricular and supraoptic nuclei, Annu. Rev. Neurosci. 6 (1983) 269–324.
[26] T.G. Waldrop, J.P. Porter, Hypothalamic involvement in respiratory and cardiovascular regulation, in: J.A. Dempsey, A.I. Pack (Eds.), Regulation of Breathing, 2nd Edition, Marcel Dekker Inc, New York, 1995, pp. 315–364. [27] E.R. Yeh, B. Erokwu, J.C. LaManna, M.A. Haxhiu, The paraventricular nucleus of the hypothalamus influences respiratory timing and activity in the rat, Neurosci. Lett. 232 (1997) 63–66. [28] K. Zhang, K. Patel, Effect of nitric oxide within the paraventricular nucleus on renal sympathetic nerve discharge: role of GABA, Am. J. Physiol. 275 (1998) R728–R734. [29] J.Q. Zheng, M. Seki, T. Hayakawa, H. Ito, K. Zyo, Descending projections from the paraventricular hypothalamic nucleus to the spinal cord: anterograde tracing study in the rat, Okajimas Folia Anat. Jpn. 72 (1995) 119–1136.
Research report
Cardiorespiratory and metabolic responses to injection of bicuculline into the hypothalamic paraventricular nucleus (PVN) of conscious rats Evelyn Schlenker*, Ludwig Barnes, Susan Hansen, Douglas Martin Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, 414 East Clark Street, Vermillion, SD 57069, USA Accepted 21 December 2000
Abstract Stimulation of the PVN increases mean arterial pressure (MAP) and heart rate (HR). However, little is known about its role in modulating ventilation. We tested the hypothesis that the stimulation of the PVN by microinjection of bicuculline methiodide (BMI), a g-aminobutyric acid (GABA)A receptor antagonist, increases ventilation in conscious rats. Oxygen consumption was also evaluated to determine if the ventilatory responses were associated with increases in metabolic rate. Male Sprague–Dawley rats were instrumented with femoral catheters to measure MAP and HR and cannulae were implanted 1 mm above the PVN. After 5 to 7 days of recovery, metabolic, ventilatory, and cardiovascular responses to artificial cerebrospinal fluid (aCSF) and BMI were evaluated. Rats were given a 50 nl unilateral microinjection of aCSF (the vehicle control) followed by 50 n1 of BMI (1 mM) into the other side. Microinjection of BMI significantly increased MAP compared to aCSF (14564 vs. 12465 mmHg, P,0.02), HR to 460617 from 362622 breaths / min (P,0.01). Ventilation increased by 300% (P50.01) by stimulating frequency of breathing (176614 compared to 79612 breaths / min, ] P,0.005) and increasing tidal volume. Concomitantly, O 2 consumption doubled (P,0.006). These data suggest that in the PVN GABA receptors may be important regulators of cardiopulmonary and metabolic function in conscious rats. 2001 Published by Elsevier Science B.V. Theme: Endocrine and autonomic regulation Topic: Respiratory regulation Keywords: Ventilation; Oxygen consumption; Blood pressure; Hypothalamus
1. Introduction Stimulation of paraventricular nucleus of the hypothalamus (PVN) by electrical or chemical methods regulates cardiovascular functions and modulates sympathetic outflow [26]. In conscious rats, previous work has shown that chemical stimulation of the PVN is associated with an increase in sympathoadrenal outflow [15] and increases in both mean arterial pressure (MAP) and heart rate (HR) [16]. In addition, stimulation of sympathetic and cardiovascular activity can also affect ventilation [26]. Moreover, direct anatomical connections have been described between the PVN and brain areas involved in regulation of *Corresponding author. Tel.: 11-605-677-5160; fax: 11-605-6776381. E-mail address: [email protected] (E. Schlenker).
ventilation, such as the nucleus tractus solitarius (NTS), nucleus ambiguus, phrenic motoneurons, and parabrachial nuclei [13,27,29]. Indeed, previous studies are consistent with a modulatory role of the PVN in the control of ventilation. Stimulation of the PVN in anesthetized rabbits was reported to increase respiratory rate [8]. Similarly, glutamate injection into the PVN increased diaphragm electromyographic activity in anesthetized rats [27]. Finally, neural activity in the PVN of conscious cats was altered during phasic respiratory events [13]. Thus, in addition to the appropriate anatomical substrates, the PVN also appears to modulate respiratory function. However, a limitation to previous work is that much of this work has been carried out under anesthesia that can markedly attenuate the responses to PVN stimulation [12]. Accordingly, this study tested the hypothesis that stimulation of the PVN would increase cardiorespiratory function. MAP, HR,
0006-8993 / 01 / $ – see front matter 2001 Published by Elsevier Science B.V. PII: S0006-8993( 01 )02011-X
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E. Schlenker et al. / Brain Research 895 (2001) 33 – 40
respiratory function and oxygen consumption were measured in conscious rats subjected to injection of bicuculline methiodide (BMI), a GABAA receptor antagonist, into the PVN.
2. Materials and methods
2.1. Surgical procedures Six adult male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighing an average of 400 g were utilized in these studies. Rats were housed in a controlled environment and allowed free access to laboratory rat chow and water. Aseptic technique was used for all surgeries. On the day of the surgery the rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg / kg) and atropine (0.4 mg / kg) and placed in a Stoelting stereotaxic apparatus. A 2 cm incision was made to expose the scalp and connective tissue was removed to expose the cranial sutures. The rats were instrumented bilaterally with 23-gauge stainless steel cannulae. The cannulae were directed bilaterally at the PVN at a 108 angle from the vertical using the following coordinates: 21.8 mm posterior from bregma, 1.6 mm lateral to the midline, and 26.3 mm ventral from dura. The animals were allowed 5 days to recover from the stereotaxic surgery. Two days prior to experimentation the rats were anesthetized with Enflurane (isoflurane). Lidocaine hydrochloride (2%; Schein) infiltration was used as a local anesthetic to supplement general anesthesia and provide postoperative analgesia at the incision sites. Cannulae were then inserted into the left femoral artery (Tygon 0.060 O.D. tipped with 28-gauge Teflon) for measurement of arterial blood pressure and into the femoral vein (PE50) for the administration of pentobarbital (150 mg / kg) to sacrifice the rats at the end of the experiments. The catheters were tunneled subcutaneously and exited at the nape of the neck. The incision was closed with surgical suture and the catheters were tied in place to prevent the rat from dislodging or pulling of the catheters. The catheters were filled with heparinized saline (25 ml / 1000 ml) to prevent clotting.
2.2. Measurement of ventilation and oxygen consumption Rats were acclimatized for 5 days in a Leucite restrainer prior to the experiment. Ventilation was evaluated using the plethysmographic technique as previously described [22]. Briefly, restrained conscious rats were placed into cylindrical Plexiglas chambers. Air entered and exited the chamber through ports. The flow rate of gases exiting the chamber was measured with a Gilmont rotameter. Chamber temperature and relative humidity were monitored with a Digetec thermometer and a Cole-Palmer hygrometer, respectively. Pressure changes related to ventilation were measured with a low-pressure Statham transducer coupled
to a Grass preamplifier and data acquisition system (Biopac Systems, Inc., Santa Barbara, CA). Ventilatory parameters included frequency of breathing, tidal volume, and it is product, ventilation. In addition, oxygen consumption (VO 2 ) was evaluated using open circuit method [22]. A Beckman OM-14 oxygen analyzer measured the fractional amount of oxygen that entered and exited the chamber. The difference in the fractional content of O 2 exiting and entering the chamber was multiplied by the flow rate to calculate VO 2 in ml / min.
2.3. Experimental protocols The rats were placed into loosely fitting Leucite restrainers to allow microinjection into the PVN and to record arterial pressure and heart rate. MAP and HR were monitored continuously by connecting the in-dwelling arterial catheter to a pressure transducer interfaced with the Biopac data acquisition unit. HR was calculated from the pulsatile blood pressure recording. The experiments were not started until the rat maintained stable (65%) blood pressure and heart rate measurements for at least 30 min. Injectors (30 gauge) extended 1.0 mm beyond the end of the guide cannulae. They were attached via PE20 polyethylene tubing to 1.0 ml Hamilton microsyringes (Hamilton Co., Reno, NV), and backfilled with artificial cerebrospinal fluid (aCSF) (vehicle) or a 1 mM solution of bicuculline methiodide (BMI) (Research Biochemicals Inc., Natick, MA) in aCSF. The volume of injectates was 50 nl. Thirty minutes prior to starting the protocol, the rat was placed in one of the Leucite restrainers with access ports for the arterial line and microinjection lines. The injectors were inserted into the PVN. Super glue was used to keep the injectors in place. A baseline period of 10 min was determined. Then aCSF was unilaterally microinjected into the PVN and responses were recorded for 20 min. Subsequently BMI was injected and metabolic and cardiopulmonary values were recorded beginning 2 min after injection and every 2 min for 10 min after which time the variables were recorded in 5 min intervals until 40 min post-injection. All procedures were approved by the Animal Care and Use Committee of the University of South Dakota and followed the NIH guidelines for the care and use of animals in laboratory research.
2.4. Data analysis Data were analyzed using a one-way analysis of variance with repeated measures to evaluate the effects of BMI administration over time. Post hoc tests consisted of the Dunnett test comparison of post-injection values with the pre-injection control. Significance was accepted as P, 0.05. Data are presented as means6S.E.M.
E. Schlenker et al. / Brain Research 895 (2001) 33 – 40
Fig. 1. Sites of injection for bicuculline (BMI) and artificial cerebrospinal fluid (aCSF) microinjections into the paraventricular nucleus of the hypothalamus (PVN). The figure is a schematic representation where BMI (stars) (n56) and aCSF (ovals) (n56) were microinjected. The black stars denote sites where BMI elicited the physiological responses described in the text. Open stars represent site at which BMI failed to elicit responses.
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150% (460620 bpm) after BMI injection, and displayed a similar pattern of response as MAP although HR only remained statistically different from baseline to the 8 min mark. BMI injection was also associated with marked changes in respiratory function. Frequency of ventilation increased from 7364 to 176614 b / min and remained significantly elevated for 15 min post-injection (Fig. 3). Tidal volume also showed an increase with BMI injection from 1.3660.21 to 2.0560.27 ml / b that plateaued between 2 and 6 min post-injection then returned to control values by 10 min post-injection (Fig. 3). Overall minute ventilation increased three-fold from 102619 to 305686 ml / min and remained significantly elevated for 8 min (Fig. 4). BMI stimulation of the PVN was also associated with an increase in oxygen consumption (maximum effect 10.1761.28 ml / min). However, the pattern of response of oxygen consumption to BMI administration was somewhat different than the other variables (Fig. 4). Oxygen consumption increased gradually reaching a peak at 6 min post-injection and then returned to baseline over the ensuing 4 min. Thus, BMI stimulated all variables, but not in the same manner nor for the same length of time.
2.5. Histology At the end of the experiments the animals were euthanized with sodium pentobarbital (150 mg / kg) and then perfused intracardially with a solution of 0.9% saline followed by 10% formaldehyde solution. The brains were then removed stored in a 10% formalin solution and cut with a sliding microtome at 60 mm coronal sections through the entire PVN region. The sections were then placed on gelatin-coated slides and allowed to air dry over night. Verification of the injection termination sites was performed under a low-power microscope using the atlas of Paxinos and Watson [19]. Injections were considered acceptable if they fell within a 0.6 mm radius of the PVN [23]. Several sites around the PVN served as anatomical controls for the BMI injections. No response was elicited from these microinjections (Fig. 1).
3. Results Baseline values for MAP, HR, oxygen consumption, ventilation, frequency and tidal volume were 12565.0 mm Hg, 352617 b.p.m., 5.5260.23 ml / min, 138.6622.8 ml / min, 8968 b / min, and 1.4760.23 ml / b, respectively. Injection of aCSF did not affect any variable relative to baseline measurements and all variables remained stable over the 20 min of measurements (see Figs. 2–4). By contrast, BMI microinjected into the PVN increased both HR and MAP within 2 min (Fig. 2). A significant elevation of MAP (maximum D51966 mmHg) was sustained for 15 min after injection and then gradually returned to baseline values by 20 min post-injection. Heart rate increased by
4. Discussion This study was undertaken to test the hypothesis that the PVN modulates respiratory function in conscious rats. The results indicate that the PVN is a pivotal region of the hypothalamus that influences not only oxygen consumption and cardiovascular parameters, but also control of breathing in conscious rats. The PVN is an important forebrain site involved in the integration of neurohumoral outflow to the cardiovascular system [25]. Previous studies have shown that stimulation of the PVN caused an increase in MAP, HR and sympathetic nervous system activity. In the present study we have confirmed the pressor and tachycardic responses to PVN stimulation in conscious rats. The responses we observed compare favorably with previous responses obtained in our hands [15,16] and those of others [10,12,20,28]. Thus, the cardiovascular outcome of the present studies is comparable previous work. In addition, we also observed that stimulation of the PVN was associated with a marked increase in respiratory function. Relatively few previous studies have addressed the role of the PVN in the control of ventilation. A separate study monitored neural activity in the PVN in freely-behaving cats and reported that PVN activity changed during phasic respiratory events suggesting a link between the PVN and respiratory function [13]. More directly, electrical stimulation the PVN in anesthetized rabbits caused a 44% increase in respiratory rate [8]. The study that most closely resembles the current work is that of Yeh and coworkers [27] who determined the effects of
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Fig. 2. Effects of bicuculline methiodide (BMI) (solid lines) and artificial cerebrospinal fluid (aCSF) (dashed lines) microinjected into the PVN on (A) mean arterial pressure and (B) heart rate (b.p.m.5beats per min). Pre-values represent mean baseline values immediately preceding the injection of either aCSF or BMI. The arrow and asterisks indicate significant (P,0.05) responses relative to the ‘pre’ values. Values are mean6S.E.M., n56.
bilateral microinjections of L-glutamate into the PVN respiratory function in anesthetized rats. They reported changes in inspiratory and expiratory times corresponding to an increase in frequency of approximately 25%. We also observed an increase in ventilatory frequency however in our study respiratory frequency doubled after BMI injection. We also recorded a 69% increase in tidal volume whereas Yeh et al. reported that peak diaphragmatic electromyographic activity (a surrogate of tidal volume) increased by approximately 100%. Thus, there are differences in the relative magnitudes of the responses of various respiratory functions to stimulation of the PVN between our study and that of Yeh et al. These could be the
result of differences between anesthetized and conscious preparations, since anesthesia may influence PVN-mediated responses [12]. On the other hand these differences may represent variations in the population of PVN neurons activated. Yeh et al. used glutamate as a stimulant, which may activate all neurons in the PVN whereas we used BMI, which should only activate those neurons tonically inhibited by GABA. Nevertheless, in the final analysis, we observed that PVN stimulation caused minute ventilation to increase to three times its baseline value. This agrees well with overall minute ventilation response (32.5) calculated from the study of Yeh et al. Thus, in addition to a clear effect on cardiovascular function, findings of the
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Fig. 3. Effects of bicuculline methiodide (BMI) (solid lines) and artificial cerebrospinal fluid (aCSF) (dashed lines) microinjected into the PVN on (A) frequency of breathing (b / m5breaths per min) and (B) tidal volume (ml / b5milliliters per breath). Pre-values represent mean baseline values immediately preceding the injection of either aCSF or BMI. Asterisks indicate significant (P,0.05) responses relative the ‘pre’ values. Values are mean6S.E.M., n56.
current study and those of others indicate that the PVN can also exert profound effects on respiratory function. Moreover, the cardiovascular and respiratory responses are interactive. We have previously shown that PVN stimulation increases MAP primarily by increasing cardiac output [14]. The increases in tidal volume and frequency recorded in the present study could facilitate venous return to support an increase in cardiac output. Thus, the PVN appears to elicit coordinated respiratory and cardiovascular responses. The pathways by which the PVN influences ventilation remain to be fully elucidated. It is known however that the PVN has extensive neural connections to other brain regions that are involved in control of respiration [26]. Zheng and coworkers [29] used anterograde tracing with Phaseolus vulgaris leucoagglutinin in rats to denote several pathways of efferent fibers from the PVN. One pathway projected to the central gray matter, Edinger–Westphal
nucleus, pedunculopontine tegmental nucleus, nucleus of the locus ceruleus and parabrachial nucleus. A second pathway had projections to the nucleus ambiguus, nucleus of the solitary tract and the dorsal motor nucleus of the vagus. The PVN also makes afferent connections with other areas such as the hippocampus and arcuate nucleus that have been shown to participate in control of breathing [6,22,25,26]. Thus the PVN may affect respiratory function via direct neural circuits to other respiratory control centers in the brain. Alternatively, the relationship between the PVN and respiration may be indirect. In view of evidence indicating that catecholamines influence ventilation [5] and that PVN stimulation increases sympathetic nerve activity [28] and circulation catecholamine concentrations [15,16], it is possible that increases in sympathetic nervous system outflow could account for the respiratory stimulation we observed. If this were the case, one would expect the
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Fig. 4. Effects of bicuculline methiodide (BMI) (solid lines) and artificial cerebrospinal fluid (aCSF) (dashed lines) microinjected into the PVN on (A) oxygen consumption and (B) ventilation. Pre-values represent mean baseline values immediately preceding the injection of either aCSF or BMI. Asterisks indicate significant (P,0.05) responses relative the ‘pre’ values. Values are mean6S.E.M., n56.
pattern of responses to be similar between the cardiovascular responses, which are mediated by the sympathetic nervous system [16], and the ventilatory responses. Indeed, the time-course of the cardiovascular and respiratory responses were quite similar suggesting that they might be mediated via similar pathways that include the sympathetic nervous system. The PVN is also well recognized as a brain site that participates in the control of metabolism [3]. Indeed we observed an increase in oxygen consumption of 200%. The PVN sends direct neural projection to brown adipose tissue (BAT), a major controller of metabolic rate in rodents [4]. Moreover, stimulation of the PVN has been observed to
increase BAT activity [2]. Similarly, increases in circulating catecholamine concentrations also stimulated BAT activity [9]. Thus, it seems reasonable that activation of BAT may have contributed significantly to the large increase in oxygen consumption observed in the present study. It could be argued that the respiratory responses we have reported were secondary to changes in metabolic rate and oxygen consumption, since the time-course of the oxygen consumption response lagged behind that for the ventilatory response. An alternative explanation is that an overshoot in the response of the respiratory system to oxygen demand, or inherent variability in the ventilatory data as reflected by the large S.E.M. contributed to these
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apparent differences in time course. Thus, the ventilatory response could include both direct and indirect effects that have different time-courses with the initial ventilatory response (at 2 min) representing a direct effect and the later time-points driven by a metabolic increase. The neurochemical signals controlling the PVN outflow to respiratory circuits is not known. Previous studies have used glutamate as a chemical stimulant. While an effective neurophysiological tool, this approach yields little information concerning neurotransmitters involved in the control of respiration since almost all neurons are responsive to glutamate. Consequently, glutamate may simultaneously stimulate excitatory and inhibitory circuits. In the present study we used BMI to stimulate the PVN. BMI has been used as an antagonist of GABAA receptors, which are abundantly found in the PVN [7]. Recently, however it has been suggested that BMI may also block calcium-activated potassium channels in some [11,17], but not all brain nuclei [24]. The use of an antagonist is advantageous since a response will only be observed if significant endogenous inhibitory tone is present. Accordingly, one possible interpretation of our findings is that the activity of PVN neurons involved in the control of respiratory drive may be tonically suppressed by endogenous GABAergic systems. Consistent with a possible role for GABAergic systems in hypothalamic control of ventilation, other studies have shown that oxygen consumption was increased by baclofen (a GABA B receptor agonist) microinjected into the 3rd ventricle in rats [1] and we also noted that microinjection of baclofen directly into the arcuate nucleus of conscious rats stimulates ventilation [21], Alternatively, if BMI was acting primarily via inhibition of calcium-activated potassium channels, our data suggest that this neural control mechanism tonically inhibits PVN control of ventilatory function. Moreover, a recent study also suggests an interaction between PVN nitric oxide and GABAergic systems in the control of cardiovascular function [28]. Whether this interaction of signaling systems is involved in PVN modulation of respiratory function remains to be determined. In any case, the data from the present study do not allow discrimination between these interesting possibilities. In conclusion, the findings of the current study suggest that inhibitory systems in the PVN modulate oxygen consumption, MAP, HR and control of breathing in conscious rats. The PVN is an important integratory center for these parameters and is ideally suited to modulate cardiorespiratory coupling in pathological states, such as hypertension associated with sleep apnea [18].
Acknowledgements This work was supported by NIH HLBI [62167 (D.M.) and the USD Foundation (E.S.).
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