Further evidence that extracellular serotonin in the rostral ventrolateral medulla modulates 5-HT1A receptor-mediated attenuation of exercise pressor reflex

We determined changes in extracellular levels of glutamate, serotonin (5-HT), norepinephrine (NE), and dopamine (DA) within rostral ventrolateral medulla (RVLM) during 5-HT_1A-receptor stimulation-mediated inhibition of cardiovascular responses to static muscle contraction using anesthetized rats. In ten rats, muscle contraction significantly increased (P<0.01) mean arterial pressure (MAP) by 29±4 mmHg, heart rate (HR) by 25±3 bpm, and glutamate levels by 4.5±0.8 ng/5 μl. Microdialysis of a 5-HT_1A receptor agonist, 8-OH-DPAT (10 mM), into the RVLM for 30 min attenuated cardiovascular responses to muscle contraction and had no effect on glutamate levels. A subsequent administration of 10 mM WAY100635, a 5-HT_1A antagonist, into the RVLM antagonized the attenuating effects of 8-OH-DPAT. In another ten rats, muscle contraction significantly increased (P<0.01) MAP and HR by 20±2 mmHg and 25±8 bpm, respectively. In addition, levels of 5-HT, NE, and DA in the RVLM significantly increased (P<0.01) by 3.6±0.3, 3.2±0.3, and 3.3±0.4 pg/10 μl, respectively. Administration of 8-OH-DPAT (10 mM) into the RVLM for 30 min attenuated cardiovascular responses to muscle contraction and had no effects on NE and DA levels. However, the drug significantly attenuated 5-HT levels following a muscle contraction. Microdialysis of 10 mM WAY100635 into the RVLM reversed both cardiovascular and 5-HT changes. These results suggest that stimulation of 5-HT_1A-receptors within the RVLM attenuates cardiovascular responses to static exercise via a reduction of extracellular 5-HT concentration and most likely not through changes in glutamate, NE or DA levels.     

Rostral ventrolateral medulla opioid receptor activation modulates glutamate release and attenuates the exercise pressor reflex

We previously reported that the administration of [D-Ala(2)]methionine enkephalinamide (DAME), an opioid receptor agonist, into the rostral (RVLM) but not into the caudal ventrolateral medulla (CVLM), attenuated increases in mean arterial pressure (MAP) and heart rate (HR) during static muscle contraction that had been blocked by prior microdialysis of the opioid receptor antagonist, naloxone [Am. J. Physiol. 274 (1998) H139-H146]. In this study, we determine whether this RVLM-mediated opioidergic-modulation of cardiovascular responses is associated with localized changes in extracellular concentrations of glutamate, an excitatory amino acid, using microdialysis techniques in anesthetized rats. Muscle contraction increased MAP and HR by 37+/-5 mmHg and 23+/-3 bpm, respectively. Extracellular glutamate concentrations, determined using HPLC-ECD, increased from 0.8+/-0.2 to 6.6+/-1.2 ng/5 microliter in the bilateral RVLM areas. Microdialysis of DAME (100 microM) for 30 min attenuated the contraction-evoked increases in MAP, HR, and glutamate levels (20+/-4 mmHg, 10+/-2 bpm, and 1.8+/-0.2 ng/5 microliter, respectively). After microdialysis of naloxone (100 microM) for 30 min into the RVLM, muscle contraction blocked the attenuations (35+/-5 mmHg, 26+/-4 bpm, and 5.8+/-1.0 ng/5 microliter, respectively). Developed muscle tensions were similar throughout the protocol (676+/-38, 678+/-37 and 687+/-37 g, respectively). These results suggest that an opioidergic receptor-mediated mechanism within the RVLM attenuates cardiovascular responses during static exercise via modulating extracellular concentrations of glutamate in the RVLM.     

Glutamate neurotransmission and nitric oxide interaction within the ventrolateral medulla during cardiovascular responses to muscle contraction

We previously reported that nitric oxide, within the RVLM and CVLM, plays an opposing role in modulating cardiovascular responses during static muscle contraction [B.J. Freda, R.S. Gaitonde, R. Lillaney, A. Ally, Cardiovascular responses to muscle contraction following microdialysis of nitric oxide precursor into ventrolateral medulla, Brain Res. 828 (1999) 60-67]. In this study, we determined whether the effects of administering L-arginine, a precursor for the synthesis of nitric oxide, and N(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase inhibitor, into the rostral (RVLM) and caudal (CVLM) ventrolateral medulla on cardiovascular responses elicited during static muscle contraction were mediated via an alteration of localized glutamate concentrations using microdialysis techniques. In experiments within the RVLM (n=8), muscle contraction increased MAP and HR by 21+/-2 mmHg and 22+/-3 bpm, respectively. Glutamate increased from 1.1+/-0.4 to 4.4+/- 0.6 ng/5 microl measured from bilateral RVLM areas. Microdialysis of L-arginine (1.0 microM) for 30 min attenuated the contraction-evoked increases in MAP, HR, and glutamate levels. After subsequent microdialysis of L-NMMA (1.0 microM) into the RVLM, contraction augmented the pressor and tachycardic responses and glutamate release. In experiments within CVLM (n=8), muscle contraction increased MAP and HR by 22+/-3 mmHg and 20+/-2 bpm, respectively. Glutamate increased from 0.8+/-0. 4 to 3.6+/-0.6 ng/5 microl measured from the CVLM. L-Arginine augmented the cardiovascular responses and glutamate release and L-NMMA attenuated all the effects. Results suggest that nitric oxide within the RVLM and CVLM plays opposing roles in modulating cardiovascular responses during static exercise via decreasing and increasing, respectively, extracellular glutamate levels.     

Cardiovascular effects of adrenocorticotropin microinjections into the rostral ventrolateral medullary pressor area of the rat

The presence of adrenocorticotropic hormone (ACTH)-immunoreactive cells and melanocortin (MC) receptors (MC4 and to a lesser extent MC3) has been demonstrated in the medullary reticular formation in the general area where rostral ventrolateral medullary pressor area (RVLM) is located. The importance of RVLM in the regulation of cardiovascular function is well established. Based on these reports, it was hypothesized that ACTH may play a role in the regulation of cardiovascular function. To test this hypothesis, experiments were carried out on artificially ventilated, adult male, urethane-anesthetized and unanesthetized mid-collicular decerebrate rats. The RVLM was identified by microinjections (100 nl) of L-glutamate (L-Glu). Microinjections (100 nl) of ACTH (0.5, 1 and 2 mmol/l) into the RVLM elicited increases in MAP and HR; tachycardic responses were relatively inconsistent. The effects of ACTH were blocked by SHU9119 and agouti-related protein (AGRP). SHU9119 (a synthetic compound) and AGRP (an endogenous peptide) are antagonists for MC4, and to a lesser extent MC3, receptors. The specificity of these antagonists for MC receptors was indicated by their lack of effect on l-Glu responses. Microinjection of ACTH into the RVLM increased the efferent discharge in the greater splanchnic nerve. It was concluded that (1) ACTH exerts excitatory effects on RVLM neurons resulting in pressor and tachycardic responses, (2) these responses were mediated via MC4 and to a lesser extent MC3 receptors in the RVLM, and (3) the pressor effects of ACTH were mediated via sympathetic activation. This is the first report showing central cardiovascular actions of ACTH.     

Effect of nitric oxide on release of glutamate in the subretrofacial nucleus (SRF) during the exercise pressor reflex in cats

The subretrofacial nucleus (SRF) has been known to play a crucial role in the expression of the exercise pressor reflex. Previously, we have reported that the release of glutamate (Glu) in the SRF was increased during muscle contraction in anesthetized cats. In this study, static muscle contraction of the triceps surae for 4 min was induced by electrical stimulation of L7 and S1 ventral roots. Endogenous release of Glu and citrulline (Cit) from the SRF was recovered by microdialysis and measured by HPLC. The microdialysis probes were also used to deliver L-arginine and L-NAME to test the effect of nitric oxide (NO) on release of Glu in the SRF and on the cardiovascular responses during muscle contraction. During control, muscle contraction significantly increased mean arterial pressure (MAP) from 98+/-8 to 151+/-9 mmHg, and the extracellular concentration of Glu from 610+/-120 to 1280+/-290 nM. Dialyzing 2 mM L-arginine into the SRF increased basal Cit concentration from 260+/-50 to 760+/-210 nM (P<0.05). During contraction after L-arginine, the increases in MAP and Glu concentration were significantly attenuated (86+/-3-124+/-6 mmHg and 300+/-60-460+/-100 nM, respectively). Dialysis of 0.5 mM L-NAME into the SRF decreased Cit concentration from 340+/-40 to 180+/-20 nM (P<0.05). During contraction after dialyzing L-NAME, the increases in MAP and Glu concentration were significantly potentiated (93+/-6-154+/-9 mmHg and 520+/-80-1290+/-380 nM, respectively). These results suggest that endogenous NO modulates the cardiovascular responses to static muscle contraction by affecting the release of Glu in the SRF.     

Cardiovascular responses to muscle contraction following microdialysis of nitric oxide precursor into ventrolateral medulla

We determined the effects of administering L-arginine, a precursor for the synthesis of nitric oxide, and L-NMMA (NG-monomethyl-L-arginine), a nitric oxide synthase blocker, into the rostral (RVLM) and caudal (CVLM) ventrolateral medulla on cardiovascular responses elicited during static contraction of the triceps surae muscle. Two microdialysis probes were inserted bilaterally into the RVLM or CVLM of anesthetized Sprague-Dawley rats using stereotaxic guides. For RVLM experiments, static muscle contraction evoked by stimulation of the tibial nerve increased mean arterial pressure (MAP) and heart rate (HR) by 29+/-3 mmHg and 44+/-7 bpm, respectively (n=8). Microdialysis of L-arginine (1.0 microM) for 30 min attenuated the contraction-evoked increases in MAP and HR. After discontinuing L-arginine, L-NMMA (1.0 microM) was microdialyzed into the RVLM for an additional 30 min followed by a muscle contraction. This contraction augmented the pressor response (37+/-4 mmHg) and HR (61+/-11 bpm) with respect to control values. For CVLM experiments, muscle contraction increased MAP and HR by 23+/-3 mmHg and 25+/-5 bpm, respectively (n=9). Microdialysis of L-arginine (1.0 microM) for 30 min potentiated the contraction-evoked increases in MAP and HR. Subsequent administration of L-NMMA (1.0 microM) into the CVLM for an additional 30 min blocked the augmented MAP and HR responses. Developed tensions did not alter during contractions throughout both RVLM and CVLM protocols. These results suggest that nitric oxide, within the RVLM and CVLM, plays an opposing role in modulating cardiovascular responses during static muscle contraction.     

The cardiovascular effects of angiotensin-(1–7) in the rostral and caudal ventrolateral medulla of the rabbit

Previous studies in the rat have indicated that the heptapeptide angiotensin-(1–7) has an excitatory action on pressor neurons in the rostral ventrolateral medulla that is equipotent to that evoked by angiotensin II, but which is mediated by separate receptors. In this study we have compared the cardiovascular effects and mechanisms of action of angiotensin-(1–7) with angiotensin II in the rostral and caudal ventrolateral medulla of the rabbit, a species which, unlike the rat, contains a high density of angiotensin receptors, similar to that observed in humans. Microinjections of angiotensin-(1–7) into the rostral and caudal ventrolateral medulla evoked dose-dependent increases and decreases, respectively, in arterial pressure and renal sympathetic nerve activity, but in comparison to angiotensin II much higher doses (approximately 50-fold higher) were required to produce cardiovascular response of similar magnitude. The cardiovascular effects of angiotensin-(1–7) were blocked by prior injection of the selective antagonist [ -Ala⁷]-Ang-(1–7) but were also blocked by the selective AT₁ receptor antagonist losartan. The results demonstrate that in the rabbit angiotensin-(1–7) can excite pressor and depressor neurons in the ventrolateral medulla, but indicate that these effects are mediated by AT₁ receptors. The much lower potency of angiotensin-(1–7) as compared to angiotensin II may be explained as a consequence of it having a much lower affinity to AT₁ receptors. Thus, in contrast to the rat, the results do not indicate that angiotensin-(1–7) has a biologically significant action in the ventrolateral medulla of the rabbit.     

Role of α1-adrenergic receptors in the intermediolateral column in mediating the pressor responses elicited by the stimulation of ventrolateral medullary pressor area

Microinjections of α1-adrenergic receptor agonists into the intermediolateral cell column of the spinal cord (IML) elicit sympathoexcitatory responses. This observation, together with the identification of projections of epinephrine-containing cells in the rostral ventrolateral medullary pressor area (VLPA) to the IML, has prompted speculation that epinephrine may mediate pressor responses to the stimulation of the VLPA. This hypothesis was tested in pentobarbital-anesthetized, artificially ventilated, male Wistar rats. A mesenteric arterial branch was cannulated for monitoring blood pressure. Pressor responses were elicited predominantly from T8–T10 by injections (1.7 nmol/20 nl) ofl-glutamate into the IML; maximum pressor responses(29.3 ± 4mmHg) were elicited from T9. Pressor responses were also elicited by injections of epinephrine into the IML at T9; maximum pressor effect(16.3 ± 1.2mmHg) was elicited by a dose of 0.05 pmol/20 nl. This effect of epinephrine at T9 was blocked by prior injections of prazosin (a selective α1-adrenergic receptor blocker; 0.125 pmol/20 nl) at the same site. Stimulation of the VLPA by unilateral microinjections of glutamate elicited pressor responses(56 ± 12mmHg). Bilateral injections of prazosin at T8–T10, in the dose ( (0.125 pmol) that blocked a maximally effective dose of epinephrine, did not block the pressor responses to subsequent injections of glutamate into the VLPA. On the other hand, bilateral microinjections of AP-7 (an NMDA receptor blocker; 1 nmol/20 nl), but not DNQX (10 pmol; a non-NMDA receptor blocker), into the IML at T8–T10 blocked the pressor effects of the subsequent injections of glutamate into the VLPA. At the dose used, AP-7 did not alter pressor responses to injections of kainic acid or AMPA into the IML at T9. These results suggest that under the experimental conditions in this study, the pressor responses following the stimulation of VLPA are not mediated by α1-adrenergic receptors in the IML. On the other hand, NMDA receptors in the IML do mediate these pressor responses.     

A role for KATP-channels in mediating the elevations of cerebral blood flow and arterial pressure by hypoxic stimulation of oxygen-sensitive neurons of rostral ventrolateral medulla

Reticulospinal sympathoexcitatory neurons of rostral ventrolateral medulla (RVL) are selectively excited by hypoxia to elevate arterial pressure (AP) and cerebral blood flow (rCBF), that are elements of the oxygen-conserving (diving) reflex. We investigated whether K_ATP⁺-channels participate in this. Tolbutamide and glibenclamide, K_ATP⁺-channel blockers, microinjected into RVL in anesthetized rats, dose-dependently and site-specifically elevated AP and rCBF and potentiated responses to hypoxemia. K_ATP⁺-channels may mediate hypoxic excitation of oxygen-sensing RVL neurons.     

Losartan, nonpeptide angiotensin II-type 1 (AT1) receptor antagonist, attenuates pressor and sympathoexcitatory responses evoked by angiotensin II andL-glutamate in rostral ventrolateral medulla

We investigated the effect of losartan, a nonpeptide angiotensin II (Ang II)-type 1 (AT₁) receptor antagonist, on the responses evoked by Ang II andL-glutamate (L-Glu) in the rostral ventrolateral medulla (RVLM). Adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were anesthetized with halothane and artificially ventilated. Responses of mean arterial pressure (MAP), heart rate (HR) and splanchnic sympathetic nerve activity (SNA) to microinjection of Ang II (100 pmol) orL-Glu (2 nmol) into the RVLM were examined following microinjection of losartan (10 pmol–10 nmol). Ang II increased MAP (16 ± 1mmHg in SHR and16 ± 1mmHg in WKY) and SNA (9 ± 1%and10 ± 1%, respectively), which were significantly (P < 0.01) attenuated by pretreatment with losartan (100 pmol − 10 nmol) in both strains. In addition, the pressor and sympathoexcitatory responses evoked byL-Glu were attenuated by losartan in a dose-dependent manner. The increases of MAP evoked byL-Glu (53 ± 6mmHg in SHR and39 ± 3mmHg in WKY) were suppressed to 5 ± 3mmHg(P < 0.01) and 4 ± 2mmHg (P < 0.01), respectively, in the presence of 10 nmol of losartan. The increase of SNA was also markedly inhibited by higher doses of losartan. The cardiovascular responses evoked byL-Glu, however, were not attenuated by pretreatment with either 1 nmol of [Sar¹, Thr⁸]-Ang II or 10 nmol of potassium acetate, suggesting that the effect of losartan onL-Glu response may not be attributed to the blockade of Ang II receptor or to the high concentration of potassium. These results indicate that the AT₁ receptor is responsible, in part, for the vasomotor action of Ang II in the RVLM and losartan has an inhibitory effect on pressor and sympathoexcitatory responses evoked byL-Glu by mechanisms other than those mediated by Ang II receptors.     

Synaptically released 5-HT modulates the activity of tonically discharging neuronal populations in the rostral ventral medulla (RVM)

There is substantial evidence for an important modulating role of monoamines (catecholamines and serotonin, 5-HT) in the rostral ventral medulla (RVM), a region which plays an important role in cardiovascular and nociceptive functions. We investigated in slices the role of endogenous monoamines in the synaptic control of the activity of rat RVM neuronal populations using intracellular recordings in the lateral RVM plus lateral aspect of nucleus paragigantocellularis lateralis. A triple-labelling protocol allowed us to identify the location of impaled neurons and their eventual monoaminergic phenotype within the serotonergic and catecholaminergic populations of the RVM. Focal electrical stimulation revealed the existence of a functional monoaminergic input onto RVM neurons which was mediated by endogenous 5-HT acting at inhibitory 5-HT1A receptors but did not involve noradrenergic neurotransmission. The slow 5-HT-mediated inhibitory postsynaptic potential (IPSP) was only observed in the regularly discharging neurons, which were found to be neither catecholaminergic nor serotonergic. The synaptic release of 5-HT was, itself, under an inhibitory control involving GABAA (gamma-aminobutyric acid) receptors. Moreover, we characterized the effect of the 5-HT-releasing agent fenfluramine on this functional 5-HT-mediated synaptic transmission. Our results show that the effect of fenfluramine is biphasic consisting of an initial prolongation of the serotonergic IPSP followed by a decrease in amplitude. Our data provide a basis for the previously reported inhibitory effects of exogenously applied serotonin agonists/antagonists on the autonomic functions controlled by the RVM. This 5-HT pathway, which functionally links the serotonergic and catecholaminergic regions, might play an important role in cardiovascular and nociceptive functions.     

Preproenkephalin mRNA is expressed by C1 and non-C1 barosensitive bulbospinal neurons in the rostral ventrolateral medulla of the rat

The autonomic regions of the thoracolumbar spinal cord receive a dense enkephalinergic (ENK) innervation from supraspinal sources, including the rostral ventrolateral medulla (RVLM). In the present study, we sought to determine whether the barosensitive bulbospinal (BSBS) neurons of the RVLM express preproenkephalin (PPE) mRNA. After injection of Fluoro-Gold (FG) into the upper thoracic spinal cord, neurons with PPE mRNA (PPE(+) neurons) were retrogradely labeled throughout the ventrolateral medulla. At the most rostral RVLM level, 29% of bulbospinal PPE+ cells were tyrosine hydroxylase-immunoreactive (TH-ir) and the latter constituted 19.4% of the bulbospinal TH-ir cells. We determined whether the bulbospinal PPE(+) RVLM neurons are barosensitive in two ways. First, we examined Fos production by FG-labeled RVLM neurons after 2 hours of hydralazine-induced hypotension (to 73 +/- 2 mm Hg) in conscious rats. Hydralazine (10 mg/kg i.v.) increased the number of Fos-ir neurons by two- to eightfold at all levels of the ventrolateral medulla examined. In the RVLM, 54% of bulbospinal PPE(+) neurons were Fos-ir, whereas such cells were more rarely found at caudal ventrolateral medullary levels. Second, we recorded individual BSBS RVLM units extracellularly in anesthetized rats and filled them juxtacellularly with biotinamide. Most biotinamide-filled neurons were PPE(+) (10 of 17), and the PPE(+) BSBS cells had a faster axonal conduction velocity than those without PPE mRNA (4.2 vs. 0.67 m/sec). Four of the 10 PPE(+) BSBS RVLM neurons were TH-ir. In summary, PPE mRNA is predominantly expressed by RVLM BSBS neurons with lightly myelinated spinal axons. PPE mRNA is present in most noncatecholaminergic BSBS neurons and also in approximately 20% of the bulbospinal C1 neurons. BSBS RVLM neurons most likely provide a major ENK input to sympathetic preganglionic neurons and PPE mRNA is the first identified positive phenotype of the non-C1 BSBS RVLM neurons.     

In vivo evidence for serotonin 5-HT2C receptor-mediated long-lasting excitability of lumbar spinal reflex and its functional interaction with 5-HT1A receptor in the mammalian spinal cord

The purpose of the present study was to investigate the 5-HT(2C) receptor-mediated effects on the spinal monosynaptic mass reflex activities and also its functional interactions with 5-HT(1A) receptors in anesthetized, acutely spinalized mammalian adult spinal cord in vivo. Intravenous administration of (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (0.1 mg/kg), an agonist of 5-HT(2A/2C) receptors, significantly increased the excitability of spinal motoneurons as reflected by an increase in the spinal monosynaptic mass reflex amplitude to 150-200% of the control. 5-HT(2A/2C) receptor-induced motoneuron excitability was slow, persistent and long-lasting for more than 2h that was significantly inhibited by 5-HT(2C) receptor specific antagonist SB 242084 administered 10 min prior to DOI. Simultaneous administration of DOI (0.1 mg/kg, i.v.) along with (+/-)-8-hydroxy dipropylaminotetraline hydrobromide (8-OH-DPAT) (0.1 mg/kg, i.v.) completely inhibited DOI-induced spinal monosynaptic mass reflex facilitation. In another separate study, administration of 8-OH-DPAT (0.1 mg/kg, i.v.) at the maximum response of DOI also inhibited the motoneuron's excitability; however, the inhibition lasted only for a period of 40-60 min after administration of 8-OH-DPAT, after which the spinal monosynaptic mass reflex amplitude reached its maximum level. These findings suggest that the 5-HT(2C) receptor is primarily involved in the mediation of the long-lasting excitability of spinal motoneurons and possibly interacts with its functional counterpart, 5-HT(1A) receptors in the mammalian adult spinal cord.     

Involvement of I(1)-imidazoline receptors in baroreceptor reflex in the caudal ventrolateral medulla of rats

There is ample evidence to show the existence of center I(1)-imidazoline receptors that are involved in the regulation of cardiovascular activities. The purpose of this study was to examine the possible role of I(1)-imidazoline receptors and alpha(2)-adrenoceptors within the caudal ventrolateral medulla (CVLM) in mediating the baroreceptor reflex in anesthetized rats. Unilateral microinjection of idazoxan (2 nmol in 50 nl), a mixed antagonist of I(1)-imidazoline receptors and alpha(2)-adrenoceptors, into the CVLM significantly (P<0.01) decreased blood pressure (BP), heart rate (HR), and the firing rate of presympathetic neurons in the rostral ventrolateral medulla (RVLM) by 21+/-6 mmHg, 25+/-5 beats per min and 3.5+/-0.9 spikes/s, respectively. Moreover, unilateral injection of idazoxan into the CVLM significantly (P<0.01) reduced the inhibitory responses of the ipsilateral RVLM presympathetic neurons evoked by stimulation of aortic nerve and elevation of BP, and partially inhibited the neuronal cardiac cycle-related rhythm. Depressor responses evoked by aortic nerve stimulation were significantly (P<0.01) attenuated 10 and 20 min after bilateral microinjection of idazoxan (2 nmol in 50 nl for each side) into the CVLM (-20+/-4 and -30+/-4 vs. -40+/-1 mmHg). However, injection of yohimbine (500 pmol in 50 nl), a selective alpha(2)-adrenoceptor antagonist, into the CVLM did not affect the resting cardiovascular activities and baroreceptor reflex. It is concluded that the CVLM I(1)-imidazoline receptors are involved in maintenance of tonic cardiovascular activities and transmission of the baroreceptor reflex.     

Inhibition of evoked glutamate release by the neuroprotective 5-HT(1A) receptor agonist BAY x 3702 in vitro and in vivo

Brain ischemia provoked by stroke or traumatic brain injury induces a massive increase in neurotransmitter release, in particular of the excitotoxin glutamate. Glutamate triggers a cascade of events finally leading to widespread neuronal cell damage and death. The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant which shows pronounced beneficial effects in various animal models of ischemic brain injury. As shown previously BAY x 3702 binds to 5-HT(1A) receptors of different species in subnanomolar range and is characterized as a full receptor agonist. In this study we investigated the influence of BAY x 3702 on potassium-evoked glutamate release in vitro and ischemia-induced glutamate release in vivo. In rat hippocampal slices BAY x 3702 inhibited evoked glutamate release in a dose-dependent manner (IC(50)=1 microM). This effect was blocked by the selective 5-HT(1A) receptor antagonist WAY 100635, indicating that BAY x 3702 specifically acts via 5-HT(1A) receptors. In vivo, release of endogenous aspartate and glutamate was measured in the cortex of rats by microdialysis before and after onset of permanent middle cerebral artery occlusion. Single dose administration of BAY x 3702 (1 microg/kg or 10 microg/kg i.v.) immediately after occlusion reduced the increase and total release of extracellular glutamate by about 50% compared to non-treated animals, whereas the extracellular aspartate levels were not significantly affected. Inhibition of glutamate release may therefore contribute to the pronounced neuroprotective efficacy of BAY x 3702 in various animal models of ischemic brain damage.     

Inhibitory effect of electroacupuncture (EA) on the pressor response induced by exercise stress

Abstract. We examined the effect of EA on the exercise stress-induced pressor response in healthy adult subjects of both sexes. Each subject was subjected to a bicycle exercise test using a ramp protocol once/week for three or four weeks. Subjects were asked to perform the following tests in random order: 1) a baseline exercise test without EA and 2) exercise after acupuncture at P 5–6, LI 4-L 7 and/or G 37–39 acupoints. Brachial systolic (SBP), diastolic (DBP), and mean blood pressures (MBP), heart rate (HR) and the rate-pressure product (RPP, systolic BP x HR/100) were measured every three min, while a 12 lead ECG was monitored continuously. We observed increases in MBP, SBP, HR and RPP in all 17 subjects during exercise. In 12 of the 17 subjects (71 %), EA for 30 min before exercise, either at Jianshi-Neiguan acupoints (P 5–6) or Hegu-Lique acupoints (LI 4-L 7), led to an increase in maximal workload, and reduced peak SBP, MBP and RPP responses to exercise; EA did not alter DBP or HR responses in these subjects. EA at control acupoints (Guangming-Xuanzhong acupoints, G 37–39) in five subjects did not alter the hemodynamic responses. Seven additional subjects were enrolled to study the effect of EA during a bicycle exercise test using a constant workload. The results were similar, in five of the seven subjects SBP, MBP and RPP after exercise were attenuated significantly by EA at P 5–6. We conclude that EA at specific acupoints improves exercise capacity and reduces the hemodynamic responses in approximately 70% of normal subjects.This project is supported by the DANA Foundation, the Susan-Sameuli Center for Integrative Medicine at UCI, the Larry K. Dodge Chair in Integrative Biology (JCL), and the General Clinical Research Center of UCI (5M61RR000827).     

5-HT(1A) receptor-mediated inhibition and 5-HT(2) as well as 5-HT(3) receptor-mediated excitation in different subdivisions of the rat amygdala

The techniques of extracellular single cell recording and microiontophoresis were used to study the effects of serotonin (5-HT) and of 5-HT(1A), 5-HT(2A/2C) and 5-HT(3) receptor agonists on the spontaneous activity of amygdaloid neurons in rats anesthetized with urethane. The background discharge rate was modified by 5-HT as well as by 5-HT agonists in about two-thirds of neurons tested in different nuclei of the amygdaloid complex. Whereas the 5-HT(2) and 5-HT(3) agonists significantly increased the neuronal discharge rate in nearly all subdivisions of the amygdala, the 5-HT(1A) agonist significantly inhibited the firing rate. Co-administration of bicuculline and 5-HT receptor agonists prevented the 8-OH-DPAT-induced increases in the firing rate in most cases tested, as well as the inhibitory effects of DOI or 2-methyl-5HT. Therefore, GABAergic interneurons seem to be involved in the mediation of serotonergic effects. The action of 5-HT agonists on the neuronal discharge rate was blocked by different receptor-specific antagonists. The results support the hypothesis that 5-HT exerts control throughout the amygdala by acting at least on 5-HT(1A), 5-HT(2A/2C) and 5-HT(3) receptors seemingly located both on projection and interneurons.     

Effects of the cold pressor test on short-term fluctuations of finger arterial blood pressure and heart rate in normal subjects

The effect of the cold pressor test on autonomic cardiovascular control was studied non-invasively by means of spectrum analysis of periodic heart rate and blood pressure fluctuations in ten normal subjects. Fast Fourier Transform algorithm based on a 256-point time series (sampling rate 2 Hz, i.e. 2-min and 8-s) was used to estimate the amplitude spectra of heart rate and blood pressure rhythmicity at the low frequency (70–140 mHz) and respiration related frequency (230–270 mHz) band. Respiration rate was controlled at 250 mHz. Auto- and cross-spectral techniques were used to determine the complex relationship between systolic blood pressure and heart rate fluctuations in the frequency domain. The spectral pattern of systolic and diastolic blood pressure showed a marked increment of the absolute and relative (100 multiplied with absolute value/total area under the curve of the amplitude spectrum from 15–500 mHz) low frequency component: control vs. cold pressor test—systolic blood pressure—absolute values (in [mmHg/Hz^1/2]): 634.4 ± 48.9 vs. 827.4 ± 69.9* relative values (in [%]): 26 ± 2 vs. 32 ± 2*; diastolic blood pressure —absolute values: 433.2 ± 42.3 vs. 537.2 ± 45.8* relative values: 35 ± 3 vs. 40 ± 2*, (average ± SEM, *P < 0.05). The cold pressor test induced no change in average heart rate; the absolute low frequency component in heart rate spectra increased clearly during the test: low frequency component (in [bpm/Hz^1/2]): 586.9 ± 89.9 vs. 712.0 ± 91.4*, while the relative low frequency component did not change: 29 ± 3 vs. 30 ± 3. There was no alteration in the respiration related frequency component in all time series and in the transfer gain and phase functions during cold pressor test. We conclude that the cold pressor test increases low frequency systolic blood pressure fluctuations which reflects an increase in sympathetic vasomotor control. Furthermore the cold pressor test does not induce change in the transfer gain and phase characteristics of modulation of heart rate by systolic blood pressure.     

Synthesis, release and receptor binding of acetylcholine in the C1 area of the rostral ventrolateral medulla: contributions in regulating arterial pressure

This study sought to determine whether release of acetylcholine (ACh) within the C1 area of nucleus reticularis rostroventrolateralis (RVL) contributes to the tonic maintenance of arterial pressure (AP) in the rat. The activity of choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh, varied 5.5-fold in micropunches of the 6 medullary regions examined. ChAT activity in the C1 area (179 +/- 35 nmol [14C]ACh formed/mg protein/60 min; n = 4) was intermediate between that of the hypoglossal nucleus (249 +/- 38; highest) and the pyramids (45 +/- 11; lowest) and equivalent to that found in the parietal cortex (147 +/- 15). Release of [3H]ACh from C1 area micropunches was increased by raising extracellular K+ concentrations (5-55 mM) and was entirely Ca2(+)-dependent. Muscarinic receptor binding density was assessed using [3H]quinuclidinyl benzylate ([3H]QNB) as ligand and a recently developed 'electronic micropunch' technique which allows measurement of quench-corrected [3H]QNB binding within corresponding cylinders of tissue obtained by the mechanical micropunch cannula. [3H]QNB binding density varied 2.6-fold: lateral reticular nucleus pars lateralis greater than C1 area greater than nucleus ambiguus = hypoglossal nucleus = pyramid = oral spinal trigeminal nucleus. In urethane-anesthetized rats, inhibition of ACh synthesis by hemicholinium-3 (HC-3, 3 nmol/50 nl), or blockade of muscarinic receptors by scopolamine (SCOP, 3 nmol/50 nl), reduced resting mean AP by 18-24 mm Hg following bilateral microinjection into the C1 area. These concentrations of HC-3 and SCOP were sufficient to attenuate by 70-80% the increase in local cholinergic neurotransmission elicited by the cholinesterase inhibitor physostigmine given systemically. High concentrations of SCOP (30-150 nmol/50 nl) lowered AP by 46-60 mm Hg. Similarly, bilateral microinjections of GABA (10 nmol/50 nl) into the C1 area markedly reduced mean AP by 51 +/- 6 mm Hg to levels normally found after transection of the spinal cord. Thus, a substantial portion of tonic sympathetic activity may be driven by activation of muscarinic receptors in the C1 area. In the spontaneously hypertensive rat (SHR), a genetic model of hypertension, neither spontaneous nor K(+)-evoked release of [3H]ACh from the C1 area differed from that of normotensive Wistar-Kyoto rats (WKY).(ABSTRACT TRUNCATED AT 400 WORDS)     

Further evidence for differential affinity states of the serotonin1A receptor in rat hippocampus.

The binding profile of [³H]8-hydroxy-2-(di-N-propylamino)-tetralin ([³H]8-OH-DPAT) to serotonin_1A (5-HT_1A) sites in rat hippocampal, frontocortical and striatal membranes has been compared. In these regions, [³H]8-OH-DPAT labels both a high and a low-affinity binding sites; the affinity values for each of the two sites are comparable in the different brain regions, but have different maximal capacity. By modifying the experimental conditions in a series of hippocampal membrane preparations, reciprocal changes in the proportion of the two sites were observed suggesting that they represent, at least in this region, different conformations or affinity states of a single receptor protein. In contrast to the lower affinity states, it appears that the high-affinity state is stabilized by coupling with a G-protein. Evidence supporting this statement is provided by addition of the guanine nucleotide Gpp(NH)p, breakage of labile disulfide bonds using N-ethylmaleimide and increasing membrane rigidity with ascorbate-induced lipid peroxidation, conditions which all reduced the density of receptors in the high-affinity state. Moreover, the high-affinity state appears to be stabilized at the expense of the lower affinity state in the presence of Mn²⁺. On the other hand, a complete shift to the low-affinity binding state was observed after a 24 h in vivo treatment with inhibitors of monoamine oxidase A (phenelzine or clorgyline) but not of monamine oxidase B (deprenyl). This disappearance of the high-affinity state with a concomitant increase in the binding capacity of the low-affinity state was reproduced by inhibiting monoamine oxidase A in vitro, as well as by reducing preincubation washout periods. Also, competitors of the [³H]8-OH-DPAT binding site, such as serotonin and unlabelled 8-OH-DPAT, display two affinity states while others like (±)-propranolol, tryptamine and spiperone recognize a single affinity component. These results suggest that the 5-HT_1A binding site may exhibit at least two different affinity states depending upon its microenvironment and the intrinsic activity of the ligand used.