Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key compone... more Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key component in the control of arterial blood pressure. Classically, C1 cells have been identified by their immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and/or phenylethanolamine N-methyltransferase (PNMT). However, no studies have simultaneously demonstrated the expression of aromatic L-amino acid decarboxylase (AADC) and dopamine -hydroxylase (DBH) in these neurons. We examined the expression and colocalization of all four enzymes in the rat ventrolateral medulla using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Retrograde tracer injected into thoracic spinal segments T2-T4 was used to identify bulbospinal neurons. Using fluorescence and confocal microscopy, most cells of the C1 group were shown to be double or triple labeled with TH, DBH, and PNMT, whereas only 65-78% were immunoreactive for AADC. Cells that lacked detectable immunoreactivity for AADC were located in the rostral C1 region, and approximately 50% were spinally projecting. Some cells in this area lacked DBH immunoreactivity (6.5-8.3%) but were positive for TH and/or PNMT. Small numbers of cells were immunoreactive for only one of the four enzymes. Numerous fibres that were immunoreactive for DBH but not for TH or PNMT were noted in the rostral C1 region. Single-cell RT-PCR analysis conducted on spinally projecting C1 neurons indicated that only 76.5% of cells that contained mRNA for TH, DBH, and PNMT contained detectable message for AADC. These experiments suggest that a proportion of C1 cells may not express all of the enzymes necessary for adrenaline synthesis.
The explosion in the use of genetically modified mouse strains to investigate function in biology... more The explosion in the use of genetically modified mouse strains to investigate function in biology has an enormous potential to expand on pharmacological studies traditionally conducted in rats. A key limitation to date is the inability to record from multiple nerves in an anaesthetised mouse for long periods. Here we describe an in vivo preparation that maintains mice in a suitable physiological state, under anaesthesia, for at least 6hr and also enables multiple cardiorespiratory recordings over that time. Using the method described, blood pressure, heart rate, phrenic nerve activity, splanchnic nerve activity and heart rate were able to be recorded for hours in an anaesthetised, paralysed and mechanically ventilated mouse. Existing anaesthetised mouse preparations are limited by difficulties in maintaining mice under anaesthesia for long periods. This time constraint therefore limits the surgical time and number of cardiorespiratory variables recorded. It also limits the type of s...
Opioid receptors are activated during severe hemorrhage, resulting in sympathoinhibition and a pr... more Opioid receptors are activated during severe hemorrhage, resulting in sympathoinhibition and a profound fall in blood pressure. This study examined the location and subtypes of opioid receptors that might contribute to hypotension after hemorrhage. Intrathecal naloxone methiodide (100 nmol) abolished the fall in blood pressure after hemorrhage (1.5% of body wt; mean arterial pressure 122 +/- 8 mmHg after naloxone methiodide vs. 46 +/- 5 mmHg in controls, P < 0. 001). Intracisternal naloxone methiodide was less effective than intrathecal naloxone methiodide, whereas intravenous naloxone methiodide, which does not cross the blood-brain barrier, did not alter the fall in blood pressure after hemorrhage. These results demonstrate that spinal opioid receptors contribute to hypotension after hemorrhage but do not exclude supraspinal effects. In separate experiments, the subtype-specific opioid antagonists ICI-174864 (delta-antagonist), norbinaltorphimine (nor-BNI; kappa-antagonist), an...
A new technique for Iocalising the centre of sites of microinjection of drugs in the brain using ... more A new technique for Iocalising the centre of sites of microinjection of drugs in the brain using the silxer inlensification of colloidal gold is described. Fhe technique is compatible with immunocytochemistL~ and in this study was used in Wistar Kyoto rats to investigate the relationship between the location of phenylelhanolamine-N-methyltransferase-like in3munoreaclivc (PNMT-LI) cell bodies in the medulla and the precise sites where microinjection of L-glutamate elicited pressor responses. ('olloidal gold provided a simple technique for localising the centre of sites of microinjection with a high degree of spatial resohition and allowed pressor sites to be precisely Ioculised in the region of ventrolalera/ medulkl with the highest density of PNMT-LI cells.
Sleep apnea is associated with repeated episodes of hypoxemia, causing marked increase in sympath... more Sleep apnea is associated with repeated episodes of hypoxemia, causing marked increase in sympathetic nerve activity and blood pressure. Considerable evidence suggests that intermittent hypoxia (IH) resulting from apnea is the primary stimulus for sympathetic overactivity in sleep apnea patients. Several IH protocols have been developed either in animals or in humans to investigate mechanisms underlying the altered autonomic regulation of the circulation. Most of these protocols involve several days (10-40 days) of IH exposure, that is, chronic intermittent hypoxia (CIH). Recent data suggest that a single session of IH exposure, that is, acute intermittent hypoxia (AIH), is already capable of increasing tonic sympathetic nerve output (sympathetic long-term facilitation, LTF) and altering chemo- and baroreflexes with or without elevation of blood pressure. This indicates that IH alters the autonomic neurocirculatory at a very early time point, although the mechanisms underlying this ...
Swallowing and breathing utilize common muscles and an anatomical passage: the pharynx. The risk ... more Swallowing and breathing utilize common muscles and an anatomical passage: the pharynx. The risk of aspiration of ingested material is minimized not only by the laryngeal adduction of the vocal folds and laryngeal elevation but also by the precise coordination of swallows with breathing. Namely, swallows: (1) are preferentially initiated in the postinspiratory/expiratory phase, (2) are accompanied by a brief apnea, and (3) are often followed by an expiration and delay of the next breath. This review summarizes the expiratory evidence on the brainstem regions comprising the central pattern generator (CPG) that produces the pharyngeal stage of swallow, how the motor acts of swallowing and breathing are coordinated, and lastly, brainstem regions where the swallowing and respiratory CPGs may interact in order to ensure &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;safe&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; swallows.
Chemical stimulation of neurons in the pontine A5 area by microinjection of L-glutamate lowers ar... more Chemical stimulation of neurons in the pontine A5 area by microinjection of L-glutamate lowers arterial blood pressure. The mechanism of this &#39;A5 depressor response&#39; is not well-established. Here, we examine the involvement of spinal cord gamma-aminobutyric acid (GABA) receptors in this depressor response in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Experiments were conducted in male WKY and age-matched SHR anaesthetised with sodium pentobarbitone and chloral hydrate. An intrathecal catheter was implanted with the tip located between T9 and L2. Three days later, rats were re-anaesthetised and 10 nl of 40 mM L-glutamate was injected into the A5 area before, during and after, blockade of spinal cord GABA-A receptors by intrathecal injection of bicuculline methiodide (1 mM in 10 microliters phosphate-buffered saline). Injection of L-glutamate (10, 20, 40, 80 mM in 10 nl) produced depressor responses that were similar in WKY (n = 6) and SHR (n = 6). Intrathecal injection of bicuculline elicited a pressor response that was greater in SHR (n = 7, 28.5 +/- 7.6% increase in mean arterial pressure) than WKY (n = 11, 11.6 +/- 3.6%, p &lt; 0.05). After bicuculline, the depressor response to injection of L-glutamate into the A5 area was eliminated in both WKY (n = 7) and SHR (n = 6). Intrathecal injection of vehicle had no effect on either resting arterial blood pressure or the depressor response to A5 stimulation. Basal blood pressure and control responses to A5 stimulation were fully restored by around 90 min after bicuculline injection in each animal. In separate groups of rats, intrathecal injection of muscimol elicited depressor responses that were greater in SHR (n = 6, -32.0 +/- 6.2%) than WKY (n = 6, -17.3 +/- 1.5%, p &lt; 0.05). Our results suggest that the A5 depressor response is due to a projection from the A5 area to the spinal cord. This projection acts directly, or through a spinal interneuron and uses GABA as a neurotransmitter. Furthermore, our results indicate a hyper-responsiveness to GABA-A receptor stimulation in SHR since intrathecal bicuculline elicited much greater increases and intrathecal muscimol elicited much greater decreases, in blood pressure in SHR than in WKY. Finally, it seems likely that the A5-spinal depressor pathway is less effective in SHR than WKY under physiological conditions since chemical stimulation of the A5 area with L-glutamate produced a comparable depressor response in both strains.
Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neuron... more Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neurons projecting to the rat adrenal medulla are immunoreactive for either the inhibitory amino acid, gamma-aminobutyric acid (GABA) or the excitatory amino acid, L-glutamate. To investigate whether or not this is true for other groups of sympathetic preganglionic neurons, and to determine whether or not the proportion of inputs containing each type of amino acid neurotransmitter is the same for different groups of sympathetic preganglionic neurons, we retrogradely labelled rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion and used post-embedding immunogold on ultrathin sections to localise GABA- and glutamate-immunoreactivity. The cell bodies and dendrites of both rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion received synapses and direct contacts from nerve fibres immunoreactive for GABA and from nerve fibres immunoreactive for glutamate. In the rat, GABA was present in 48.9% of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion, and glutamate was present in 51.7% of inputs. Double immunogold labelling for glutamate and GABA on the same section, as well as labelling of consecutive serial sections for the two antigens, indicated that GABA and glutamate occur in separate populations of nerve fibres that provide input to rat sympathetic preganglionic neurons projecting to the superior cervical ganglion. We now have shown that GABA or glutamate is present in virtually all of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion and in essentially all of the inputs to sympathetic preganglionic neurons supplying the adrenal medulla. These findings are consistent with the hypothesis that all fast synaptic transmission in central autonomic pathways may be mediated by either excitatory or inhibitory amino acids. Furthermore, we showed a statistically significant difference in the proportion of glutamate-immunoreactive inputs between sympathetic preganglionic neurons projecting to the superior cervical ganglion and sympathoadrenal neurons (data from Llewellyn-Smith et al. [Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1992. Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res. 581, 67-80]), with preganglionics supplying the adrenal medulla receiving more excitatory inputs than those supplying the superior cervical ganglion. This increased excitatory input to sympathoadrenal neurons may explain the predominant activation of these neurons following baroreceptor unloading.
In anesthetized cats, we recorded intracellularly from 26 hypoglossal motoneurons which were anti... more In anesthetized cats, we recorded intracellularly from 26 hypoglossal motoneurons which were antidromically activated following electrical stimulation of either the medial or lateral branches of the hypoglossal nerve. Twenty-one of these neurons were protruder motoneurons 6 of which had inspiratory activity. Three of the protruder motoneurons with inspiratory activity were filled with Neurobiotin and found to be closely apposed to substance P-like immunoreactive nerve terminals. q
In this study we aimed to determine whether the levels of gene expression for phenylethanolamine-... more In this study we aimed to determine whether the levels of gene expression for phenylethanolamine-Nmethyltransferase (PNMT), noradrenaline transporter (NAT), ␣ 1A -receptor (␣ 1A -R), and ␣ 2A -receptor (␣ 2A -R) vary with resting systolic blood pressure in spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) or Sprague-Dawley (SD) rats. Sites examined included central and peripheral regions associated with the control of arterial pressure. Twenty week old SD (nϭ6), WKY (nϭ6), and SHR (nϭ6) were used. Systolic blood pressure was measured using tail cuff plethysmography 2 weeks before tissue extraction. RNA was isolated and reverse-transcribed into cDNA. Gene expression levels were measured, using quantitative real time PCR, relative to the expression of GAPDH. PNMT, NAT, and ␣ 1A -R mRNA expression was significantly greater in SHR tissue samples compared with normotensives. In the rostral ventrolateral medulla, PNMT mRNA in SHR was 3 times greater than that in WKY (SHR: 0.82Ϯ0.02%; WKY: 0.29Ϯ0.02%). The amount of ␣ 2A -R mRNA was significantly lower in SHR compared with normotensives. For example, the level of ␣ 2A -R mRNA in spinal cord of SHR was 3 times less than that found in WKY (SHR: 1.85Ϯ0.04%; WKY: 3.26Ϯ0.07%). PNMT, NAT, and ␣ 1A -R mRNA levels were positively correlated with systolic blood pressure in all central tissue investigated. Conversely, ␣ 2A -R mRNA levels in central sites were negatively correlated with systolic blood pressure. Clearly, a decrease in central ␣ 2A -R and an increase in ␣ 1A -R is consistent with the elevated blood pressure and sympathetic activity observed in SHR. (Hypertension. 2002;40:342-347.)
In urethane-anaesthetised artificially ventilated Sprague-Dawley rats, bilateral microinjection o... more In urethane-anaesthetised artificially ventilated Sprague-Dawley rats, bilateral microinjection of the divalent cation nickel chloride (Ni 2؉ ; 50 mM, 50 nl) into the rostral ventrolateral medulla elicited a dramatic inhibition of splanchnic sympathetic nerve activity (؊44؎6%) and a marked depressor response (؊35؎7 mmHg).
The specific role of the ⌬ opioid receptor (DOR), in opioid-induced respiratory depression in the... more The specific role of the ⌬ opioid receptor (DOR), in opioid-induced respiratory depression in the ventral respiratory group (VRG) is largely unknown. Here, we sought to determine (1) the relationship between DOR-immunoreactive (ir) boutons, bulbospinal and functionally identified respiratory neurons in the VRG and (2) the effects of microinjection of the selective DOR agonist, D-Pen 2,5-enkephalin (DPDPE), into different subdivisions of the VRG, on phrenic nerve discharge and mean arterial pressure.
The effects of hyperoxic hypercapnia (5, 10 or 15% CO 2 in O 2 ) on splanchnic sympathetic nerve ... more The effects of hyperoxic hypercapnia (5, 10 or 15% CO 2 in O 2 ) on splanchnic sympathetic nerve activity (sSNA) and sympathetic reflexes such as the somato-sympathetic reflex or baroreflex were studied in urethane anaesthetised, paralysed, artificially ventilated and vagotomized Sprague-Dawley rats. Hypercapnia caused a small increase in mean arterial blood pressure (MAP) in the 10% CO 2 group and a fall in heart rate (HR) in all three groups. sSNA increased in all three groups. Phrenic frequency and amplitude increased during hypercapnia, with frequency adapting back towards baseline during the CO 2 exposure. The somato-sympathetic reflex was attenuated in the 5% CO 2 group and abolished in the 10 and 15% CO 2 groups, whereas there was little effect on the sSNA baroreflex. Hypercapnia significantly affects phrenic nerve activity (PNA), sSNA and selectively inhibits the somato-sympathetic reflex with little effect on the sSNA baroreflex.
Central apnea is common in the premature newborn. To explain the upper airway findings in differe... more Central apnea is common in the premature newborn. To explain the upper airway findings in different clinical conditions characterized by central apnea, we made single unit recordings from laryngeal motoneurons during normal and hyperventilation. Posterior cricoarytenoid (n = 4) and cricothyroid (n = 4) motoneurons displayed an inspiratory pattern during normal ventilation and remained synchronous with phrenic nerve discharge (PND) during hyperventilation. Laryngeal constrictor motoneurons (LCon) displayed a post-inspiratory pattern during normal ventilation, but lost phasic activity during early hyperventilation (the period after the onset of hyperventilation but before cessation of PND; n = 12). There was a nearly linear relationship between the post-inspiratory activity and strength of PND. Six LCon motoneurons remained silent throughout hyperventilation, while the other six developed a tonic activity during cessation of PND. Further analysis suggested that the silent and tonic LCon motoneurons are likely to share a similar mechanism in their post-inspiratory pattern generation, but differ from each other in their responses to CO 2 inputs. In addition, strong inhibition of the LCon tonic activity by the early return of PND could be an important factor in recovery following a period of apnea. Failure of this inspiratory inhibition to occur might explain certain clinical situations, where obstructive apnea supervenes following a period of central apnea. Crown
Chronic intravenous angiotensin II (Ang II) has been widely used to establish centrally mediated ... more Chronic intravenous angiotensin II (Ang II) has been widely used to establish centrally mediated hypertension in experimental animals, and disruption of Ang II activity is a frontline treatment for hypertensive disease. However, the acute central actions of circulating Ang II are poorly understood. We examined the effects of intravenous pressor doses of Ang II on autonomic activity in anaesthetized rats under neuromuscular blockade, and compared baroinhibition evoked by Ang II pressor ramps to equipressor responses evoked by phenylephrine (PE). Baroinhibition of splanchnic sympathetic nerve activity was attenuated during Ang II trials compared with PE, and rats remained sensitive to electrical stimulation of the aortic depressor nerve at higher arterial pressures during Ang II trials. This was not due to a direct effect of Ang II on aortic nerve baroreceptors. In a separate series of experiments, we provide direct evidence that bulbospinal barosensitive neurones in the rostral ventrolateral medulla are differentially sensitive to pressure ramps evoked by Ang II or PE vasoconstriction. Nineteen out of 41 units were equally sensitive to increased arterial pressure evoked by Ang II or PE. In 17 of 41 units, barosensitivity was attenuated during Ang II trials, and in five of 41 cases units that had previously been barosensitive increased their firing rate during Ang II trials. These results show, for the first time, that circulating Ang II acutely modulates central cardiovascular control mechanisms. We suggest that this results from activation by Ang II of a central pathway originating at the circumventricular organs.
Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key compone... more Adrenergic (C1) neurons located in the rostral ventrolateral medulla are considered a key component in the control of arterial blood pressure. Classically, C1 cells have been identified by their immunoreactivity for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and/or phenylethanolamine N-methyltransferase (PNMT). However, no studies have simultaneously demonstrated the expression of aromatic L-amino acid decarboxylase (AADC) and dopamine -hydroxylase (DBH) in these neurons. We examined the expression and colocalization of all four enzymes in the rat ventrolateral medulla using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Retrograde tracer injected into thoracic spinal segments T2-T4 was used to identify bulbospinal neurons. Using fluorescence and confocal microscopy, most cells of the C1 group were shown to be double or triple labeled with TH, DBH, and PNMT, whereas only 65-78% were immunoreactive for AADC. Cells that lacked detectable immunoreactivity for AADC were located in the rostral C1 region, and approximately 50% were spinally projecting. Some cells in this area lacked DBH immunoreactivity (6.5-8.3%) but were positive for TH and/or PNMT. Small numbers of cells were immunoreactive for only one of the four enzymes. Numerous fibres that were immunoreactive for DBH but not for TH or PNMT were noted in the rostral C1 region. Single-cell RT-PCR analysis conducted on spinally projecting C1 neurons indicated that only 76.5% of cells that contained mRNA for TH, DBH, and PNMT contained detectable message for AADC. These experiments suggest that a proportion of C1 cells may not express all of the enzymes necessary for adrenaline synthesis.
The explosion in the use of genetically modified mouse strains to investigate function in biology... more The explosion in the use of genetically modified mouse strains to investigate function in biology has an enormous potential to expand on pharmacological studies traditionally conducted in rats. A key limitation to date is the inability to record from multiple nerves in an anaesthetised mouse for long periods. Here we describe an in vivo preparation that maintains mice in a suitable physiological state, under anaesthesia, for at least 6hr and also enables multiple cardiorespiratory recordings over that time. Using the method described, blood pressure, heart rate, phrenic nerve activity, splanchnic nerve activity and heart rate were able to be recorded for hours in an anaesthetised, paralysed and mechanically ventilated mouse. Existing anaesthetised mouse preparations are limited by difficulties in maintaining mice under anaesthesia for long periods. This time constraint therefore limits the surgical time and number of cardiorespiratory variables recorded. It also limits the type of s...
Opioid receptors are activated during severe hemorrhage, resulting in sympathoinhibition and a pr... more Opioid receptors are activated during severe hemorrhage, resulting in sympathoinhibition and a profound fall in blood pressure. This study examined the location and subtypes of opioid receptors that might contribute to hypotension after hemorrhage. Intrathecal naloxone methiodide (100 nmol) abolished the fall in blood pressure after hemorrhage (1.5% of body wt; mean arterial pressure 122 +/- 8 mmHg after naloxone methiodide vs. 46 +/- 5 mmHg in controls, P < 0. 001). Intracisternal naloxone methiodide was less effective than intrathecal naloxone methiodide, whereas intravenous naloxone methiodide, which does not cross the blood-brain barrier, did not alter the fall in blood pressure after hemorrhage. These results demonstrate that spinal opioid receptors contribute to hypotension after hemorrhage but do not exclude supraspinal effects. In separate experiments, the subtype-specific opioid antagonists ICI-174864 (delta-antagonist), norbinaltorphimine (nor-BNI; kappa-antagonist), an...
A new technique for Iocalising the centre of sites of microinjection of drugs in the brain using ... more A new technique for Iocalising the centre of sites of microinjection of drugs in the brain using the silxer inlensification of colloidal gold is described. Fhe technique is compatible with immunocytochemistL~ and in this study was used in Wistar Kyoto rats to investigate the relationship between the location of phenylelhanolamine-N-methyltransferase-like in3munoreaclivc (PNMT-LI) cell bodies in the medulla and the precise sites where microinjection of L-glutamate elicited pressor responses. ('olloidal gold provided a simple technique for localising the centre of sites of microinjection with a high degree of spatial resohition and allowed pressor sites to be precisely Ioculised in the region of ventrolalera/ medulkl with the highest density of PNMT-LI cells.
Sleep apnea is associated with repeated episodes of hypoxemia, causing marked increase in sympath... more Sleep apnea is associated with repeated episodes of hypoxemia, causing marked increase in sympathetic nerve activity and blood pressure. Considerable evidence suggests that intermittent hypoxia (IH) resulting from apnea is the primary stimulus for sympathetic overactivity in sleep apnea patients. Several IH protocols have been developed either in animals or in humans to investigate mechanisms underlying the altered autonomic regulation of the circulation. Most of these protocols involve several days (10-40 days) of IH exposure, that is, chronic intermittent hypoxia (CIH). Recent data suggest that a single session of IH exposure, that is, acute intermittent hypoxia (AIH), is already capable of increasing tonic sympathetic nerve output (sympathetic long-term facilitation, LTF) and altering chemo- and baroreflexes with or without elevation of blood pressure. This indicates that IH alters the autonomic neurocirculatory at a very early time point, although the mechanisms underlying this ...
Swallowing and breathing utilize common muscles and an anatomical passage: the pharynx. The risk ... more Swallowing and breathing utilize common muscles and an anatomical passage: the pharynx. The risk of aspiration of ingested material is minimized not only by the laryngeal adduction of the vocal folds and laryngeal elevation but also by the precise coordination of swallows with breathing. Namely, swallows: (1) are preferentially initiated in the postinspiratory/expiratory phase, (2) are accompanied by a brief apnea, and (3) are often followed by an expiration and delay of the next breath. This review summarizes the expiratory evidence on the brainstem regions comprising the central pattern generator (CPG) that produces the pharyngeal stage of swallow, how the motor acts of swallowing and breathing are coordinated, and lastly, brainstem regions where the swallowing and respiratory CPGs may interact in order to ensure &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;safe&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; swallows.
Chemical stimulation of neurons in the pontine A5 area by microinjection of L-glutamate lowers ar... more Chemical stimulation of neurons in the pontine A5 area by microinjection of L-glutamate lowers arterial blood pressure. The mechanism of this &#39;A5 depressor response&#39; is not well-established. Here, we examine the involvement of spinal cord gamma-aminobutyric acid (GABA) receptors in this depressor response in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Experiments were conducted in male WKY and age-matched SHR anaesthetised with sodium pentobarbitone and chloral hydrate. An intrathecal catheter was implanted with the tip located between T9 and L2. Three days later, rats were re-anaesthetised and 10 nl of 40 mM L-glutamate was injected into the A5 area before, during and after, blockade of spinal cord GABA-A receptors by intrathecal injection of bicuculline methiodide (1 mM in 10 microliters phosphate-buffered saline). Injection of L-glutamate (10, 20, 40, 80 mM in 10 nl) produced depressor responses that were similar in WKY (n = 6) and SHR (n = 6). Intrathecal injection of bicuculline elicited a pressor response that was greater in SHR (n = 7, 28.5 +/- 7.6% increase in mean arterial pressure) than WKY (n = 11, 11.6 +/- 3.6%, p &lt; 0.05). After bicuculline, the depressor response to injection of L-glutamate into the A5 area was eliminated in both WKY (n = 7) and SHR (n = 6). Intrathecal injection of vehicle had no effect on either resting arterial blood pressure or the depressor response to A5 stimulation. Basal blood pressure and control responses to A5 stimulation were fully restored by around 90 min after bicuculline injection in each animal. In separate groups of rats, intrathecal injection of muscimol elicited depressor responses that were greater in SHR (n = 6, -32.0 +/- 6.2%) than WKY (n = 6, -17.3 +/- 1.5%, p &lt; 0.05). Our results suggest that the A5 depressor response is due to a projection from the A5 area to the spinal cord. This projection acts directly, or through a spinal interneuron and uses GABA as a neurotransmitter. Furthermore, our results indicate a hyper-responsiveness to GABA-A receptor stimulation in SHR since intrathecal bicuculline elicited much greater increases and intrathecal muscimol elicited much greater decreases, in blood pressure in SHR than in WKY. Finally, it seems likely that the A5-spinal depressor pathway is less effective in SHR than WKY under physiological conditions since chemical stimulation of the A5 area with L-glutamate produced a comparable depressor response in both strains.
Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neuron... more Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neurons projecting to the rat adrenal medulla are immunoreactive for either the inhibitory amino acid, gamma-aminobutyric acid (GABA) or the excitatory amino acid, L-glutamate. To investigate whether or not this is true for other groups of sympathetic preganglionic neurons, and to determine whether or not the proportion of inputs containing each type of amino acid neurotransmitter is the same for different groups of sympathetic preganglionic neurons, we retrogradely labelled rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion and used post-embedding immunogold on ultrathin sections to localise GABA- and glutamate-immunoreactivity. The cell bodies and dendrites of both rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion received synapses and direct contacts from nerve fibres immunoreactive for GABA and from nerve fibres immunoreactive for glutamate. In the rat, GABA was present in 48.9% of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion, and glutamate was present in 51.7% of inputs. Double immunogold labelling for glutamate and GABA on the same section, as well as labelling of consecutive serial sections for the two antigens, indicated that GABA and glutamate occur in separate populations of nerve fibres that provide input to rat sympathetic preganglionic neurons projecting to the superior cervical ganglion. We now have shown that GABA or glutamate is present in virtually all of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion and in essentially all of the inputs to sympathetic preganglionic neurons supplying the adrenal medulla. These findings are consistent with the hypothesis that all fast synaptic transmission in central autonomic pathways may be mediated by either excitatory or inhibitory amino acids. Furthermore, we showed a statistically significant difference in the proportion of glutamate-immunoreactive inputs between sympathetic preganglionic neurons projecting to the superior cervical ganglion and sympathoadrenal neurons (data from Llewellyn-Smith et al. [Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1992. Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res. 581, 67-80]), with preganglionics supplying the adrenal medulla receiving more excitatory inputs than those supplying the superior cervical ganglion. This increased excitatory input to sympathoadrenal neurons may explain the predominant activation of these neurons following baroreceptor unloading.
In anesthetized cats, we recorded intracellularly from 26 hypoglossal motoneurons which were anti... more In anesthetized cats, we recorded intracellularly from 26 hypoglossal motoneurons which were antidromically activated following electrical stimulation of either the medial or lateral branches of the hypoglossal nerve. Twenty-one of these neurons were protruder motoneurons 6 of which had inspiratory activity. Three of the protruder motoneurons with inspiratory activity were filled with Neurobiotin and found to be closely apposed to substance P-like immunoreactive nerve terminals. q
In this study we aimed to determine whether the levels of gene expression for phenylethanolamine-... more In this study we aimed to determine whether the levels of gene expression for phenylethanolamine-Nmethyltransferase (PNMT), noradrenaline transporter (NAT), ␣ 1A -receptor (␣ 1A -R), and ␣ 2A -receptor (␣ 2A -R) vary with resting systolic blood pressure in spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) or Sprague-Dawley (SD) rats. Sites examined included central and peripheral regions associated with the control of arterial pressure. Twenty week old SD (nϭ6), WKY (nϭ6), and SHR (nϭ6) were used. Systolic blood pressure was measured using tail cuff plethysmography 2 weeks before tissue extraction. RNA was isolated and reverse-transcribed into cDNA. Gene expression levels were measured, using quantitative real time PCR, relative to the expression of GAPDH. PNMT, NAT, and ␣ 1A -R mRNA expression was significantly greater in SHR tissue samples compared with normotensives. In the rostral ventrolateral medulla, PNMT mRNA in SHR was 3 times greater than that in WKY (SHR: 0.82Ϯ0.02%; WKY: 0.29Ϯ0.02%). The amount of ␣ 2A -R mRNA was significantly lower in SHR compared with normotensives. For example, the level of ␣ 2A -R mRNA in spinal cord of SHR was 3 times less than that found in WKY (SHR: 1.85Ϯ0.04%; WKY: 3.26Ϯ0.07%). PNMT, NAT, and ␣ 1A -R mRNA levels were positively correlated with systolic blood pressure in all central tissue investigated. Conversely, ␣ 2A -R mRNA levels in central sites were negatively correlated with systolic blood pressure. Clearly, a decrease in central ␣ 2A -R and an increase in ␣ 1A -R is consistent with the elevated blood pressure and sympathetic activity observed in SHR. (Hypertension. 2002;40:342-347.)
In urethane-anaesthetised artificially ventilated Sprague-Dawley rats, bilateral microinjection o... more In urethane-anaesthetised artificially ventilated Sprague-Dawley rats, bilateral microinjection of the divalent cation nickel chloride (Ni 2؉ ; 50 mM, 50 nl) into the rostral ventrolateral medulla elicited a dramatic inhibition of splanchnic sympathetic nerve activity (؊44؎6%) and a marked depressor response (؊35؎7 mmHg).
The specific role of the ⌬ opioid receptor (DOR), in opioid-induced respiratory depression in the... more The specific role of the ⌬ opioid receptor (DOR), in opioid-induced respiratory depression in the ventral respiratory group (VRG) is largely unknown. Here, we sought to determine (1) the relationship between DOR-immunoreactive (ir) boutons, bulbospinal and functionally identified respiratory neurons in the VRG and (2) the effects of microinjection of the selective DOR agonist, D-Pen 2,5-enkephalin (DPDPE), into different subdivisions of the VRG, on phrenic nerve discharge and mean arterial pressure.
The effects of hyperoxic hypercapnia (5, 10 or 15% CO 2 in O 2 ) on splanchnic sympathetic nerve ... more The effects of hyperoxic hypercapnia (5, 10 or 15% CO 2 in O 2 ) on splanchnic sympathetic nerve activity (sSNA) and sympathetic reflexes such as the somato-sympathetic reflex or baroreflex were studied in urethane anaesthetised, paralysed, artificially ventilated and vagotomized Sprague-Dawley rats. Hypercapnia caused a small increase in mean arterial blood pressure (MAP) in the 10% CO 2 group and a fall in heart rate (HR) in all three groups. sSNA increased in all three groups. Phrenic frequency and amplitude increased during hypercapnia, with frequency adapting back towards baseline during the CO 2 exposure. The somato-sympathetic reflex was attenuated in the 5% CO 2 group and abolished in the 10 and 15% CO 2 groups, whereas there was little effect on the sSNA baroreflex. Hypercapnia significantly affects phrenic nerve activity (PNA), sSNA and selectively inhibits the somato-sympathetic reflex with little effect on the sSNA baroreflex.
Central apnea is common in the premature newborn. To explain the upper airway findings in differe... more Central apnea is common in the premature newborn. To explain the upper airway findings in different clinical conditions characterized by central apnea, we made single unit recordings from laryngeal motoneurons during normal and hyperventilation. Posterior cricoarytenoid (n = 4) and cricothyroid (n = 4) motoneurons displayed an inspiratory pattern during normal ventilation and remained synchronous with phrenic nerve discharge (PND) during hyperventilation. Laryngeal constrictor motoneurons (LCon) displayed a post-inspiratory pattern during normal ventilation, but lost phasic activity during early hyperventilation (the period after the onset of hyperventilation but before cessation of PND; n = 12). There was a nearly linear relationship between the post-inspiratory activity and strength of PND. Six LCon motoneurons remained silent throughout hyperventilation, while the other six developed a tonic activity during cessation of PND. Further analysis suggested that the silent and tonic LCon motoneurons are likely to share a similar mechanism in their post-inspiratory pattern generation, but differ from each other in their responses to CO 2 inputs. In addition, strong inhibition of the LCon tonic activity by the early return of PND could be an important factor in recovery following a period of apnea. Failure of this inspiratory inhibition to occur might explain certain clinical situations, where obstructive apnea supervenes following a period of central apnea. Crown
Chronic intravenous angiotensin II (Ang II) has been widely used to establish centrally mediated ... more Chronic intravenous angiotensin II (Ang II) has been widely used to establish centrally mediated hypertension in experimental animals, and disruption of Ang II activity is a frontline treatment for hypertensive disease. However, the acute central actions of circulating Ang II are poorly understood. We examined the effects of intravenous pressor doses of Ang II on autonomic activity in anaesthetized rats under neuromuscular blockade, and compared baroinhibition evoked by Ang II pressor ramps to equipressor responses evoked by phenylephrine (PE). Baroinhibition of splanchnic sympathetic nerve activity was attenuated during Ang II trials compared with PE, and rats remained sensitive to electrical stimulation of the aortic depressor nerve at higher arterial pressures during Ang II trials. This was not due to a direct effect of Ang II on aortic nerve baroreceptors. In a separate series of experiments, we provide direct evidence that bulbospinal barosensitive neurones in the rostral ventrolateral medulla are differentially sensitive to pressure ramps evoked by Ang II or PE vasoconstriction. Nineteen out of 41 units were equally sensitive to increased arterial pressure evoked by Ang II or PE. In 17 of 41 units, barosensitivity was attenuated during Ang II trials, and in five of 41 cases units that had previously been barosensitive increased their firing rate during Ang II trials. These results show, for the first time, that circulating Ang II acutely modulates central cardiovascular control mechanisms. We suggest that this results from activation by Ang II of a central pathway originating at the circumventricular organs.
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Papers by Paul Pilowsky