Microinjection Of DL-homocysteic Acid Research Articles

The pedunculopontine tegmentum controls renal sympathetic nerve activity and cardiorespiratory activities in nembutal-anesthetized rats.

Elevated renal sympathetic nerve activity (RSNA) accompanies a variety of complex disorders, including obstructive sleep apnea, heart failure, and chronic kidney disease. Understanding pathophysiologic renal mechanisms is important for determining why hypertension is both a common sequelae and a predisposing factor of these disorders. The role of the brainstem in regulating RSNA remains incompletely understood. The pedunculopontine tegmentum (PPT) is known for regulating behaviors including alertness, locomotion, and rapid eye movement sleep. Activation of PPT neurons in anesthetized rats was previously found to increase splanchnic sympathetic nerve activity and blood pressure, in addition to altering breathing. The present study is the first investigation of the PPT and its potential role in regulating RSNA. Microinjections of DL-homocysteic acid (DLH) were used to probe the PPT in 100-μm increments in Nembutal-anesthetized rats to identify effective sites, defined as locations where changes in RSNA could be evoked. A total of 239 DLH microinjections were made in 18 rats, which identified 20 effective sites (each confirmed by the ability to evoke a repeatable sympathoexcitatory response). Peak increases in RSNA occurred within 10–20 seconds of PPT activation, with RSNA increasing by 104.5 ± 68.4% (mean ± standard deviation) from baseline. Mean arterial pressure remained significantly elevated for 30 seconds, increasing from 101.6 ± 18.6 mmHg to 135.9 ± 36.4 mmHg. DLH microinjections also increased respiratory rate and minute ventilation. The effective sites were found throughout the rostal-caudal extent of the PPT with most located in the dorsal regions of the nucleus. The majority of PPT locations tested with DLH microinjections did not alter RSNA (179 sites), suggesting that the neurons that confer renal sympathoexcitatory functions comprise a small component of the PPT. The study also underscores the importance of further investigation to determine whether sympathoexcitatory PPT neurons contribute to adverse renal and cardiovascular consequences of diseases such as obstructive sleep apnea and heart failure.

ElPBN neurons regulate rVLM activity through elPBN-rVLM projections during activation of cardiac sympathetic afferent nerves.

The external lateral parabrachial nucleus (elPBN) within the pons and rostral ventrolateral medulla (rVLM) contributes to central processing of excitatory cardiovascular reflexes during stimulation of cardiac sympathetic afferent nerves (CSAN). However, the importance of elPBN cardiovascular neurons in regulation of rVLM activity during CSAN activation remains unclear. We hypothesized that CSAN stimulation excites the elPBN cardiovascular neurons and, in turn, increases rVLM activity through elPBN-rVLM projections. Compared with controls, in rats subjected to microinjection of retrograde tracer into the rVLM, the numbers of elPBN neurons double-labeled with c-Fos (an immediate early gene) and the tracer were increased after CSAN stimulation (P < 0.05). The majority of these elPBN neurons contain vesicular glutamate transporter 3. In cats, epicardial bradykinin and electrical stimulation of CSAN increased the activity of elPBN cardiovascular neurons, which was attenuated (n = 6, P < 0.05) after blockade of glutamate receptors with iontophoresis of kynurenic acid (Kyn, 25 mM). In separate cats, microinjection of Kyn (1.25 nmol/50 nl) into the elPBN reduced rVLM activity evoked by both bradykinin and electrical stimulation (n = 5, P < 0.05). Excitation of the elPBN with microinjection of dl-homocysteic acid (2 nmol/50 nl) significantly increased basal and CSAN-evoked rVLM activity. However, the enhanced rVLM activity induced by dl-homocysteic acid injected into the elPBN was reversed following iontophoresis of Kyn into the rVLM (n = 7, P < 0.05). These data suggest that cardiac sympathetic afferent stimulation activates cardiovascular neurons in the elPBN and rVLM sequentially through a monosynaptic (glutamatergic) excitatory elPBN-rVLM pathway.

Postnatal maturation of the spinal-bulbo-spinal loop: brainstem control of spinal nociception is independent of sensory input in neonatal rats.

The rostroventral medial medulla (RVM) is part of a rapidly acting spino-bulbo-spinal loop that is activated by ascending nociceptive inputs and drives descending feedback modulation of spinal nociception. In the adult rat, the RVM can facilitate or inhibit dorsal horn neuron inputs but in young animals descending facilitation dominates. It is not known whether this early life facilitation is part of a feedback loop. We hypothesized that the newborn RVM functions independently of sensory input, before the maturation of feedback control. We show here that noxious hind paw pinch evokes no fos activation in the RVM or the periaqueductal gray at postnatal day (P) 4 or P8, indicating a lack of nociceptive input at these ages. Significant fos activation was evident at P12, P21, and in adults. Furthermore, direct excitation of RVM neurons with microinjection of DL-homocysteic acid did not alter the net activity of dorsal horn neurons at P10, suggesting an absence of glutamatergic drive, whereas the same injections caused significant facilitation at P21. In contrast, silencing RVM neurons at P8 with microinjection of lidocaine inhibited dorsal horn neuron activity, indicating a tonic descending spinal facilitation from the RVM at this age. The results support the hypothesis that early life descending facilitation of spinal nociception is independent of sensory input. Since it is not altered by RVM glutamatergic receptor activation, it is likely generated by spontaneous brainstem activity. Only later in postnatal life can this descending activity be modulated by ascending nociceptive inputs in a functional spinal-bulbo-spinal loop.

Nucleus raphé pallidus participates in midbrain-medullary cardiovascular sympathoinhibition during electroacupuncture

We have shown that electroacupuncture (EA) inhibits sympathoexcitatory rostral ventrolateral medulla (rVLM) neurons and reflex responses following activation of a long-loop pathway in the arcuate nucleus and ventrolateral periaqueductal gray (vlPAG). Additionally, EA at P 5-6 acupoints (overlying the median nerve) activates serotonin-containing neurons in the nucleus raphé pallidus (NRP), which, in turn, inhibit rVLM neurons. Although direct projections from the vlPAG to the rVLM exist, it is uncertain whether an indirect pathway through the NRP serves an important role in vlPAG-rVLM cardiovascular modulation. Therefore, the splanchnic nerve (SN) was stimulated to induce cardiovascular sympathoexcitatory reflexes, and EA was applied at P 5-6 acupoints in α-chloralose-anesthetized cats. A single-barreled recording electrode was inserted into the NRP or rVLM. Microinjection of DL-homocysteic acid (DLH) into the vlPAG increased the NRP neuronal response to SN stimulation (5 ± 1 to 12 ± 2 spikes/30 stim). Likewise, EA at P 5-6 for 30 min increased the NRP response to SN stimulation (3 ± 1 to 10 ± 2 spikes/30 stim), an effect that could be blocked by microinjection of kynurenic acid (KYN) into the caudal vlPAG. Furthermore, the reflex increase in blood pressure induced by application of bradykinin to the gallbladder and the rVLM cardiovascular presympathetic neuronal response to SN stimulation was inhibited by injection of DLH into the vlPAG, a response that was reversed by injection of KYN into the NRP. These results indicate that EA activates the vlPAG, which excites the NRP to, in turn, inhibit rVLM presympathetic neurons and reflex cardiovascular sympathoexcitatory responses.

Nucleus Raphe Pallidus in EA inhibition of rostral ventrolateral medulla and cardiovascular excitatory responses

EA at P5‐6 acupoints (median nerve) excites the arcuate nucleus and ventrolateral periaqueductal gray (vlPAG), and inhibits sympathetic cardiovascular rostral ventrolateral medulla (rVLM) neurons and associated reflex responses. The present study investigated the role of the NRP in EA inhibition of rVLM neurons and whether EA and activity from the vlPAG to the rVLM passes through the NRP. In [alpha]‐chloralose anesthetized cats the splanchnic nerve (SN) was stimulated to induce cardiovascular sympathoexcitatory reflexes and EA was applied at P 5‐6 acupoints. A recording electrode was inserted into the NRP or rVLM. Microinjection of dl‐homocysteic acid (DLH), but not normal saline (NS), into vlPAG increased the NRP neuronal response to SN stimulation (5±1 to 12±1 spikes/30 stim). EA at P 5‐6 for 30 min increased the NRP response to SN stimulation (5±1 to 14±3 spikes/30 stim). EA excitation of the NRP response was blocked by microinjection of kynurenic acid (KYN) into the caudal vlPAG. Furthermore, the reflex increase in BP induced by application of bradykinin on gallbladder and the rVLM neuronal response to SN stimulation was inhibited (~58%) by injection of DLH into the vlPAG, a response that could be blocked by injection of KYN into the NRP. Thus, EA activates the arcuate and vlPAG, which excites the NRP and, in turn, inhibits the rVLM activity and sympathoexcitatory cardiovascular reflex responses. (HL‐63313)

Selective inhibition from the anterior hypothalamus of C- versus A-fibre mediated spinal nociception

Modulation of spinal nociception from the anterior hypothalamus/preoptic area (AH/POA), and consequent alterations in the pain experience may contribute to integrated responses brought into play during fear or stress and as part of the sickness response. This study was designed to compare the effects of descending control from AH/POA on A- versus C-fibre-evoked spinal nociception, since any differential control is of behavioural and clinical importance given that A-fibre and C-fibre nociceptors convey different qualities of the pain signal (first and second pain, respectively), and play different roles in the development and maintenance of chronic pain states. In anaesthetised rats, electromyographic responses were recorded to monitor thresholds of withdrawal to slow (2.5 °C s −1) or fast (7.5 °C s −1) rates of skin heating of the hindpaw, to preferentially activate C- or A-nociceptors, respectively. Neuronal activation by microinjection of dl-homocysteic acid at sites within a specific region of AH/POA, lateral area of the anterior hypothalamus (LAAH), significantly increased response thresholds to slow heating rates ( p < 0.02, n = 11), but not those to fast rates of heating ( p = 0.48, n = 10). Injection of DLH adjacent to LAAH ( n = 9) had no significant effect on responses to slow ( n = 8) or fast ( n = 9) rates of skin heating. The functional significance of differential descending control of spinal processing of C- and A-nociceptive inputs is discussed with respect to roles both of the LAAH in pain processing, and of C- and A-nociceptive inputs in acute and chronic pain.

Midbrain control of spinal nociception discriminates between responses evoked by myelinated and unmyelinated heat nociceptors in the rat

Descending control of spinal nociception is a major determinant of normal and chronic pain. Myelinated (A-fibre) and unmyelinated (C-fibre) nociceptors convey different qualities of the pain signal (first and second pain, respectively), and they play different roles in the development and maintenance of chronic pain states. It is of considerable importance, therefore, to determine whether descending control has differential effects on the central processing of A- vs. C-nociceptive input. In anaesthetised rats, biceps femoris EMG was recorded to monitor the thresholds and encoding properties of responses evoked by fast (7.5 °C s −1) or slow (2.5 °C s −1) rates of skin heating of the dorsal surface of a hindpaw to preferentially activate myelinated or unmyelinated heat nociceptors, respectively. Activation of neurones in the periaqueductal grey (PAG) by microinjection of dl-homocysteic acid (DLH) or bicuculline (BIC) significantly increased response thresholds to slow rates of heating ( P < 0.001), but not those to fast rates of heating ( P > 0.05). The ability of the EMG to encode the stimulus intensity of fast rates of skin heating remained intact and unaltered ( r 2 = 0.99, P < 0.001) following BIC but not DLH injection. In contrast, encoding of the stimulus intensity of slow rates of skin heating was abolished following BIC and DLH injection. The functional significance of differential descending control of the central processing of C- and A-nociceptive inputs is discussed with respect to role of the PAG in mediating antinociception as part of active coping strategies in emergency situations and the role of C- and A-nociceptive inputs in animal models of chronic pain.

A mapping study of cardiorespiratory responses to chemical stimulation of the midline medulla oblongata in ventilated and freely breathing rats.

The aim of this study was to examine the cardiorespiratory effects of chemically stimulating neurons in the midline medulla oblongata (MM) of artificially ventilated and freely breathing anesthetized rats. Earlier studies reported that stimulation of the MM elicits increases or decreases in mean arterial pressure (MAP) and phrenic nerve activity, depending on the mode and site of stimulation, anesthetic, and species. In the first series of experiments, rats were anesthetized with urethane, artificially ventilated, paralyzed, and bilaterally vagotomized. The rostrocaudal extent of the MM was mapped by microinjections of DL-homocysteic acid or L-glutamate (both 100 mM, 100 nl), and, in line with previous studies, most injections produced only small responses in MAP, heart rate, and splanchnic sympathetic nerve activity. Increases in respiratory parameters were evoked in caudal regions. However, activation of a discrete region of the MM at the level of the caudal pole of the facial nucleus (CP7) consistently caused a dramatic reduction in phrenic nerve amplitude and/or frequency and, in six rats, produced a prolonged apnea. The second series of experiments was carried out on freely breathing pentobarbitone sodium-anesthetized rats, with a diaphragmatic electromyogram used to monitor respiratory activity. Respiratory activity could again be abolished at CP7 after microinjections of glutamate (100 mM, 50 nl); however, these responses were accompanied by large decreases in MAP and moderate reductions in heart rate. This depression of respiratory activity may be due to activation of propriobulbar inhibitory neurons that project to known respiratory centers in the brain stem.

Focal CO2/H+ alters phrenic motor output response to chemical stimulation of cat pre-Botzinger complex in vivo.

Microinjection of dl-homocysteic acid (DLH), a glutamate analog, into the pre-Bötzinger complex (pre-BötC) can produce tonic excitation of phrenic nerve discharge. Although this DLH-induced tonic excitation can be modified by systemic hypercapnia, the role of focal increases in pre-BötC CO(2)/H(+) in this modulation of the DLH-induced response remains to be determined. Therefore, we examined the effects of unilateral microinjection of DLH (10 mM; 10-20 nl) into the pre-BötC before and during increased focal pre-BötC CO(2)/H(+) (i.e., focal tissue acidosis) in chloralose-anesthetized, vagotomized, mechanically ventilated cats. Focal tissue acidosis was produced by blockade of carbonic anhydrase with either focal acetazolamide (AZ) or methazolamide (MZ) microinjection. For these experiments, sites were selected in which unilateral microinjection of DLH into the pre-BötC produced a nonphasic tonic excitation of phrenic nerve discharge (n = 10). Microinjection of 10-20 nl AZ (50 microM) or MZ (50 microM) into these 10 sites in the pre-BötC increased the amplitude and/or frequency of eupneic phrenic bursts, as previously reported. Subsequent microinjection of DLH produced excitation in which phasic respiratory bursts were superimposed on tonic discharge. These DLH-induced phasic respiratory bursts had an increased frequency compared with the preinjection baseline frequency (P < 0.05). These findings demonstrate that modulation of phrenic motor activity evoked by DLH-induced activation of the pre-BötC is influenced by focal CO(2)/H(+) chemosensitivity in this region. Furthermore, these findings suggest that focal increases in pre-BötC CO(2)/H(+) may have contributed to the modulation of the DLH-induced responses previously observed during systemic hypercapnia.

Influence of respiratory network drive on phrenic motor output evoked by activation of cat pre-Bötzinger complex

We have previously demonstrated that microinjection of dl-homocysteic acid (DLH), a glutamate analog, into the pre-Bötzinger complex (pre-BötC) can produce either phasic or tonic excitation of phrenic nerve discharge during hyperoxic normocapnia. Breathing, however, is influenced by input from both central and peripheral chemoreceptor activation. This influence of increased respiratory network drive on pre-BötC-induced modulation of phrenic motor output is unclear. Therefore, these experiments were designed to examine the effects of chemical stimulation of neurons (DLH; 10 mM; 10-20 nl) in the pre-BötC during hyperoxic modulation of CO2 (i.e., hypercapnia and hypocapnia) and during normocapnic hypoxia in chloralose-anesthetized, vagotomized, mechanically ventilated cats. For these experiments, sites were selected in which unilateral microinjection of DLH into the pre-BötC during baseline conditions of hyperoxic normocapnia [arterial PCO2 (PaCO2) = 37-43 mmHg; n = 22] produced a tonic (nonphasic) excitation of phrenic nerve discharge. During hypercapnia (PaCO2 = 59.7 +/- 2.8 mmHg; n = 17), similar microinjection produced excitation in which phasic respiratory bursts were superimposed on varying levels of tonic discharge. These DLH-induced phasic respiratory bursts had an increased frequency compared with the preinjection baseline frequency (P < 0.01). In contrast, during hypocapnia (PaCO2 = 29.4 +/- 1.5 mmHg; n = 11), microinjection of DLH produced nonphasic tonic excitation of phrenic nerve discharge that was less robust than the initial (normocapnic) response (i.e., decreased amplitude). During normocapnic hypoxia (PaCO2 = 38.5 +/- 3.7; arterial Po2 = 38.4 +/- 4.4; n = 8) microinjection of DLH produced phrenic excitation similar to that seen during hypercapnia (i.e., increased frequency of phasic respiratory bursts superimposed on tonic discharge). These findings demonstrate that phrenic motor activity evoked by chemical stimulation of the pre-BötC is influenced by and integrates with modulation of respiratory network drive mediated by input from central and peripheral chemoreceptors.

Taste responses of neurons in the hamster solitary nucleus are modulated by the central nucleus of the amygdala.

Previous studies have shown a modulatory influence of forebrain gustatory areas, such as the gustatory cortex and lateral hypothalamus, on the activity of taste-responsive cells in the nucleus of the solitary tract (NST). The central nucleus of the amygdala (CeA), which receives gustatory afferent information, also exerts descending control over taste neurons in the parabrachial nuclei (PbN) of the pons. The present studies were designed to investigate the role of descending amgydaloid projections to the NST in the modulation of gustatory activity. Extracellular action potentials were recorded from 109 taste-responsive cells in the NST of urethan-anesthetized hamsters and analyzed for a change in excitability following electrical and chemical stimulation of the CeA. Electrical stimulation of the CeA orthodromically modulated 36 of 109 (33.0%) taste-responsive NST cells. An excitatory response was observed in 33 (30.28%) cells. An initial decrease in excitability to electrical stimulation of the CeA, suggestive of postsynaptic inhibition, was observed in three (2.75%) NST taste cells. NST cells modulated by the CeA were significantly less responsive to taste stimuli than cells that were not. Many of these cells were under the modulatory influence of the contralateral CeA (28/36 = 77.8%) as well as the ipsilateral (22/36 = 61.1%); 14 (38.9%) were excited bilaterally. Latencies for excitation were longer after ipsilateral than after contralateral CeA stimulation. Microinjection of DL-homocysteic acid (DLH) into the CeA mimicked the effect of electrical stimulation on each of the nine cells tested: DLH excited eight and inhibited one of these electrically activated NST cells. Application of subthreshold electrical stimulation to the CeA during taste trials increased the taste responses of every CeA-responsive NST cell (n = 7) tested with this protocol. These effects would enhance taste discriminability by increasing the signal-to-noise ratio of taste-evoked activity.

Role of the pre-Bötzinger complex in respiratory rhythm generation in vivo: influence of respiratory network drive

We have previously demonstrated that microinjection of DL-homocysteic acid (DLH), a glutamate analog, into the pre-Botzinger complex (pre-BotC) can produce either phasic or tonic excitation of phrenic nerve discharge. Our initial findings were obtained during hyperoxic normocapnia; thus, the influence of respiratory network drive, including intrinsic pre-BotC neuronal excitability, on the DLH-induced modulation of phrenic motor output requires clarification. We, therefore, examined the effects of chemical stimulation of the pre-BotC during increased respiratory network drive elicited by hypercapnia, hypoxia (ie, peripheral chemoreflex), or focal pre-BotC tissue acidosis in chloralose-anesthetized, vagotomized, mechanically ventilated cats. For these experiments, we selected sites in which unilateral microinjection of DLH into the pre-BotC during hyperoxic normocapnia (PaCO2 = 37–43 mmHg) produced a non-rhythmic tonic excitation of phrenic nerve discharge. During hypercapnia (PaCO2 = 59.7 ± 2.8 mmHg; n = 17), similar microinjection produced excitation in which respiratory rhythmic oscillations were superimposed on varying levels of tonic discharge. A similar pattern of modulation was observed in response to microinjection of DLH into the pre-BotC during normocapnic hypoxia (PaCO2 = 38.5 ± 3.7; PaO2 = 38.4 ± 4.4; n = 8) and during focal pre-BotC tissue acidosis (n = 7). Further, during increased respiratory network drive, these DLH-induced respiratory rhythmic oscillations had an increased frequency compared to the preinjection baseline frequency of phrenic bursts (P < 0.05). These findings demonstrate that tonic inspiratory motor activity evoked by chemical stimulation of the pre-BotC is influenced by and integrates with modulation of respiratory network drive mediated by input from both central and peripheral chemoreceptors, as well as focal pre-BotC CO2/H+ chemosensitivity.

Inhibitory effects evoked from both the lateral and ventrolateral periaqueductal grey are selective for the nociceptive responses of rat dorsal horn neurones

In rats anaesthetized with alphaxalone/alphadolone a comparative study was made of the inhibitory effects on dorsal horn neurones evoked by chemical stimulation at identified pressor and depressor sites in the lateral and ventrolateral periaqueductal grey (PAG). Stimulating micropipettes were inserted stereotaxically into the lateral or ventrolateral PAG at sites where microinjection of dl-homocysteic acid (DLH) evoked increases or decreases respectively in mean arterial blood pressure. The effects of DLH microinjection at these sites were tested against the responses of dorsal horn neurones to noxious and innocuous stimuli applied to their cutaneous receptive fields. Single unit extracellular recordings were made from 15 Class 1 (low-threshold) and 37 Class 2 (wide dynamic range) dorsal horn neurones in laminae II–VI of the lower lumbar spinal cord. The responses of Class 1 neurones to innocuous prodding of their receptive fields were unaffected by neuronal activation in either the lateral or ventrolateral PAG. The nociceptive (noxious pinch/heat) responses of most Class 2 neurones were strongly inhibited by chemical stimulation in either sector of the PAG. The low threshold (prod) responses of the same neurones were generally unaffected or only weakly inhibited by identical stimulation, regardless of stimulation site. No significant differences were found between the effects of lateral vs. ventrolateral PAG stimulation on the responses of dorsal horn neurones. These results do not support the view that dorsal horn neurones may be inhibited with different selectivities by hyper- and hypotensive regions of the PAG.

Blockade of glutamate receptors in CVLM and NTS attenuates airway dilation evoked from parabrachial region.

Previous work from our laboratory has shown that stimulation of cell bodies and dendrites in the medial and lateral parabrachial nuclei dilates the airways. The sites participating in the pathway mediating this airway response are not known. Two likely candidates are the caudal ventrolateral medulla (CVLM) and the nucleus tractus solitarii (NTS). Using chloralose-anesthetized cats, we assessed the airway dilation evoked from the parabrachial region before and during bilateral blockade of the NTS or the CVLM. The airway dilation arising from stimulation of the parabrachial region was evoked by microinjection of DL-homocysteic acid (25 nl, 100 mM). Bilateral blockade of the NTS or CVLM, achieved by microinjection of kynurenic acid (50 nl, 100 mM), reversibly attenuated the airway dilation in every cat tested. On average, kynurenic acid-induced blockade of the NTS caused a more complete attenuation of the dilation evoked from the parabrachial region than did blockade of the CVLM. Bilateral microinjection of cobalt chloride (50 nl, 50 mM) into the CVLM gave inconclusive results, attenuating the airway dilation evoked from the parabrachial region in six cats and potentiating it in three others. We conclude that the CVLM and the NTS participate in the airway dilation arising from the parabrachial region.

Gastric relaxation in response to chemical stimulation of the area postrema in the rabbit

Microinjections of DL-homocysteic acid into the area postrema (AP) of anesthetized rabbits provoked gastric relaxations associated with small changes in blood pressure and marked excitatory effects on respiration. Both gastric and cardiovascular effects failed to occur after bilateral vagotomy. Comparable gastric relaxations were induced before and after treatment with atropine or atropine and guanethidine. The AP appears to play a role in gastric motility via vagus nerves and noradrenergic noncholinergic intramural inhibitory neurons.

Chemical activation of caudal medullary expiratory neurones alters the pattern of breathing in the cat.

1. The purpose of this work was to ascertain whether the activation of caudal expiratory neurones located in the caudal part of the ventral respiratory group (VRG) may affect the pattern of breathing via medullary axon collaterals. 2. We used microinjections of DL-homocysteic acid (DLH) to activate this population of neurones in pentobarbitone-anaesthetized, vagotomized, paralysed and artificially ventilated cats. Both phrenic and abdominal nerve activities were monitored; extracellular recordings from medullary and upper cervical cord respiratory neurones were performed. 3. DLH (160 mM) microinjected (10-30 nl for a total of 1.6-4.8 nmol) into the caudal VRG, into sites where expiratory activity was encountered, provoked an intense and sustained activation of the expiratory motor output associated with a corresponding period of silence in phrenic nerve activity. During the progressive decline of the activation of abdominal motoneurones, rhythmic inspiratory activity resumed, displaying a decrease in frequency and a marked reduction or the complete suppression of postinspiratory activity as its most consistent features. 4. Medullary and upper cervical cord inspiratory neurones exhibited inhibitory responses consistent with those observed in phrenic nerve activity, while expiratory neurones in the caudal VRG on the side contralateral to the injection showed excitation patterns similar to those of abdominal motoneurones. On the other hand, in correspondence to expiratory motor output activation, expiratory neurones of the Bötzinger complex displayed tonic discharges whose intensity was markedly lower than the peak level of control breaths. 5. Bilateral lignocaine blockades of neural transmission at C2-C3 affecting the expiratory and, to a varying extent, the inspiratory bulbospinal pathways as well as spinal cord transections at C2-C3 or C1-C2, did not suppress the inhibitory effect on inspiratory neurones of either the ipsi- or contralateral VRG in response to DLH microinjections into the caudal VRG. 6. The results show that neurones within the column of caudal VRG expiratory neurones promote inhibitory effects on phrenic nerve activity and resetting of the respiratory rhythm. We suggest that these effects are mediated by medullary bulbospinal expiratory neurones, which may, therefore, have a function in the control of breathing through medullary axon collaterals.

The rostral hypothalamus: an area for the integration of autonomic and sensory responsiveness

1. An investigation has been made of the effects of chemical stimulation of neurons in the rostral hypothalamus on cardiovascular and respiratory parameters and on a nociceptive viscerosomatic reflex (reflex activity recorded from a lumbar spinal nerve in response to electrical stimulation of visceral afferent fibers in the splanchnic nerve) in alphaxalone/alphadolone-anesthetised rats. 2. Neurons were stimulated by microinjection of DL-homocysteic acid (DLH) at ventromedial forebrain sites between 2 mm caudal and 3 mm rostral to bregma. Any changes were monitored in viscerosomatic reflex activity and in the rate and depth of respiration, arterial blood pressure, heart rate, and hindlimb muscle blood flow. 3. Viscero-somatic reflex activity was depressed to varying degrees after injection of DLH at 20/96 sites and this was always accompanied by some form of cardiovascular change. At 8 sites inhibition of the reflex was accompanied by a cardiovascular response that included an initial pressor response (type I response) and at the other 12 sites there was an initial depressor response (type II response). Injection of DLH at a further 30 sites evoked a depressor response and no change in viscerosomatic reflex activity and at the remaining 45 sites there was no detectable change in any of the variables measured. 4. The degree of inhibition evoked from type I sites (mean 90%) was significantly greater (P < 0.001) than that evoked from type II sites (mean 58%). Type I responses were only evoked from an extremely localized region in the rostral anterior hypothalamus that corresponds to the lateral area of the anterior hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Respiratory responses to electrical and chemical stimulation of the area postrema in the rabbit.

1. The respiratory role of the area postrema (AP) has been investigated in pentobarbitone- or alpha-chloralose-anaesthetized, vagotomized, paralysed and artificially ventilated rabbits, by means of electrical stimulation and microinjections of DL-homocysteic acid (DLH). Phrenic nerve activity was used as an index of central respiratory drive. 2. Bipolar electrical or chemical stimulation (microinjections of DLH, 5-30 nl; 160 mM) of the caudal compact portion of the AP provoked excitatory effects on the inspiratory motor output, without apparent changes in the arterial blood pressure. 3. Depressant effects on inspiratory activity, accompanied on some occasions by changes in arterial blood pressure (as a rule, increases > or = 30 mmHg) were induced by DLH microinjections in close neighbouring areas (including the medial part of the nucleus tractus solitarii) or in the IV ventricle. 4. These results support a role for the AP in the neural control of respiration. The findings are discussed in connection with other autonomic functions to which the AP has been reported to contribute, in different animal species.

Activation of neurons in the rostral ventrolateral medulla increases bronchomotor tone in dogs

Previous work from this laboratory has demonstrated that the chemical activation of cell bodies in the caudal ventrolateral medulla of chloralose-anesthetized dogs decreased bronchomotor tone by withdrawing cholinergic input to airway smooth muscle. In the present study we determined the bronchomotor responses to microinjection of DL-homocysteic acid (100 mM; 25-50 nl) into the rostral ventrolateral (RVL) medulla of chloralose-anesthetized dogs. Total lung resistance was used as a functional index of bronchomotor tone. Microinjection of DL-homocysteic acid into the 20 sites located in the lateral aspect of the RVL medulla increased both total lung resistance [from 6.5 +/- 0.4 to 9.1 +/- 0.8 (SE) cmH2O.l-1.s; P less than 0.05] and mean arterial pressure (from 125 +/- 5 to 148 +/- 8 mmHg; P less than 0.05). Microinjection of this amino acid into nine sites located in the medial aspect of the RVL medulla increased mean arterial pressure (from 130 +/- 6 to 153 +/- 6 mmHg; P less than 0.05) but had no effect on total lung resistance. We confirmed in three sites that the increase in total lung resistance evoked by microinjection of DL-homocysteic acid was accompanied by an increase in tracheal smooth muscle tension. The increase in total lung resistance evoked by DL-homocysteic acid was not affected by beta-adrenergic blockade but was abolished by muscarinic blockade.

Hypothalamic influences on viscero‐somatic neurones in the lower thoracic spinal cord of the anaesthetized rat.

1. Single unit electrical activity has been recorded from thirty-four viscero-somatic neurones in the dorsal horn of the lower thoracic spinal cord (T9-T11) of chloralose-anaesthetized rats. All neurones were driven by natural and/or electrical stimulation within their somatic receptive fields and gave excitatory responses to electrical stimulation of the ipsilateral splanchnic nerve. Descending influences on these neurones were tested by electrical and chemical (microinjections of DL-homocysteic acid) stimulation of sites in the rostral hypothalamus. 2. The electrical activity of most viscero-somatic neurones (64%) was inhibited by electrical stimulation at sites throughout the anterior hypothalamus-preoptic region. In any one cell, responses to stimulation of visceral and somatic afferent fibres were inhibited to the same extent and any on-going activity was also depressed. Only one cell was driven by the conditioning stimulus and the electrical activity of the remaining cells (n = 7) was unaffected. 3. At certain hypothalamic sites the effects of electrical conditioning stimulation on the responses of viscero-somatic neurones were compared with those of local microinjection of DL-homocysteic acid. Electrical stimulation at all sites tested (n = 7) led to an inhibition of on-going and evoked neuronal activity. At two hypothalamic sites, both located in the ventral part of the preoptic area, microinjection of DL-homocysteic acid resulted in a complete abolition of the responses to the test stimuli and in a cessation of any on-going activity. Microinjection of DL-homocysteic acid at the remaining five sites had no detectable influences on dorsal horn activity. 4. The results of this study include the first description of input properties of viscero-somatic neurones in the lower thoracic spinal cord of the rat. In addition, these results demonstrate that transmission of visceral and somatic information through these neurones can be modulated by pathways that originate in the anterior hypothalamus-preoptic region of the ventromedial forebrain.