Tomonori Takazawa

Glycinergic and GABAergic tonic inhibition fine tune inhibitory control in regionally distinct subpopulations of dorsal horn neurons.

Inhibition mediated by glycine and GABA in the spinal cord dorsal horn is essential for controlling sensitivity to painful stimuli. Loss of inhibition results in hyperalgesia, a sensitized response to a painful stimulus, and allodynia, a pain‐like response to an innocuous stimulus like touch. The latter is due, in part, to disinhibition of an excitatory polysynaptic pathway linking low threshold touch input to pain projection neurons. This critical impact of disinhibition raises the issue of what regulates the activity of inhibitory interneurons in the dorsal horn under non‐pathological conditions. We have found that inhibitory neurons throughout lamina I–III, identified by the GAD67 promoter‐driven EGFP, are tonically inhibited by glycine or GABA in a regionally distinct way that is mirrored by their inhibitory synaptic input. This tonic inhibition strongly modifies action potential firing properties. Surprisingly, we found that inhibitory neurons at the lamina II/III border are under tonic glycinergic control and receive synapses that are predominantly glycinergic. Futhermore, this tonic glycinergic inhibition remains strong as the mice mature postnatally. Interestingly, GlyT1, the glial glycine transporter, regulates the strength of tonic glycinergic inhibition of these glycine‐dominant neurons. The more dorsal lamina I and IIo inhibitory neurons are mainly under control by tonic GABA action and receive synapses that are predominantly GABAergic. Our work supports the hypothesis that tonic glycine inhibition controls the inhibitory circuitry deep in lamina II that is likely to be responsible for separating low threshold input from high threshold output neurons of lamina I.

Synaptic pathways and inhibitory gates in the spinal cord dorsal horn

Disinhibition in the dorsal horn accompanies peripheral nerve injury and causes the development of hypersensitivity to mild stimuli. This demonstrates the critical importance of inhibition in the dorsal horn for maintaining normal sensory signaling. Here we show that disinhibition induces a novel polysynaptic low‐threshold input onto lamina I output neurons, suggesting that inhibition normally suppresses a preexisting pathway that probably contributes to abnormal pain sensations such as allodynia. In addition, we show that a significant proportion of superficial dorsal horn inhibitory neurons are activated by low‐threshold input. These neurons are well situated to contribute to suppressing low‐threshold activation of pain output neurons in lamina I. We further discuss several aspects of inhibition in the dorsal horn that might contribute to suppressing pathological signaling.

Pre- and postsynaptic inhibitory control in the spinal cord dorsal horn

Sensory information transmitted to the spinal cord dorsal horn is modulated by a complex network of excitatory and inhibitory interneurons. The two main inhibitory transmitters, GABA and glycine, control the flow of sensory information mainly by regulating the excitability of dorsal horn neurons. A presynaptic action of GABA has also been proposed as an important modulatory mechanism of transmitter release from sensory primary afferent terminals. By inhibiting the release of glutamate from primary afferent terminals, activation of presynaptic GABA receptors could play an important role in nociceptive and tactile sensory coding, while changes in their expression or function could be involved in pathological pain conditions, such as allodynia.

Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells

Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.

Exercise-induced cerebral deoxygenation among untrained trekkers at moderate altitudes.

The pathophysiology of altitude-related disorders in untrained trekkers has not been clarified. In the present study, the effects of workload on cardiovascular parameters and regional cerebral oxygenation were studied in untrained trekkers at altitudes of 2700 m and 3700 m above sea level. We studied 6 males and 4 females at each altitude, and their average ages were 31.3+/-7.1 y at 2700 m and 31.2+/-6.8 y at 3700 m, respectively. The resting values of heart rate and mean blood pressure were not significantly different at 2700 m and 3700 m than at sea level. However, increases in these values after exercise were more prominent at high altitudes (heart rate increase = 51.6% at 2700 m and 70.4% at 3700 m; mean blood pressure increase: 19.0% at 2700 m and 17.2% at 3700 m). In addition, post-exercise blood lactate concentration was significantly higher at 3700 m than at sea level or at 2700 m (i.e., 7.6 mM at 3700 m, 3.8 mM at 2700 m, and 4.17 mM at 0 m, respectively). Exercise induced an acute reduction in the arterial oxygen saturation value (SpO2) at 2700 m and 3700 m (i.e., 11.2% reduction at 2700 m and 9.4% at 3700 m), whereas no changes were observed at sea level. The resting values of regional oxygen saturation (rSO2)--measured by a near infra-red spectrophotometer at sea level, 2700 m, and 3700 m-were nearly identical. Exercise at sea level did not reduce this value. In contrast, we observed a decrease in rSO2 after subjects exercised at 2700 m and 3700 m (i.e., 26.9% at 2700 m and 48.1% at 3700 m, respectively). The rSO2 measured 2 min and 3 min after exercise at 3700 m was significantly higher than the preexercise value. From these observations, we concluded that alterations in cardiovascular parameters were apparent only after an exercise load occurred at approximately 3000 m altitude. Acute reduction in cerebral regional oxygen saturation might be a primary cause of headache and acute mountain sickness among unacclimatized trekkers.

Three suspected cases of sugammadex-induced anaphylactic shock

BackgroundSugammadex has a unique mechanism of action and is widely used because of its safety and efficacy. A few recent reports have described allergic reactions to clinical doses of sugammadex. We hereby describe another series of cases of possible anaphylaxis to sugammadex.Case presentationWe present three suspected cases of sugammadex-induced anaphylactic shock, including a 13-year-old boy who underwent laparoscopic appendectomy, a 75-year-old woman who underwent left knee arthroplasty, and a 34-year-old man who underwent left pansinectomy for sinobronchitis. All three patients received general anesthesia with rocuronium and their tracheas were intubated. Shortly after injection of sugammadex for reversal of rocuronium, all of them experienced a decrease in blood pressure along with mucocutaneous erythema. In the most severe case, reintubation after extubation was required due to difficulty in manual ventilation. All patients recovered with anti-allergic therapy. On later investigation, all three patients had a positive skin reaction to sugammadex.ConclusionOur results suggest that physicians using sugammadex should be aware of the possibility of sugammadex-induced anaphylaxis.

Relationship between afterhyperpolarization profiles and the regularity of spontaneous firings in rat medial vestibular nucleus neurons

Our previous in vivo and in vitro whole‐cell patch‐clamp recording studies demonstrated that neurons in the medial vestibular nucleus (MVN) could be characterized on the basis of three electrophysiological properties: afterhyperpolarization (AHP) profile; firing pattern; and response pattern to hyperpolarizing current pulses. In the present study, to clarify which types of the classified MVN neurons correspond to neurons with regular or irregular firing, we investigated their spike discharge patterns using whole‐cell patch‐clamp recording in both in vivo and in vitro preparations. The discharge regularity was related to AHP profiles, and we found that: (i) the coefficient of variation (CV) of interspike intervals during spike discharges was smaller in neurons exhibiting AHP with a slow component [AHP(s+)] than in those without a slow component [AHP(s−)], or with a slow AHP component preceded by afterdepolarization (ADP) [AHP(s+) with ADP]; (ii) the blockade of Ca2+‐dependent K+ channels by 100 nm apamin abolished the slow component and increased the CV in neurons exhibiting AHP(s+); and (iii) the modulation of firing (firing gain) in response to ramp current was larger in neurons exhibiting AHP(s−) than in the other two neuronal types. These results suggest that neurons exhibiting AHP(s+) are regularly discharging neurons with small firing gains to stimulus, neurons exhibiting AHP(s+) with ADP are irregularly discharging neurons with small firing gains, and neurons exhibiting AHP(s−) are irregularly discharging neurons with large firing gains. The regular firing of neurons exhibiting AHP(s+) is attributed to the activation of apamin‐sensitive Ca2+‐dependent K+ channels.

The interaction of noradrenaline with sevoflurane on GABA(A) receptor-mediated inhibitory postsynaptic currents in the rat hippocampus

Little is known about the interaction of noradrenaline with volatile anesthetics in inhibitory synaptic transmission. The purpose of the present study was to investigate the interactions of noradrenaline and sevoflurane on inhibitory synaptic transmission mediated by GABA(A) receptors in the rat hippocampus. Pharmacologically isolated GABA(A) receptor-mediated IPSCs were recorded with whole-cell patch-clamp techniques in pyramidal neurons of the CA1 region of rat hippocampal slices. The actions of noradrenaline, noradrenaline analog, sevoflurane, and the interactions of these agents on the frequency and kinetics of spontaneous GABA(A) receptor-mediated IPSCs were studied. Noradrenaline (10 microM) caused an increase in the frequency of action potential-dependent sIPSCs. These effects were completely reversed by the addition of tetrodotoxin (1 microM), suggesting that noradrenaline produces the discharge of GABAergic interneurons innervating on pyramidal cells via adrenoceptors. Although sevoflurane (0.40 mM, 20 min) slightly depressed the amplitude of sIPSCs, sevoflurane significantly prolonged the decay time constant to 451.1 +/- 89.0% of control (n = 9, P < 0.001) without affecting the rise time. In addition, sevoflurane increased the frequency of sIPSCs up to 3-fold. However, pretreatment of cadmium, multiple Ca channel blocker, abolished sevoflurane effects on the frequency whereas the effects on the decay were still observed. Application of both noradrenaline and sevoflurane produced a significant increase of the IPSC frequency than that of noradrenaline alone or sevoflurane alone with prolonged decays. These results provide evidence that both agents have additive effects on GABAergic synaptic transmission at the central nervous system via different mechanisms.

Sugammadex and rocuronium-induced anaphylaxis

Perioperative anaphylaxis is a life-threatening clinical condition that is typically the result of drugs or substances used for anesthesia or surgery. The most common cause of anaphylaxis during anesthesia is reportedly neuromuscular blocking agents. Of the many muscle relaxants that are clinically available, rocuronium is becoming popular in many countries. Recent studies have demonstrated that succinylcholine (but also rocuronium use) is associated with a relatively high rate of IgE-mediated anaphylaxis compared with other muscle relaxant agents. Sugammadex is widely used for reversal of the effects of steroidal neuromuscular blocking agents, such as rocuronium and vecuronium. Confirmed cases of allergic reactions to clinical doses of sugammadex have also been recently reported. Given these circumstances, the number of cases of hypersensitivity to either sugammadex or rocuronium is likely to increase. Thus, anesthesiologists should be familiar with the epidemiology, mechanisms, and clinical presentations of anaphylaxis induced by these drugs. In this review, we focus on the diagnosis and treatment of anaphylaxis to sugammadex and neuromuscular blocking agents. Moreover, we discuss recent studies in this field, including the diagnostic utility of flow cytometry and improvement of rocuronium-induced anaphylaxis with the use of sugammadex.

Inhibition Mediated by Glycinergic and GABAergic Receptors on Excitatory Neurons in Mouse Superficial Dorsal Horn Is Location-Specific but Modified by Inflammation

The superficial dorsal horn is the synaptic termination site for many peripheral sensory fibers of the somatosensory system. A wide range of sensory modalities are represented by these fibers, including pain, itch, and temperature. Because the involvement of local inhibition in the dorsal horn, specifically that mediated by the inhibitory amino acids GABA and glycine, is so important in signal processing, we investigated regional inhibitory control of excitatory interneurons under control conditions and peripheral inflammation-induced mechanical allodynia. We found that excitatory interneurons and projection neurons in lamina I and IIo are dominantly inhibited by GABA while those in lamina IIi and III are dominantly inhibited by glycine. This was true of identified neuronal subpopulations: neurokinin 1 receptor-expressing (NK1R+) neurons in lamina I were GABA-dominant while protein kinase C gamma-expressing (PKCγ+) neurons at the lamina IIi–III border were glycine-dominant. We found this pattern of synaptic inhibition to be consistent with the distribution of GABAergic and glycinergic neurons identified by immunohistochemistry. Following complete Freunds adjuvant injection into mouse hindpaw, the frequency of spontaneous excitatory synaptic activity increased and inhibitory synaptic activity decreased. Surprisingly, these changes were accompanied by an increase in GABA dominance in lamina IIi. Because this shift in inhibitory dominance was not accompanied by a change in the number of inhibitory synapses or the overall postsynaptic expression of glycine receptor α1 subunits, we propose that the dominance shift is due to glycine receptor modulation and the depressed function of glycine receptors is partially compensated by GABAergic inhibition. SIGNIFICANCE STATEMENT Pain associated with inflammation is a sensation we would all like to minimize. Persistent inflammation leads to cellular and molecular changes in the spinal cord dorsal horn, including diminished inhibition, which may be responsible for enhance excitability. Investigating inhibition in the dorsal horn following peripheral inflammation is essential for development of improved ways to control the associated pain. In this study, we have elucidated regional differences in inhibition of excitatory interneurons in mouse dorsal horn. We have also discovered that the dominating inhibitory neurotransmission within specific regions of dorsal horn switches following peripheral inflammation and the accompanying hypersensitivity to thermal and mechanical stimuli. Our novel findings contribute to a more complete understanding of inflammatory pain.