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We have previously reported that microinjections of endomorphin-2 (E-2; an endogenous mu-receptor agonist) into the medial subnucleus of the NTS (mNTS) elicit depressor and bradycardic responses via activation of ionotropic glutamate receptors located on secondary mNTS-neurons. Based on this report, it was hypothesized that activation of secondary mNTS neurons by E-2 may result in an exaggeration of baroreflex responses. In order to test this hypothesis, baroreflex responses were studied in adult, urethane-anesthetized, artificially ventilated, male Wistar rats before and after the microinjections of E-2 into the mNTS. Baroreceptors were stimulated by applying pressure increments (80-100 mm Hg) in the carotid sinus and by electrical stimulation (stimulus intensity: 0.5 V, frequencies 5, 10, and 25 pulses/s, pulse duration: 1 ms) of the aortic nerve for 30-s periods. Baroreceptor stimulation elicited depressor and bradycardic responses. Microinjections (100 nl) of E-2 (0.4 mmol/l) into the mNTS attenuated the baroreflex responses. Microinjections of naloxone (an opioid receptor antagonist) into the mNTS (0.5 mmol/l) did not alter baroreflex responses. Based on these results, it was concluded that activation of mu-opioid receptors in the mNTS attenuates baroreflex responses. Possible mechanisms for excitatory effects of E-2 in the mNTS resulting in depressor and bradycardic responses, on one hand, and inhibitory effects resulting in attenuation of baroreflex responses, on the other, are discussed.

Cardiovascular effects of activation of opioid receptor like receptors (ORL1 receptors) in the nucleus ambiguus were studied in urethane-anesthetized, adult male Wistar rats. Microinjections of nociceptin (0.31, 0.62, 1.25 and 2.25 mmol/L) into the nucleus ambiguus elicited increases in heart rate (17.5 ± 4, 33.3 ± 2.9, 16.5 ± 1.5 and 13.9 ± 2.7 beats/min, respectively) which were blocked by an ORL1 receptor antagonist. These results indicate that activation of ORL1 receptors in the nucleus ambiguus elicits tachycardia.

Although several agonists and antagonists for different subtypes of metabotropic glutamate receptors (mGLURs) have become available in recent years, detailed information regarding their selectivity is not complete in the in vivo animal models. The purpose of the present investigation was to study the cardiovascular effects of microinjections of some of these mGLUR agonists and antagonists into the nucleus tractus solitarius (nTS). Microinjections (100 nl) of EC(50) concentrations of 3,5-DHPG (0.005 mM; mGLUR(1) agonist), APDC (17.3 mM; mGLUR(2/3) agonist), PPG (11.7 mM; mGLUR(8) agonist) and L-AP(4) (1 mM; mGLUR(4) agonist) into the nucleus tractus solitarius of urethane-anesthetized male Wistar rats elicited depressor and bradycardic responses which may be mediated by pre- and/or postsynaptic mechanisms. The blocking effect of mGLUR antagonists used here was not specific for any one type of glutamate receptors (GLURs). For example, AIDA (50 mM; mGLUR(1) antagonist) blocked the effects of EC(50) concentrations of 3,5-DHPG, and PPG. LY341495 (135 mM; mGLUR(2/3) antagonist) blocked all of the mGLURs and ionotropic GLURs. EGLU, APICA and MCCG (250 mM each; mGLUR(2/3) antagonists) blocked the effects of APDC, NMDA and AMPA. CPPG (80 mM) and MSOP (125 mM), mGLUR(4) antagonists, blocked the effects of 3,5-DHPG, PPG and L-AP(4.) D-AP7 (NMDA receptor antagonist) and NBQX (a non-NMDA receptor antagonist) did not alter the responses of any of the mGLUR agonists. The data presented may be useful in assessing the role of metabotropic and ionotropic GLURs in mediating different cardiovascular reflexes.

Respiratory and cardiovascular responses to microinjections (10 nl) of l -glutamate (10 mM) into the dorsal medulla were studied in spontaneously breathing urethane-anesthetized, adult male Wistar rats. A total of 10 patterns of respiratory and cardiovascular responses were observed: (1) hypotension alone; (2) hypotension and bradycardia; (3) hypotension and apnea; (4) hypotension, bradycardia, and apnea; (5) apnea alone; (6) hypotension and fast and shallow breathing; (7) hypotension, bradycardia, and fast and shallow breathing; (8) fast and shallow breathing alone; (9) sighs; and (10) increase in BP and HR accompanied with fast and shallow breathing. The sites from which a combination of hypotension, bradycardia, and apnea was elicited, occupied a region in the medial subnucleus of nucleus tractus solitarius (nTS), the reticular formation just ventral to it, and the dorsal motor nucleus of vagus. The sites from which hypotension alone or a combination of hypotension and apnea were elicited occupied the margins of the medial subnucleus of nTS. The sites from which apnea alone was elicited were located in the ventrolateral part of nTS and the reticular formation just ventral to it. In the commissural subnucleus of nTS, the responses comparable to those elicited by peripheral chemoreceptor stimulation (i.e., increase in BP, HR, and respiratory rate) were located in a midline region just caudal to the calamus scriptorius, the sites from which sighs were elicited were located slightly lateral and deeper, the sites from which fast and shallow breathing were elicited were located in the dorsal portion, slightly lateral to the midline. These results are expected to prove useful in studies in which microinjection technique is used to identify transmitters/receptors involved in mediating respiratory and cardiovascular reflex responses.

The carotid body chemoreceptor afferents have been reported to project to a discrete area located in the nucleus commissuralis of nucleus tractus solitarius [A. Vardhan et al., Am. J. Physiol., 264 (1993) R41-R50]. The afore-mentioned study was done in spontaneously breathing rats and the afferents and efferents located in the chest wall and the respiratory tract of these animals were intact. In order to exclude the role, if any, of these afferents and efferents, in the present experiments respiratory changes were monitored by recording the phrenic nerve activity instead of tracheal airflow. Experiments were carried out in pentobarbital-anesthetized, bilaterally vagotomized, paralyzed and artificially ventilated rats with a pneumothorax. The carotid body chemoreceptors were stimulated with tracheal administration of nitrogen for 7-10 s. The chemoreceptor stimulation induced an increase in the frequency and amplitude of phrenic nerve bursts. A decrease in the duration of inspiratory (T1), expiratory (TE) and total cycles (TTOT) was observed in the phrenic nerve activity. Inhibition of neuronal cell bodies by microinjections of muscimol (140 pmol/20 nl) into a discrete area in the commissural subnucleus of the nucleus tractus solitarius (coordinates in mm: 0.3 rostral to 0.5 caudal, 0 to 0.5 lateral and 0.3 to 0.5 deep with respect to the calamus scriptorius), attenuated the phrenic nerve responses to the carotid body stimulation. On the other hand, control injections of saline (0.9%) into this site did not alter the phrenic nerve response to the carotid body stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Microinjections of alpha 1-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) of L-glutamate into the IML; maximum pressor responses (29.3 +/- 4 mmHg) were elicited from T9. Pressor responses were also elicited by injections of epinephrine into the IML at T9; maximum pressor effect (16.3 +/- 1.2 mmHg) 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 alpha 1-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 +/- 12 mmHg). 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of cardio-pulmonary vagal afferent C fiber endings by right atrial injections of phenylbiguanide (PBG, 40 μg/kg) elicited apnea, bradycardia and hypotension. These responses were abolished by bilateral vagotomy low in the neck. Stimulation of neurons in a specific nucleus tractus solitarius site (0.5 mm rostral, 0.5 mm lateral and 0.5 mm deep with reference to the calamus scriptorius) by injections of L -glutamate produced responses similar to those following the stimulation of cardio-pulmonary vagal afferent C fiber endings by PBG. Inhibition of neurons in this NTS site by injections of muscimol abolished the responses to PBG. Specific blockade of NMDA receptors by microinjections of AP-7 (100 pmol) or non-NMDA receptors (KA and AMPA) by injections of DNQX (10–25 pmol) into this NTS site did not block the responses to PBG. Microinjections of kynurenic acid (1–4 nmol) into this NTS site blocked the responses to right atrial injections of PBG. These results indicate that: (1) blockade of either NMDA receptors or non-NMDA receptors (KA and AMPA) alone in the above-mentioned NTS site does not abolish the responses to the stimulation of cardio-pulmonary vagal afferent C fiber endings; (2) it is necessary to block NMDA as well as non-NMDA receptors in this NTS site for abolishing the responses to the stimulation of these nerve endings.

Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson's disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro. The same hSNCA silencing sequence (shRNA) was used in both vectors, but in one vector, the shRNA was embedded in a microRNA backbone and driven by a pol II promoter, and in the other the shRNA was not embedded in a microRNA and was driven by a pol III promoter. Both vectors silenced hSNCA to the same extent in 293T cells transfected with hSNCA. In DA PC12 cells, neither vector decreased expression of rat SNCA, tyrosine hydroxylase (TH), dopamine transporter (DAT) or the vesicular monoamine transporter (VMAT). However, the mir30 embedded vector was significantly less toxic to both PC12 and SH-SY5Y cells. Our in vitro data suggest that this miRNA-embedded silencing vector may be ideal for chronic in vivo SNCA gene silencing in DA neurons.

In anesthetized (chloralose and urethane), paralyzed and artificially ventilated rats, the neurons in the ventrolateral medullary depressor area (VLDA) were chemically stimulated by microinjections of L-glutamate (2.5-5 nmole in 100 nl of 0.9% sodium chloride solution) and the cerebral blood flow (CBF) was determined using a combination of labeled microspheres (57Co, 113Sn and 46Sc). Unilateral chemical stimulation of the VLDA (n = 11) produced a significant (P less than 0.05) decrease in CBF of the cerebral cortex ipsilateral to the stimulated VLDA; the CBF was 41 +/- 5 (mean +/- S.E.M.) and 29 +/- 4 ml.min-1.(100 g)-1 before and during the chemical stimulation of VLDA. The decrease in CBF was not due to the decrease in arterial blood pressure (ABP) caused by the chemical stimulation of the VLDA because the CBF during the chemical stimulation of the VLDA was significantly smaller (P less than 0.01) than the CBF during controlled hemorrhagic hypotension (n = 10). In another group of rats (n = 6), moderate hypertension was induced by blood transfusion. Unilateral chemical stimulation of the VLDA in these rats decreased ABP but it remained within normotensive range. A significant (P less than 0.05) decrease in CBF (from 46 +/- 12 to 29 +/- 7 ml.min-1.(100 g)-1) and a significant (P less than 0.01) increase in cerebrovascular resistance (from 2.7 +/- 0.4 to 4.3 +/- 0.6 mmHg per [ml.min-1.(100 g)-1]) was observed in the ipsilateral cerebral cortex of these rats. Chemical stimulation of the VLDA did not affect the reactivity of the cerebral vessels to hypercapnea (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac responses to microinjections of norepinephrine (NE) into the intermediolateral column of the spinal cord (IML) at T2 level were studied in pentobarbital-anesthetized, immobilized and artificially ventilated, male Wistar rats. For describing the effects of NE conveniently, the doses of NE were divided into two ranges. The small dose-range consisted of 20 nl volumes of 50, 75 and 100 micromolar (μM) solutions (i.e. 1, 1.5 and 2 pmole in 20 nl, respectively). The larger dose-range consisted of 20 nl volumes of 2.5, 25, 40 and 50 millimolar (mM) solutions (i.e. 0.05, 0.5, 0.8 and 1 nmole in 20 nl, respectively). Injections of small doses of NE (1–2 pmole) into the IMLincreased heart rate (HR); intravenous injections of these doses did not alter either blood pressure (BP) or HR. Larger doses of NE (0.05–1 nmole) elicited adecrease in HR; intravenous injections of these doses increased HR and BP. Maximum increase in HR was produced by injections of 1.5 pmole of NE into the IML; this effect was blocked by prior injections of prazosin (an α−1adrenergic receptor antagonist; 50 pmole) but not idazoxan (an α−2 adrenergic receptor blocker; 10 pmole) into the IML. Maximum decrease in HR was elicited by injections of 0.8 nmole of NE into the IML; this effect was blocked by idazoxan (10 pmole) but not prazosin (50 pmole). Microinjections of idazoxan (10 pmole) alone increased HR while prazosin (50 pmole) alone was ineffective. Intravenous injections of chlorisondamine (a ganglion blocker) blocked the increase in HR elicited by injections of 1.5 pmole of NE into the IML. Injections of NE, prazosin or idazoxan into the IML did not alter the responses to subsequent injections of glutamate at the same site. The effects of NE remained unchanged in midcollicular decerebrate rats. Vehicles in which NE, prazosin or idazoxan were dissolved, did not elicit a response when injected into the IML. Injections of NE outside the IML failed to elicit any response. These results suggest that NE may have a role to play as a neuromodulator/neurotransmitter in the IML. The pathways involved in these actions of NE in the IML remain to be identified.

In anesthetized (chloralose and urethane), paralyzed and artificially ventilated rats, the neurons in the nucleus tractus solitarius (NTS) were chemically stimulated by microinjections of L-glutamate and the cerebral blood flow (CBF) was determined using a combination of labeled microspheres (either 57Co, 113Sn and 46Sc or 141Ce, 85Sr and 46Sc). Unilateral chemical stimulation of the NTS (n = 14) decreased CBF significantly in most brain areas. The decrease in CBF was not due to the decrease in arterial blood pressure (ABP) because the CBF of the whole cerebral cortex during the chemical stimulation of the NTS was significantly smaller (P less than 0.05) than the CBF during controlled hemorrhagic hypotension (n = 10). In another group of rats (n = 6), moderate hypertension was induced by blood transfusion. Unilateral chemical stimulation of the NTS in these rats decreased ABP but it remained within normotensive range. A significant (P less than 0.05) decrease in CBF (from 62 +/- 28 (mean +/- S.D.) to 48 +/- 23 ml.min-1.(100 g)-1) and increase in cerebrovascular resistance (from 1.9 +/- 1.2 to 2.6 +/- 1.2 mm Hg per [ml.min-1.(100 g)-1]) was observed in the whole cerebral cortex of these rats. Chemical stimulation of the NTS did not affect the reactivity of the cerebral vessels to hypercapnea (n = 5). These results suggest that the cell bodies within the NTS may play a role in the control of cerebral circulation.

Immunoreactivity for nociceptin, an endogenous ligand for the ORL1 opioid receptors, has been reported in the nucleus tractus solitarius (nTS). A midline area in the commissural subnucleus (nCom) of nTS is the site of peripheral chemoreceptor projections. This investigation was carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats, to study the cardiovascular effects of the activation of ORL1 receptors in a midline area of the nCom. Microinjections (30 nl) of nociceptin (0.15-0.62 mM) into the nCom elicited depressor and bradycardic responses. Prior microinjections of [N-Phe(1)]-nociceptin-(1-13)-NH(2) (4.5 mM), a specific antagonist for ORL1 opioid receptors, into the nCom blocked the effects of nociceptin (0.31 mM, the maximally effective concentration), but not endomorphin-2 (0.6 mM; an endogenous ligand for micro -opioid receptors). On of other hand, naloxone (0.125 mM; an antagonist for classical opioid receptors) did not block the effects of nociceptin, while it did block the effects of endomorphin-2. The blockade of nociceptin effects by [N-Phe(1)]-nociceptin-(1-13)-NH(2) and endomorphin-2 by naloxone, was not due to some nonspecific effects because the responses to L-Glu (5 mM) remained unaltered after the microinjection of these antagonists. These results indicate that activation of ORL1 receptors in the nCom may play a role in the regulation of cardiovascular function.

Relatively few studies have been done to characterize cardiovascular responses to the chemical stimulation of sites located in the medullary lateral tegmental field (LTF) and most of them have been carried out in anesthetized animals. Our experiments were carried out in decerebrated, artificially ventilated, adult male Wistar rats. In the LTF, two types of cardiovascular responses were elicited. One type consisted of pressor responses accompanied by bradycardia. Such responses were elicited from a region 0.4 mm caudal to 0.8 mm rostral to the calamus scriptorius (CS); maximum responses were elicited from a site 0.6 mm rostral to the CS, 1.2 mm lateral to the midline and 1.2 mm deep from the dorsal medullary surface. Another type consisted of pressor responses without any change in heart rate; such responses were elicited from a region 1-1.6 mm rostral to the CS. Nucleus ambiguus (nAmb) and dorsal motor nucleus of the vagus (nDMX) and the reticular formation surrounding these areas were the main sites from which bradycardia (accompanied by either no or small changes in BP) was elicited. In the nAmb, maximum bradycardia was elicited from a site 0.6 mm rostral to the CS, 1.8 mm lateral to the midline and 2.4 mm deep from the dorsal medullary surface. In the nDMX, most prominent bradycardic responses were elicited at 0-0.6 mm rostral to the CS, and 0.6 mm lateral to the midline and 1 mm deep from the dorsal medullary surface. Cardiovascular effects elicited from sites in other well-known areas, such as the rostral ventrolateral medullary pressor area (RVLM) and caudal ventrolateral medullary depressor area (CVLM), and the nucleus tractus solitarius (nTS) were also included for comparison of different responses. These results are expected to prove useful in studies in which the microinjection technique is used to characterize cardiovascular responses.

Microinjections of L-glutamate were used to identify the pre-Botzinger complex in urethane-anesthetized, immobilized, bilaterally vagotomized, artificially ventilated, adult male Wistar rats. Unilateral microinjections (20-30 nl) of L-glutamate into the pre-Botzinger complex on either side elicited a bilateral continuous phrenic nerve discharge superimposed on which was an increase in burst-frequency. Neurokinin-1 receptor immunoreactivity in the semi-compact region of the nucleus ambiguus and the area immediately ventral to it indicated that the site of microinjections was in the general region of pre-Botzinger complex. Unilateral microinjections of glycine into the pre-Botzinger complex caused an inhibition of phrenic nerve activity bilaterally in a concentration-dependent manner. At lower concentrations (1 and 3 mM) phrenic nerve burst-frequency as well as burst-amplitude were decreased. At higher concentrations (6 mM), complete bilateral cessation of phrenic nerve activity was observed. The effects of glycine were prevented by a prior microinjection of strychnine (0.5 mM) into the pre-Botzinger complex. The specificity of strychnine as an antagonist for glycine receptors was established by its lack effect on GABA(A) receptors; muscimol was used as a GABA(A) receptor agonist. Unilateral microinjections of muscimol (0.01 and 0.1 mM) into previously identified pre-Botzinger complex also caused a bilateral decrease in phrenic nerve burst-frequency and burst-amplitude. At higher concentrations (0.3 and 1 mM) muscimol microinjections into the pre-Botzinger elicited a complete bilateral cessation of phrenic nerve activity. The effects of muscimol were not altered by prior microinjections of strychnine (0.5 mM) at the same site. These results demonstrate pharmacologically the presence of glycine receptors in the pre-Botzinger complex. The role of these receptors in the regulation of respiration remains to be elucidated.

This study focuses on the role of nicotinic receptors located in the caudal ventrolateral medullary depressor area (CVLM) in regulating/modulating cardiovascular function. Blood pressure and heart rate were monitored by standard techniques in urethane-anesthetized, artificially ventilated, adult male Wistar rats. Multi-barreled glass-micropipettes (tip size 20-40 microm) were used to make microinjections (100 nl) into the CVLM. Concentrations of nicotine ranging from 250 micromto 10 mM were microinjected unilaterally into the CVLM. The maximum depressor and bradycardic responses were elicited by a 1 mM concentration of nicotine. Sequential microinjections of mecamylamine (1 mM), an antagonist for nicotinic receptors containing alpha3beta4 subunits, then alpha-bungarotoxin (1 microm), an antagonist for nicotinic receptors containing alpha-7 subunits, were made into the CVLM. Microinjecting a combination of a nicotinic receptor blocker and toxin resulted in the complete blockade of the cardiovascular responses induced by nicotine (1 mM, 100 nl). These results indicate that: (1) nicotinic receptors are present in the CVLM; (2) activation of these receptors results in depressor and bradycardic responses; (3) for a complete blockade of nicotine-induced cardiovascular responses, it is necessary to use a combination of mecamylamine and alpha-bungarotoxin; (4) since mecamylamine and alpha-bungarotoxin are known to block nicotinic receptors containing alpha3beta4 and alpha-7 subunits, respectively, two different subtypes of nicotinic receptors (one which contains a combination of alpha3beta4 subunits, and one which contains alpha-7 subunits) must be present in the CVLM; and (5) it is not clear whether these two subtypes of nicotinic receptor are located on the same or different populations of CVLM-neurons.

Microinjections (50 nl) of carbachol (2.5–500 μM) into the phrenic motor nucleus (PMN) of anesthetized rats caused a dose-dependent decrease in the phrenic nerve (PN) burst-amplitude. Prior microinjections of pirenzepine and methoctramine (1 mM, each) into the PMN, in separate experiments, significantly attenuated the carbachol-induced inhibition of PN activity. These results suggest that inhibition of PN activity induced by microinjections of carbachol into the PMN is mediated via M 1 and M 2 receptors. Since pirenzepine and methoctramine microinjections into the PMN did not alter the control PN activity, it was concluded that in anesthetized rats cholinergic inputs to the PMN, if any, are not tonically active. It is possible that muscarinic receptors in the PMN come into play only under specific conditions such as activation of a reflex mechanism which alters PN activity. These hypotheses remain to be tested.

Although, area postrema (AP) as been implicated in the regulation of cardiovascular function, there is no consensus regarding the type of responses elicited by stimulation of this brain structure. Microinjections (50 nl) of smaller concentrations of excitatory amino acid receptor agonists (e.g., NMDA, KA and trans-ACPD, 10 microM each) into the AP elicited pressor and tachycardic responses in unanesthetized decerebrate as well as urethane-anesthetized rats. Microinjections of higher concentrations (e.g., 50 microM NMDA) of excitatory amino acids (EAAs) into the AP elicited an initial pressor and tachycardic response which was followed by a depressor and bradycardic response; when high concentrations of NMDA were microinjected into the AP, enough concentration may have reached the nucleus tractus solitarius (nTS) to elicit depressor and bradycardic responses. Alternatively, high concentrations of NMDA may excite known projections from AP to the nTS.

Phrenic nerve (PN) responses to unilateral microinjections of L-glutamate (L-Glu, 5 mM) or N-methyl-D-aspartic acid (NMDA, 1 mM) into different subregions of ventral respiratory neuronal group (VRG) were studied in urethane-anesthetized, immobilized, and artificially ventilated, adult male Wistar rats. A 50-nl volume of microinjection was used in all the subregions of VRG except in Pre-Botzinger complex (Pre-BotC) where a 20-nl volume was used. Unilateral microinjections of L-Glu or NMDA into the Botzinger complex (BotC) and caudal VRG (cVRG), caused a transient cessation of phrenic nerve (PN) activity. Expiratory neurons, abundant in BotC and cVRG, were excited by stimulation of cardiopulmonary receptors while their responses to carotid chemoreceptor stimulation were variable. Microinjections of L-Glu or NMDA into the Pre-BotC caused an increase in the PN background discharge (this response was unique to Pre-BotC) superimposed on which was an increase in the PN burst frequency. Microinjections of L-Glu or NMDA into the rostral VRG (rVRG) caused an increase in the frequency and amplitude of PN bursts. Inspiratory neurons, abundant in Pre-BotC and rVRG, were excited and inhibited by activation of carotid chemoreceptors and cardiopulmonary receptors, respectively. The coordinates for the location of different subregions of VRG were as follows (reference points are listed in parentheses). BotC: 1.6-2.6 mm rostral (calamus scriptorius), 1.7-2.7 mm lateral (midline), and 2.3-2.8 mm deep (dorsal surface of medulla); Pre-BotC: 1.4-1.6 mm rostral, 1. 8-2.5 mm lateral, and 2.3-2.8 mm deep; rVRG: 0.4-1.4 mm rostral, 1. 6-2.5 mm lateral, and 2.3-2.8 mm deep; and cVRG: 0.5 mm caudal to 0. 5 mm rostral, 1.0-2.2 mm lateral, and 2.1-2.6 mm deep. A detailed map of the subregions of VRG, functionally identified by L-Glu and NMDA-microinjections, has been presented. These data are likely to prove useful in future studies on respiratory reflex mechanisms.

In anesthetized rats, microinjections of excitatory amino acids (EAAs) into the nucleus tractus solitarii (nTS), in a region located immediately rostral to the calamus scriptorius (CS), have been generally reported to elicit depressor and bradycardic responses. On the other hand, in conscious freely moving rats, similar microinjections have been reported to elicit pressor and bradycardic responses. These divergent results have been attributed to the effect of anesthetics. A reinvestigation of the effects of EAAs into the nTS in unanesthetized animals became necessary in order to resolve this controversy. The microinjection technique used in freely moving conscious rats suffers from several technical limitations; for example, microinjections cannot be delivered stereotaxically. In order to avoid these limitations, the present experiments were carried out in unanesthetized supracollicular decerebrate rats. A systematic mapping of nTS in these rats, using microinjections of the solutions of EAAs in artificial cerebrospinal (aCSF) fluid, confirmed that depressor and bradycardic responses are elicited from all the sites in the nTS extending from the CS to a level about 1 mm rostral to it. Pressor responses were elicited by microinjections of l-glutamate (l-Glu) only from the chemoreceptor projection site (a region of the commissural subnucleus, 0.1-0.5 mm caudal to the CS, 0-0.5 mm lateral to the midline and 0.4-0.5 mm deep from the medullary surface). The pressor responses elicited from the aforementioned site were accompanied with bradycardia; this response may be due to diffusion of l-Glu to the dorsal motor nucleus of vagus because the bradycardia disappeared when the depth of the microinjection was reduced to 0.3, instead of 0.5 mm, from the dorsal medullary surface. When urethane was administered intravenously in unanesthetized decerebrate rats, the responses to microinjections of l-Glu remained unchanged, i.e., depressor and bradycardic responses were elicited from all the sites in the nTS extending from the CS to a level about 1 mm rostral to it and pressor and tachycardic responses were elicited from the chemoreceptor projection site. These observations indicated that there is no anesthetic-induced qualitative alteration of the cardiovascular responses to microinjections of EAAs into the nTS.

The neurotransmission of bulbospinal respiratory drive is believed to involve primarily non-NMDA receptors located in the phrenic motonucleus (PMN). This conclusion is based on studies carried out mainly on in vitro brainstem-spinal cord preparations of the neonatal rat. The present study was undertaken to investigate the transmitter/receptor mechanisms in the PMN which are involved in the neurotransmission of inspiratory drive, using an in vivo adult rat model. Microinjections of glutamate, NMDA and AMPA into the PMN elicited an increase in the phrenic nerve (PN) background discharge. These injections did not alter significantly the frequency of spontaneously occurring PN bursts confirming that mechanisms responsible for respiratory rhythm reside in the supraspinal structures. Microinjections of an NMDA receptor blocker (AP-7), in concentrations that did not alter the responses to a non-NMDA receptor agonist (AMPA), reduced the PN amplitude significantly. Similarly, microinjections of a potent non-NMDA receptor blocker (NBQX), in concentrations that did not alter responses to NMDA, reduced the PN amplitude significantly. Sequential microinjections, within an interval of 5 min, of AP-7 and NBQX into the PMN, resulted in a dramatic reduction in the spontaneous PN bursts. The reduction of PN amplitude started immediately after the microinjection of AP-7 and NBQX, either alone or in combination, and reached a maximum within 5-10 min. These results indicate that, unlike in the neonatal rat, both NMDA and non-NMDA receptors located in the PMN play a significant role in the neurotransmission of the inspiratory drive in the adult rat.

In urethane-anesthetized, paralyzed and artificially ventilated rats, the neurons in the rostral ventrolateral medullary pressor area (VLPA) were chemically stimulated by microinjections of L-glutamate (1.7-5.0 nmole in 100 nl of 0.9% sodium chloride solution) and the cerebral blood flow (CBF) was determined using a combination of labeled microspheres (57Co, 113Sn and 46Sc). In one group of rats (n = 11), unilateral chemical stimulation of the VLPA produced a significant (P less than 0.01) increase in arterial blood pressure (ABP), a significant (P less than 0.05) decrease in CBF, and a significant (P less than 0.01) increase in cerebrovascular resistance (CVR) in the cerebral cortex ipsilateral to the stimulated VLPA. The CBF was 52 +/- 3 (mean +/- S.E.M.) and 48 +/- 4 ml.min-1.(100 g)-1 before and during the chemical stimulation of VLPA; the CVR was 1.9 +/- 0.1 and 2.6 +/- 0.3 mmHg per ml.min-1.(100 g)-1 before and during the stimulation. In order to measure CBF at normotension, moderate hypotension was induced by controlled hemorrhage in another group of rats (n = 8). Unilateral chemical stimulation of the VLPA in these rats increased ABP but it remained within normotensive range. The CBFs of ipsilateral and contralateral cerebral cortices decreased significantly (P less than 0.05) from 57 +/- 14 to 41 +/- 9 and from 50 +/- 12 to 39 +/- 9 ml.min-1.(100 g)-1, respectively. The CVRs of ipsilateral and contralateral cortices increased significantly (P less than 0.05) from 2.6 +/- 0.6 to 3.5 +/- 0.8 and from 2.7 +/- 0.5 to 3.5 +/- 0.8 mmHg/[ml.min-1.(100 g)-1], respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravenous injections of clonidine produce an initial transient increase in blood pressure followed by a long-lasting hypotension and bradycardia. The initial pressor response is due to activation of vascular α 1 -adrenergic receptors while the hypotension and bradycardia are caused by the central actions of clonidine. Although, hypothalamus, nucleus tractus solitarius (NTS), ventrolateral medulla and the intermediolateral cell column of the thoracolumbar spinal cord (IML) have been implicated, the exact site of these actions of clonidine in the central nervous system is not established. The results of this investigation suggest that the pressor area in the ventrolateral medulla (VLPA) is the site of hypotensive and bradycardic actions of intravenously administered clonidine. This conclusion is based on the observation that microinjections of idazoxan, a specific α 2 -adrenergic receptor blocker, into the VLPA prevented and reversed the hypotension and bradycardia despite the fact that other proposed sites of these actions (NTS, hypothalamus and IML) were intact and accessible to intravenously administered clonidine.

Sympathoexcitatory cardiovascular responses to the microinjections of l -glutamate into the intermediolateral cell column (IML) of the upper thoracic cord (C 8 to T 4 ) were studied. Mean arterial pressure (MAP), heart rate (HR), the rate of increase in the left ventricular pressure (d P /d t ) and contractility index were monitored in immobilized and artificially ventilated male Wistar rats anesthetized with pentobarbital or isofluorane. On the right side, microinjections (10–20 nl) of l -glutamate (0.9–1.77 nmol in 0.9% sodium chloride solution, pH 7.4) into the IML at T 2 level produced marked tachycardic responses with relatively small changes in contractility. On the left side, similar microinjections produced marked increase in d P /d t and contractility index with relatively small increase in HR. On either side, the responses were smaller at T 1 and T 3 level and absent at C 8 and T 4 level. No changes in blood pressure were observed with microinjections of l -glutamate on either side. Microinjections of N -methyl- d -aspartic acid (NMDA), 1–100 pmol, into the IML elicited responses similar to those of l -glutamate. These amino acids failed to evoked any response when microinjected into the adjacent areas (e.g. 0.5 mm lateral or medial to the IML). The effects of glutamate and NMDA in the IML were blocked by microinjections of glutamic acid diethylester (GDEE) and d -2-amino-7-phosphonoheptanoic acid (D-AP7), respectively. Control microinjections of physiological saline into the IML produced no responses. These results indicate that excitatory amino acids, in small doses and volumes, can be used to identify cardiac sympathoexcitatory neuronal pools in the IML. This preparation may prove useful in characterizing pharmacological actions of various putative neurotransmitters in this region of the spinal cord.

Cardiovascular responses to the microinjections of cholinergic agonists into the intermediolateral cell column (IML) of the spinal cord at T1-T3 level were studied. Mean arterial pressure (MAP), heart rate (HR), the rate of increase in the left ventricular pressure (dp/dt) and contractility index (CI) were monitored in immobilized and artificially ventilated male Wistar rats either anesthetized with pentobarbital or decerebrated at mid-collicular level. Microinjections (20 nl) of carbachol (110-660 pmol) into the right IML elicited a marked increase in HR and a small increase in CI. A marked increase in the CI with relatively little effect on the HR was observed when carbachol was injected into the left IML. The cardioacceleratory effects of carbachol, but not those of L-glutamate, were blocked by prior microinjections of scopolamine (18 nmol) into the IML. Intravenous injections of chlorisondamine (a ganglion blocker) also blocked these effects of carbachol. Spinal transections at C4 or T6 level did not alter these responses. Microinjections of acetylcholine (0.01-1 nmol) into the right IML also produced tachycardic effects. The responses to acetylcholine were blocked by prior injections of a muscarinic receptor blocker (atropine hemisulfate, 0.2 nmol). Microinjections of a selective M2 muscarinic receptor agonist, cis-methyldioxolane (CD; 0.2-0.8 nmol), but not those of a relatively selective M1 receptor agonist (McN-A343; 2-3 nmol), into the right IML elicited an increase in HR. Previous microinjections of a selective competitive M2 receptor antagonist (AFDX-116; 0.8 nmol), but not those of a potent selective M1 receptor antagonist (pirenzepine; 2 nmol), into the IML blocked the effects of CD. Nicotine (0.25-1 nmol) when injected into the right IML also produced positive chronotropic effects. These responses were blocked by prior microinjections of hexamethonium (5 nmol). The above-mentioned results suggest that cholinoceptive neurons, interneurons or terminals are located in the areas of IML which control cardiac functions. Muscarinic as well as nicotinic receptors are present in this area. Muscarinic receptors are predominantly of the M2 type. The physiological significance of the presence of cholinergic receptors in this area in controlling cardiac functions remains to be established.

Opiate receptor stimulation by microinjections of a δ-receptor agonist, d -Ala 2 - d -Leu 5 -enkephalin (DADLE) into the caudal depressor (A 1 ) area of the ventrolateral medulla produced a hypertensive response which was prevented as well as reversed by the blockade of cholinergic receptors in the rostral pressor (C 1 ) area. These results suggest that the hypertensive responses to opiates in the A 1 area are mediated via cholinergic mechanisms in the rostral C 1 area of the ventrolateral medulla and acetylcholine may be the neurotransmitter released in the ventrolateral pressor area.