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.

Effects of riluzole on spinal seizure-like activity in the brainstem-spinal cord preparation of newborn rat.

Riluzole blocks persistent Na+ current, inhibits generation of neuronal bursts and decreases glutamate-induced excitotoxicity. In previous studies of respiratory activity, riluzole suppressed inspiratory-related burst generation activity in rat slice or en bloc preparations. We examined riluzole's effects on inspiratory burst generation and drug-induced seizure-like activity in newborn rat en bloc preparations. Medulla-spinal cord preparations from postnatal day 0-3 Wistar rats were isolated under deep isoflurane anesthesia and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25-26°C. Inspiratory activity was monitored from the fourth cervical ventral root. Seizure-like activity was induced by application of 20µM DL-threo-ß-benzyloxyasparatate (TBOA, a glutamate uptake blocker preferentially acting on astrocytes) or coadministration of GABAA antagonist bicuculline (10µM) and glycine antagonist strychnine (10µM). Pretreatment and co-application with 10µM riluzole abolished the seizure-like burst activity induced by TBOA or bicuculline/strychnine. N-methyl-d-aspartic acid receptor antagonist MK801 (10µM) also depressed this activity. Riluzole may attenuate excessive glutamate action involved in pathological hyperexcitability of motor neurons with no major effect on generation of respiratory activity. Riluzole at the optimal dose could be a potential treatment to protect drug-induced epileptic brain tissue from excitotoxic damage without inducing respiratory suppression.

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.

Effects of riluzole on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat.

The persistent sodium channel is an important pacemaker component in rhythm generation. In the present study, we examined the effects of a persistent sodium channel blocker, riluzole on pre-inspiratory (Pre-I) and inspiratory neurons in the rostral medulla as well as on 4th cervical ventral root (C4)-inspiratory activity in brainstem-spinal cord preparations. Preparations were isolated from postnatal day 0-3 Wistar rats and were superfused with artificial cerebrospinal fluid, equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25-26 °C. The C4 inspiratory burst rate decreased in a dose-dependent manner (50-200 µM) after 15 min application of riluzole. Riluzole caused a strong reduction in the drive potential of Pre-I neurons but not of inspiratory neurons. After washout, C4 inspiratory burst gradually changed into an episodic pattern, in which one burst consisted of 3-9 short separate bursts. Riluzole also depressed the induction of repetitive firing induced by depolarizing stimulation. Under voltage clamp conditions, riluzole suppressed the negative-slope component of Pre-I neurons. Riluzole also depressed the intrinsic burst generation of Pre-I neurons in low calcium and high magnesium solution. Our findings indicate that the burst generation of Pre-I neurons is more sensitive than inspiratory burst generation to riluzole and thus suggested that persistent sodium channels have an important role in the burst generation of Pre-I neurons and are involved in the primary respiratory rhythm generation.

Layer-by-layer assembled titania tubular nanostructures at different assembly conditions.

Layer-by-layer (LbL) deposition of poly(L-lysine) (PLL) dissolved in different solutions and a water-soluble titania precursor, titanium(IV) bis(ammonium lactate) dihydroxide (TiBALDH) to form multilayer films on the wall of polycarbonate (PC) membrane pores was performed to prepare nanostructured titania-PLL composite and pure anatase and rutile titania tubes. A battery of analytical techniques was utilized to characterize and compare the structures, crystal phases, and photocatalytic properties of the titania tubes. In different solutions conditions, PLL which adopts secondary conformations (i.e., alpha-helix and random coil) and has varying interactions with different counterions (i.e., chloride and phosphate ions) can influence PLL/TiBALDH deposition and, in turn, results in the titania materials with different nanostructures and phtocatalytic properties. The influence of LbL assembly condition, deposition cycle, and polypeptide molecular weight on photocatalytic properties of resultant anatase titania tubes were further explored and these materials are promising photocatalyst with the advantage of easily handling and recycling. This reported approach may provide a facile and general way to prepare organic-inorganic composite and other inorganic materials with different compositions, structures, and properties for various applications.