Effects of aconitine on the respiratory activity of brainstem-spinal cord preparations isolated from newborn rats.

Aconitine is a sodium channel opener, but its effects on the respiratory center are not well understood. We investigated the dose-dependent effects of aconitine on central respiratory activity in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 0.5-5 µM aconitine caused an increase in respiratory rhythm and decrease in the inspiratory burst amplitude of the fourth cervical ventral root (C4). Separate application of aconitine revealed that medullary neurons were responsible for the respiratory rhythm increase, and neurons in both the medulla and spinal cord were involved in the decrease of C4 amplitude by aconitine. A local anesthetic, lidocaine (100 µM), or a voltage-dependent sodium channel blocker, tetrodotoxin (0.1 µM), partially antagonized the C4 amplitude decrease by aconitine. Tetrodotoxin treatment tentatively decreased the respiratory rhythm, but lidocaine tended to further increase the rhythm. Treatment with 100 µM riluzole or 100 µM flufenamic acid, which are known to inhibit respiratory pacemaker activity, did not reduce the respiratory rhythm enhanced by aconitine + lidocaine. The application of 1 µM aconitine depolarized the preinspiratory, expiratory, and inspiratory motor neurons. The facilitated burst rhythm of inspiratory neurons after aconitine disappeared in a low Ca2+/high Mg2+ synaptic blockade solution. We showed the dose-dependent effects of aconitine on respiratory activity. The antagonists reversed the depressive effects of aconitine in different manners, possibly due to their actions on different sites of sodium channels. The burst-generating pacemaker properties of neurons may not be involved in the generation of the facilitated rhythm after aconitine treatment.

Effect of cisplatin on respiratory activity in neonatal rats.

One side effect of cisplatin, a cytotoxic platinum anticancer drug, is peripheral neuropathy; however, its central nervous system effects remain unclear. We monitored respiratory nerve activity from the C4 ventral root in brainstem and spinal cord preparations from neonatal rats (P0-3) to investigate its central effects. Bath application of 10-100 µM cisplatin for 15-20 min dose-dependently decreased the respiratory rate and increased the amplitude of C4 inspiratory activity. These effects were not reversed after washout. In separate perfusion experiments, cisplatin application to the medulla decreased the respiratory rate, and application to the spinal cord increased the C4 burst amplitude without changing the burst rate. Application of other platinum drugs, carboplatin or oxaliplatin, induced no change of respiratory activity. A membrane potential analysis of respiratory-related neurons in the rostral medulla showed that firing frequencies of action potentials in the burst phase tended to decrease during cisplatin application. In contrast, in inspiratory spinal motor neurons, cisplatin application increased the peak firing frequency of action potentials during the inspiratory burst phase. The increased burst amplitude and decreased respiratory frequency were partially antagonized by riluzole and picrotoxin, respectively. Taken together, cisplatin inhibited respiratory rhythm via medullary inhibitory system activation and enhanced inspiratory motor nerve activity by changing the firing property of motor neurons.

Optogenetic Approach to Local Neuron Network Analysis of the Medullary Respiratory Center.

Using an optogenetic approach, we analyzed a local neuron network of the respiratory center in the medulla of a brainstem-spinal cord preparation isolated from neonatal rat. We developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch) or one of the step-function channelrhodopsin variants (ChRFR) under the control of Phox2b promoter-enhancer regions. Then, in en bloc preparations from 0- to 2-day-old Tg neonatal rats, we analyzed membrane potential changes of medullary respiratory-related neurons in response to photostimulation of the rostral ventral medulla. The photostimulation-induced inhibition or facilitation of the respiratory rhythm in Arch-expressing or ChRFR-expressing Tg rat preparations, respectively. Selective photoactivation of Phox2b-positive neurons expressing ChRFR in the rostral ventrolateral medulla of a neonatal rat en bloc preparation induced membrane potential changes of respiratory-related neurons that were dependent on heterogeneous properties of synaptic connections in the respiratory center. We concluded that the optogenetic approach is a powerful method of verifying a hypothetical model of local networks among respiratory-related neurons in the rostral ventrolateral medulla of neonatal rat.

Optogenetic analysis of respiratory neuronal networks in the ventral medulla of neonatal rats producing channelrhodopsin in Phox2b-positive cells.

Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem-spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.

Effects of eugenol on respiratory burst generation in newborn rat brainstem-spinal cord preparations.

Eugenol is contained in several plants including clove and is used as an analgesic drug. In the peripheral and central nervous systems, this compound modulates neuronal activity through action on voltage-gated ionic channels and/or transient receptor potential channels. However, it is unknown whether eugenol exerts any effects on the respiratory center neurons in the medulla. We examined the effects of eugenol on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat (P0-P3). The preparations were superfused by artificial cerebrospinal fluid at 25-26 °C, and inspiratory C4 ventral root activity was monitored. Membrane potentials of respiratory neurons were recorded in the parafacial region of the rostral ventrolateral medulla. Bath application of eugenol (0.5-1 mM) decreased respiratory rhythm accompanied by strong inhibition of the burst activity of pre-inspiratory neurons. After washout, respiratory rhythm partly recovered, but the inspiratory burst duration was extremely shortened, and this continued for more than 60 min after washout. The shortening of C4 inspiratory burst by eugenol was not reversed by capsazepine (TRPV1 antagonist) or HC-030031 (TRPA1 antagonist), whereas the depression was partially blocked by GABAA antagonist bicuculline and glycine antagonist strychnine or GABAB antagonist phaclofen. A spike train of action potentials in respiratory neurons induced by depolarizing current pulse was depressed by application of eugenol. Eugenol decreased the negative slope conductance of pre-inspiratory neurons, suggesting blockade of persistent Na+ current. These results suggest that changes in both membrane excitability and synaptic connections are involved in the shortening of respiratory neuron bursts by eugenol.

Erythropoietin, a putative neurotransmitter during hypoxia, is produced in RVLM neurons and activates them in neonatal Wistar rats.

Recent studies indicate that erythropoietin (EPO) is present in many areas of the brain and is active in the restoration of impaired neurons. In this study, we examined the presence of EPO and its role in bulbospinal neurons in the rostral ventrolateral medulla (RVLM). Hypoxia is often accompanied by a high blood pressure (BP). We hypothesized that EPO is produced in response to hypoxia in RVLM neurons and then activates them. To investigate whether RVLM neurons are sensitive to EPO, we examined the changes in the membrane potentials (MPs) of bulbospinal RVLM neurons using the whole cell patch-clamp technique during superfusion with EPO. A brainstem-spinal cord preparation was used for the experiments. EPO depolarized the RVLM neurons, and soluble erythropoietin receptor (SEPOR), an antagonist of EPO, hyperpolarized them. Furthermore, hypoxia-depolarized RVLM neurons were significantly hyperpolarized by SEPOR. In histological examinations, the EPO-depolarized RVLM neurons showed the presence of EPO receptor (EPOR). The RVLM neurons that possessed EPORs showed the presence of EPO and hypoxia-inducible factor (HIF)-2α. We also examined the levels of HIF-2α and EPO messenger RNA (mRNA) in the ventral sites of the medullas (containing RVLM areas) in response to hypoxia. The levels of HIF-2α and EPO mRNA in the hypoxia group were significantly greater than those in the control group. These results suggest that EPO is produced in response to hypoxia in RVLM neurons and causes a high BP via the stimulation of those neurons. EPO may be one of the neurotransmitters produced by RVLM neurons during hypoxia.

Inhibitory effects of eugenol on putative nociceptive response in spinal cord preparation isolated from neonatal rats.

Eugenol is contained in several plants including clove and is thought to exert an analgesic effect. It has been suggested that the slow ventral root potential induced by ipsilateral dorsal root stimulation in the isolated (typically lumbar) spinal cord of newborn rats reflects the nociceptive response, and this in vitro experimental model is useful to assess the actions of analgesics. To further elucidate neuronal mechanisms of eugenol-induced analgesia, we examined the effects of extracellularly applied eugenol on the nociceptive spinal reflex response. To evaluate the effects of eugenol on putative nociceptive responses, the ipsilateral fifth lumbar (L5) dorsal root was stimulated using a glass suction electrode, and the induced reflex responses were recorded from the L5 and twelfth thoracic (Th12) ventral roots in spinal cord preparations (Th10-L5) from newborn rats (postnatal day 0-3). We found that eugenol (0.25-1.0 mM) caused dose-dependent attenuation of the reflex response and also depressed spontaneous ventral root activity. We also found that the slow ventral root potential was further divided into two components: initial and late components. A lower concentration of eugenol selectively depressed the late component. The inhibitory effects by 1.0 mM eugenol were not reversed by 10 µM capsazepine (TRPV1 antagonist) or 40 µM HC-030031 (TRPA1 antagonist). The depressive effect of eugenol on the reflex response was also confirmed by optical recordings using voltage-sensitive dye. Our report provides additional evidence on the basic neuronal mechanisms of eugenol to support its clinical use as a potential analgesic treatment.

Effects of a TRPV1 agonist capsaicin on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats.

The heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channels are expressed in the peripheral and central nervous systems. However, there is no report on how the activation of TRPV1 causes the modulation of neuronal activity in the medullary respiratory center. We examined effects of capsaicin, a specific agonist of TRPV1 channels, on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats. Capsaicin induced a biphasic response in the respiratory rhythm (a transient decrease followed by an increase in the C4 rate). The second-phase excitatory effect (but not the initial inhibitory effect) in the biphasic response was partly blocked by capsazepine or AMG9810 (TRPV1 antagonists). Capsaicin caused strong desensitization. After its washout, the strength of C4 burst inspiratory activity was augmented once per four to five respiratory cycles. The preinspiratory and inspiratory neurons showed tonic firings due to membrane depolarization during the initial inhibitory phase. In the presence of TTX, capsaicin increased the fluctuation of the membrane potential of the CO2-sensitive preinspiratory neurons in the parafacial respiratory group (pFRG), accompanied by slight depolarization. The C4 inspiratory activity did not stop, even 60-90 min after the application of 50/100 µM capsaicin. Voltage-sensitive dye imaging demonstrated that the spatiotemporal pattern of the respiratory rhythm generating networks after application of capsaicin (50 µM, 70-90 min) was highly similar to the control. A histochemical analysis using TRPV1 channel protein antibodies and mRNA demonstrated that the TRPV1 channel-positive cells were widely distributed in the reticular formation of the medulla, including the pFRG. Our results showed that the application of capsaicin in the medulla has various influences on the respiratory center: transient inhibitory and subsequent excitatory effects on the respiratory rhythm and periodical augmentation of the inspiratory burst pattern. The effects of capsaicin were partially blocked by TRPV1 antagonists but could be also induced at least partially via the non-specific action. Our results also suggested a minor contribution of the TRPV1 channels to central chemoreception.

The Effects of Lidocaine on Central Respiratory Neuron Activity and Nociceptive-Related Responses in the Brainstem-Spinal Cord Preparation of the Newborn Rat.

BACKGROUND: Lidocaine is widely used in the clinical setting as a local anesthetic and antiarrhythmic drug. Although it has been suggested that lidocaine exerts inhibitory effects on the central and peripheral neurons, there are no reports on its effects on central respiratory activity in vertebrates. In this study, we examined the effects of lidocaine on respiratory rhythm generation and nociceptive response in brainstem-spinal cord preparations from the newborn rats. METHODS: Preparations were isolated from Wistar rats (postnatal day 0-3) and superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25°C to 26°C. We examined the effects of lidocaine on the fourth cervical ventral root (C4)-inspiratory activity and on the preinspiratory and inspiratory neurons in the rostral medulla. We also examined the effects on the C4/C5 reflex responses induced by ipsilateral C7/C8 dorsal root stimulation, which are thought to be related to the nociceptive response. RESULTS: The application of low doses of lidocaine (10-20 µM) resulted in a slight increase of the C4 burst rate, whereas high doses of lidocaine (100-400 µM) decreased the burst rate in a dose-dependent manner, eventually resulting in the complete cessation of respiratory rhythm. High doses of lidocaine decreased the burst duration and negative slope conductance of preinspiratory neurons, suggesting that lidocaine blocked persistent Na+ current. After the burst generation of the respiratory neurons ceased, depolarizing current stimulation continued to induce action potentials; however, the induction of the spike train was depressed because of strong adaptation. A low dose of lidocaine (20 µM) depressed C4/C5 spinal reflex responses. CONCLUSIONS: Our findings indicate that lidocaine depressed nociception-related responses at lower concentrations than those that induced respiratory depression. Our report provides the basic neuronal mechanisms to support the clinical use of lidocaine, which shows antinociceptive effects with minimal side effects on breathing.

Midline section of the medulla abolishes inspiratory activity and desynchronizes pre-inspiratory neuron rhythm on both sides of the medulla in newborn rats.

Each half of the medulla contains respiratory neurons that constitute two generators that control respiratory rhythm. One generator consists of the inspiratory neurons in the pre-Bötzinger complex (preBötC); the other, the pre-inspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG), rostral to the preBötC. We investigated the contribution of the commissural fibers, connecting the respiratory rhythm generators located on the opposite side of the medulla to the generation of respiratory activity in brain stem-spinal cord preparation from 0- to 1-day-old rats. Pre-I neuron activity and the facial nerve and/or first lumbar (L1) root activity were recorded as indicators of the pFRG-driven rhythm. Fourth cervical ventral root (C4) root and/or hypoglossal (XII) nerve activity were recorded as indicators of preBötC-driven inspiratory activity. We found that a midline section that interrupted crossed fibers rostral to the obex irreversibly eliminated C4 and XII root activity, whereas the Pre-I neurons, facial nerve, and L1 roots remained rhythmically active. The facial and contralateral L1 nerve activities were synchronous, whereas right and left facial (and right and left L1) nerves lost synchrony. Optical recordings demonstrated that pFRG-driven burst activity was preserved after a midline section, whereas the preBötC neurons were no longer rhythmic. We conclude that in newborn rats, crossed excitatory interactions (via commissural fibers) are necessary for the generation of inspiratory bursts but not for the generation of rhythmic Pre-I neuron activity.

Long-lasting facilitation of respiratory rhythm by treatment with TRPA1 agonist, cinnamaldehyde.

The transient receptor potential (TRP) channels are widely distributed in the central nervous system (CNS) and peripheral nervous system. We examined the effects of TRP ankyrin 1 (TRPA1) agonists (cinnamaldehyde and allyl isothiocyanate) on respiratory rhythm generation in brainstem-spinal cord preparations from newborn rats [postnatal days 0-3 (P0-P3)] and in in situ-perfused preparations from juvenile rats (P11-P13). Preparations were superfused with modified Krebs solution at 25-26°C, and activity of inspiratory C4 ventral root (or phrenic nerve) was monitored. In the newborn rat, an in vitro preparation of cinnamaldehyde (0.5 mM) induced typically biphasic responses in C4 rate: an initial short increase and subsequent decrease, then a gradual recovery of rhythm during 15 min of bath application. After washout, the respiratory rhythm rate further increased, remaining 200% of control for >120 min, indicating long-lasting facilitation. Allyl isothiocyanate induced effects similar to those of cinnamaldehyde. The long-lasting facilitation of respiratory rhythm was partially antagonized by the TRPA1 antagonist HC-030031 (10 µM). We obtained similar long-lasting facilitation in an in situ-perfused reparation from P11-P13 rats. On the basis of results from transection experiments of the rostral medulla and whole-cell recordings from preinspiratory neurons in the parafacial respiratory group (pFRG), we suggest that the rostral medulla, including the pFRG, is important to the induction of long-lasting facilitation. A histochemical analysis demonstrated a wide distribution of TRPA1 channel-positive cells in the reticular formation of the medulla, including the pFRG. Our findings suggest that TRPA1 channel activation could induce long-lasting facilitation of respiratory rhythm and provide grounds for future study on the roles of TRPA1 channels in the CNS.

Effects of linalool on respiratory neuron activity in the brainstem-spinal cord preparation from newborn rats.

Linalool and linalyl acetate are major components of lavender essential oil. These substances possess many biological activities, such as anti-inflammatory activity, analgesic and anxiolytic effects, and anticonvulsant properties, and they also induce modulation of neuronal activity in the autonomic nervous system. However, there are no reports of the direct effects of linalool on respiratory activity. In the present study, we analyzed the effects of linalool and linalyl acetate on central respiratory activity in the brainstem-spinal cord preparation isolated from newborn rats. Linalool dose-dependently decreased the rate of respiratory activity. This effect was reversed by bicuculline, suggesting that linalool enhanced inhibitory synaptic connections via GABAA receptors. In addition, linalool reduced the coefficient of variation of inspiratory burst intervals and thus could work to stabilize the respiratory rhythm. Linalyl acetate did not cause inhibitory effects as observed in linalool treatment. Linalool depressed burst activity of pre-inspiratory neurons in the medullary respiratory networks and increased the amplitude of inspiratory inhibitory postsynaptic potentials of pre-inspiratory neurons. We concluded that linalool caused inhibitory effects on respiratory rhythm generation mainly through activation of presynaptic GABAA receptors of pre-inspiratory neurons.

SGLT2 and SGLT1 inhibitors suppress the activities of the RVLM neurons in newborn Wistar rats.

Hypertension is well-known to often coexist with diabetes mellitus (DM) in humans. Treatment with sodium-glucose cotransporter 2 (SGLT2) inhibitors has been shown to decrease both the blood glucose and the blood pressure (BP) in such patients. Some reports show that SGLT2 inhibitors improve the BP by decreasing the activities of the sympathetic nervous system. Therefore, we hypothesized that SGLT2 inhibitors might alleviate hypertension via attenuating sympathetic nervous activity. Combined SGLT2/SGLT1 inhibitor therapy is also reported as being rather effective for decreasing the BP. In this study, we examined the effects of SGLT2 and SGLT1 inhibitors on the bulbospinal neurons of the rostral ventrolateral medulla (RVLM). To investigate whether bulbospinal RVLM neurons are sensitive to SGLT2 and SGLT1 inhibitors, we examined the changes in the neuronal membrane potentials (MPs) of these neurons using the whole-cell patch-clamp technique during superfusion of the cells with the SGLT2 and SGLT1 inhibitors. A brainstem-spinal cord preparation was used for the experiments. Our results showed that superfusion of the RVLM neurons with SGLT2 and SGLT1 inhibitor solutions induced hyperpolarization of the neurons. Histological examination revealed the presence of SGLT2s and SGLT1s in the RVLM neurons, and also colocalization of SGLT2s with SGLT1s. These results suggest the involvement of SGLT2s and SGLT1s in regulating the activities of the RVLM neurons, so that SGLT2 and SGLT1 inhibitors may inactivate the RVLM neurons hyperpolarized by empagliflozin. SGLT2 and SGLT1 inhibitors suppressed the activities of the bulbospinal RVLM neurons in the brainstem-spinal preparations, suggesting the possibilities of lowering BP by decreasing the sympathetic nerve activities. RVLM, rostral ventrolateral medulla. IML, intralateral cell column. aCSF, artificial cerebrospinal fluid.

Impaired response to hypoxia in the respiratory center is a major cause of neonatal death of the PACAP-knockout mouse.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide expressed widely in nervous tissues. PACAP-knockout ((-/-)) mice display a sudden infant death syndrome (SIDS)-like phenotype, although the underlying physiological mechanism to explain this remains unclear. Here, we report on the presence of abnormal respiratory activity in PACAP(-/-) mice under hypoxic conditions, which provides a basis for the SIDS-like phenotype. PACAP(-/-) mice display a lowered baseline respiratory activity compared with wild-type animals, and an abnormal response to hypoxia. More specifically, PACAP(-/-) mice at postnatal day 7 showed respiratory arrest in response to hypoxia. In contrast, their response to hypercapnic conditions was the same as that of wild-type mice. Histological and real-time PCR analyses indicated that the catecholaminergic system in the medulla oblongata was impaired in PACAP(-/-) mice, suggesting that endogenous PACAP affects respiratory centers in the medulla oblongata via its action on the catecholaminergic system. We propose that disruption of this system is involved in the SIDS-like phenotype of PACAP(-/-) mice. Thus, disorders of the catecholaminergic system involved with O(2) sensing could be implicated in underlying neuronal mechanisms responsible for SIDS.

Involvement of cannabinoid receptors in depression of the putative nociceptive response in spinal cord preparations isolated from neonatal rats.

A metabolite of acetaminophen, AM404, which is an anandamide transporter inhibitor, induces analgesia mainly via activation of transient receptor potential channel 1 in the spinal cord, although the role of cannabinoid receptors remains to be studied. The ventral root reflex response induced by stimulation of the dorsal root in in vitro preparations of rat spinal cord is useful to assess the effect of analgesics. We analyzed the effects of AM404 and cannabinoid receptor antagonist AM251 on reflex responses in lumbar spinal cord preparations from newborn rats and found that the amplitude of the slow ventral root potential after administration of 10 µM AM404 was not significantly changed, whereas 10 µM AM251 significantly increased the amplitude. Administration of the cannabinoid receptor 1 agonist WIN55,212-2 (10 µM) did not significantly affect the reflex response. We suggest that endogenous cannabinoids in the spinal cord are involved in the antinociceptive mechanism through suppressive effects.

Cannabinoid receptors involved in descending inhibition on spinal seizure-like activity in the phrenic output.

Seizure-like burst activities are induced by blockade of GABAA and/or glycine receptors in various spinal ventral roots of brainstem-spinal cord preparation from neonatal rodents. We found that this is not applicable to the phrenic nerve and that a new inhibitory descending pathway may suppress seizure-like activity in the phrenic nerve. Experiments were performed in brainstem-spinal cord preparation from newborn rats (age: 0-1 day). Left phrenic nerve and right C4 activities were recorded simultaneously. When GABAA and glycine receptors were blocked by 10 µM bicuculline and 10 µM strychnine (Bic+Str), seizure-like burst activities appeared in the fourth cervical ventral root (C4) but not the phrenic nerve. After making a transverse section at C1, the inspiratory burst activity disappeared from both C4 and the phrenic nerve, whereas seizure-like activity appeared in both nerves. We hypothesized that inhibitory descending pathways other than those via GABAA and/or glycine receptors (from the medulla to the spinal cord) work to avoid disturbance of regular respiratory-related diaphragm contraction by seizure-like activity. We found that cannabinoid receptor antagonist, AM251 was effective for the induction of seizure-like activity by Bic+Str in the phrenic nerve in brainstem-spinal cord preparation. Cannabinoid receptors may be involved in this descending inhibitory system.

Cell Responses of the Ventrolateral Medulla to PAR1 Activation and Changes in Respiratory Rhythm in Newborn Rat En Bloc Brainstem-Spinal Cord Preparations.

Proteinase-activated receptor-1 (PAR1) is expressed in astrocytes of various brain regions, and its activation is involved in the modulation of neuronal activity. Here, we report effects of PAR1 selective agonist TFLLR on respiratory rhythm generation in brainstem-spinal cord preparations. Preparations were isolated from newborn rats (P0-P4) under deep isoflurane anesthesia and were transversely cut at the rostral medulla. Preparations were superfused with artificial cerebrospinal fluid (25-26 °C), and inspiratory C4 ventral root activity was monitored. The responses to TFLLR of cells close to the cut surface were detected by calcium imaging or membrane potential recordings. Application of 10 µM TFLLR (4 min) induced a rapid and transient increase of calcium signal in cells of the ventrolateral respiratory regions of the medulla. More than 88% of responding cells (223/254 cells from 13 preparations) were also activated by low (0.2 mM) K+ solution, suggesting that they were astrocytes. Immunohistochemical examination demonstrated that PAR1 was expressed on many astrocytes. Respiratory-related neurons in the medulla were transiently hyperpolarized (-1.8 mV) during 10 µM TFLLR application, followed by weak membrane depolarization after washout. C4 burst rate decreased transiently in response to application of TFLLR, followed by a slight increase. The inhibitory effect was partially blocked by 50 µM theophylline. In conclusion, activation of astrocytes via PAR1 resulted in a decrease of inspiratory C4 burst rate in association with transient hyperpolarization of respiratory-related neurons. After washout, slow and weak excitatory responses appeared. Adenosine may be partially involved in the inhibitory effect of PAR1 activation.

Postsynaptic mechanisms of CO(2) responses in parafacial respiratory neurons of newborn rats.

The parafacial respiratory group (pFRG) in the rostral ventrolateral medulla of the newborn rat is predominantly composed of pre-inspiratory (Pre-I) neurons and is involved in respiratory rhythm generation. The subgroup located close to the ventral surface (at least partially overlapping the retrotrapezoid nucleus, RTN) expresses the Phox2b transcription factor and responds to hypercapnic stimulation with strong depolarization, which suggests it has a role in central chemoreception. Although a CO(2) response of pFRG/RTN neurons has been confirmed in the presence of tetrodotoxin (TTX), it is unknown whether the depolarization involved in this response is induced by a direct postsynaptic response of pFRG/RTN neurons or by any presynaptic components mediated by Ca(2+)-dependent mechanisms. In this study, we examined the effects of ATP or substance P receptor antagonists on hypercapnic responses of rostral pFRG/RTN neurons. We tested effects of Cd(2+) and low Ca(2+)-high Mg(2+) in the presence of TTX. The experiments were performed in in vitro brainstem­spinal cord preparations from newborn rats in which Pre-I neurons reflect the discharge pattern of the pFRG. We found that ATP receptor and substance P receptor antagonists do not block membrane potential responses to hypercapnic stimulation (2%→8%) of pFRG/RTN neurons in the rostral parafacial region.Moreover, rostral pFRG/RTN neurons were depolarized by hypercapnia under conditions where the contribution of presynaptic components was inhibited in the presence of TTX and Cd(2+) or in a low Ca(2+)-high Mg(2+) solution containing TTX and Cd(2+). All cases (except some cases in a low Ca(2+)-high Mg(2+) solution) of membrane depolarization by hypercapnic stimulation were accompanied with an increase in input resistance. These neurons were predominantly Phox2b immunoreactive. Our findings suggest that the response of pFRG/RTN neurons to hypercapnia is induced by direct action on the postsynaptic membrane via closing of K(+) channels.

Glycine and GABAA receptors suppressively regulate the inspiratory-related calcium rise in the thoracic inspiratory cells of the neonatal rat.

We previously demonstrated that in an isolated brainstem-spinal cord preparation from neonatal rats, a local bath application of strychnine (a broad antagonist of glycine and GABAA receptors) to the spinal cord enhances thoracic inspiratory motor activity. Herein, to investigate the involvement of the inspiratory spinal interneurons that provide excitatory input to the motoneuron, we conducted calcium imaging using this preparation. Oregon Green 488 BAPTA-1 AM, a fluorescent calcium indicator, was injected into the ventromedial surface of the thoracic cord. In all cells that showed inspiratory-related fluorescence changes > 2% of the baseline fluorescence intensity, the inspiratory-related fluorescence change decreased when the focal depth was deepened. The application of strychnine to the spinal cord increased the inspiratory-related intracellular calcium rise in these cells. These results suggest that the enhancement of inspiratory interneuron activity could be involved in this enhancement of inspiratory motor activity.