Masaharu Mizuno

Direct GABAergic and Glycinergic Inhibition of the Substantia Gelatinosa from the Rostral Ventromedial Medulla Revealed by In Vivo Patch-Clamp Analysis in Rats

Stimulation of the rostral ventromedial medulla (RVM) is believed to exert analgesic effects through the activation of the serotonergic system descending to the spinal dorsal horn; however, how nociceptive transmission is modulated by the descending system has not been fully clarified. To investigate the inhibitory mechanisms affected by the RVM, an in vivo patch-clamp technique was used to record IPSCs from the substantia gelatinosa (SG) of the spinal cord evoked by chemical (glutamate injection) and electrical stimulation (ES) of the RVM in adult rats. In the voltage-clamp mode, the RVM glutamate injection and RVM-ES produced an increase in both the frequency and amplitude of IPSCs in SG neurons that was not blocked by glutamate receptor antagonists. Serotonin receptor antagonists were unexpectedly without effect, but a GABAA receptor antagonist, bicuculline, or a glycine receptor antagonist, strychnine, completely suppressed the RVM stimulation-induced increase in IPSCs. The RVM-ES-evoked IPSCs showed fixed latency and no failure at 20 Hz stimuli with a conduction velocity of >3 m/s (3.1–20.7 m/s), suggesting descending monosynaptic GABAergic and/or glycinergic inputs from the RVM to the SG through myelinated fibers. In the current-clamp mode, action potentials elicited by noxious mechanical stimuli applied to the receptive field of the ipsilateral hindlimb were suppressed by the RVM-ES in more than half of the neurons tested (63%; 10 of 16). These findings suggest that the RVM-mediated antinociceptive effects on noxious inputs to the SG may be exerted preferentially by the direct GABAergic and glycinergic pathways to the SG.

Slow oscillation of membrane currents mediated by glutamatergic inputs of rat somatosensory cortical neurons: In vivo patch-clamp analysis

Using in vivo patch‐clamp technique, the slow oscillation of membrane currents was characterized by its synaptic nature, correlation with electroencephalogram (EEG) and responses to different anesthetic agents, in primary somatosensory cortex (SI) neurons in urethane‐anesthetized rats. In more than 90% of the SI neurons, the slow oscillation of the inward currents (0.1–2.5 Hz) with the duration of several hundreds of a millisecond was observed at the holding membrane potential of −70 mV. The reversal potential of the inward currents was approximately 0 mV and was suppressed by application of an α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) receptor antagonist. In most cases (> 90%) the inward current was synchronized with positive wave of the surface EEG recorded from ipsilateral and even contralateral cortical regions. The frequency as well as duration of the slow oscillation decreased by a volatile anesthetic agent, isoflurane (1.5–5.0%), and excitatory postsynaptic currents (EPSCs) were almost abolished at the highest concentration. Intraperitoneal injection of pentobarbital (25 mg/kg) also decreased the frequency of the slow oscillation without affecting short EPSCs. When γ‐aminobutyric acid A (GABAA) receptors were activated by local microinjection of muscimol (3 × 10−3 m, 1–10 µL) into the thalamus, the frequency of the slow oscillation markedly decreased, but was not abolished completely. These findings suggest that the slow oscillation of the inward currents is generated by the summation of glutamatergic EPSCs, and affected by isoflurane and pentobarbital differently. In addition, GABAergic system in the thalamus can affect the frequency, but is not essentially implicated in the genesis of the slow oscillation.

Intracellular Na+ and K+ shifts induced by contractile activities of rat skeletal muscles.

The effects of direct and indirect electrical stimulation on intracellular potassium and sodium contents ([K]i and [Na]i, respectively) in rat soleus muscle (SOL) and extensor digitorum longus muscle (EDL) were investigated under in vivo conditions. The changes of [K]i and [Na]i contents in both muscles which were stimulated indirectly reached respective values at 30 min or 1 hr after the beginning of stimulation, whereas those of EDL stimulated with 60 Hz changed gradually through 2 hr stimulation. The shifts of [K]i and [Na]i in EDL occurred during the twitch contraction at considerably lower frequency stimulation (0.5-10 Hz), whereas those in SOL were observed during the tetanus contraction at high frequency stimulation (10-40 Hz). The difference of change in cationic shifts between EDL and SOL under low frequency stimulation was reduced by ouabain treatment, though the difference was still significant. When the muscles were indirectly stimulated 6000 times at 1, 5, 10 and 20 Hz, the cationic shifts in EDL were greater than those in SOL, extending over all frequencies. It was concluded that such a difference in ionic shift between contracting EDL and SOL may be primarily due to the difference in unidirectional ionic fluxes per stimulation and, secondly, to the difference in Na(+)-K+ pump activity.

Separate Inhibitory and Excitatory Components Underlying Receptive Field Organization in Superficial Medullary Dorsal Horn Neurons

Extracellular recording has shown that dorsal horn neurons can have an inhibitory surround outside their excitatory receptive field, but cannot reveal inhibitory inputs within the excitatory field, or show the underlying excitatory and inhibitory synaptic inputs that determine net output. To study the underlying components of receptive field organization, in vivo patch-clamp recording was used to compare the size and distribution of subthreshold, suprathreshold, and inhibitory fields, in neurons in the mouse superficial medullary dorsal horn that were characterized by their responses to noxious and innocuous mechanical facial stimulation. Subthreshold excitatory fields typically extended some distance beyond the borders of the suprathreshold field, and also commonly exhibited broader stimulus selectivity, in that the majority of nociceptive-specific neurons exhibited subthreshold responses to brush. Separate voltage-clamp recording of excitatory and inhibitory inputs using different holding potentials revealed that inhibition could be evoked from both within and outside the excitatory field. In nociceptive neurons, inhibition tended to be maximal at the excitatory receptive field center, and was usually greater for pinch than brush, although the selectivity for pinch versus brush was not as great as with excitatory responses. Based on current data on dorsal horn organization, we propose that the localized peak of inhibition at the excitatory field center could be mediated by local interneurons, while the more widespread surrounding inhibition may depend on supraspinal circuitry.

Categorization of biologically significant objects, food and gender, in rhesus monkeys: I. Behavioral study

Macaque monkeys have a highly evolved visual system comparable to that of humans. One of the important visual functions is performing discriminations among biologically significant objects such as food or heterosexual partners. In the present study, we examined whether rhesus monkeys could categorize two-dimensional images related to food or gender using a visual discrimination task. Three rhesus monkeys were trained to make distinctions of food from non-food items, and between male and female monkeys, using 60 or more different pictures of each category. After more than 9 months of training, the monkeys discriminated a variety of foods from non-food and different males from females with more than 80% accuracy, even when the stimuli were used for the first time or presented only once in a session. The proportion of correct responses and response latencies showed better performance in discrimination of food/non-food than that of gender. The results suggest that rhesus monkeys are able to perform visual discrimination of highly abstract biologically significant categories with better performance in a food-related category than a gender-related one, using two-dimensional visual information.

Three‐dimensional organization of local excitatory and inhibitory inputs to neurons in laminae III–IV of the spinal dorsal horn

•  Axons of sensory neurons that detect painful and non‐painful stimulation of body tissues project centrally to the dorsal horn of the spinal cord, where they are partially segregated in the superficial and deep laminae, respectively. •  Interneuronal connections between superficial and deep laminae could potentially modulate sensory transmission and contribute to alterations that occur under conditions of pain hypersensitivity. •  This study used a localized stimulation technique (laser scanning photostimulation) for high‐resolution mapping of local interneuronal synaptic connections to laminae III–IV neurons, combined with intracellular staining for morphological analysis, in an in vitro‘slice’ preparation of the rat lumbar spinal cord. •  Synaptic input from superficial laminae (I–II) was received by laminae III–IV neurons with long dorsal dendrites, supporting the idea that interlaminar connectivity is mediated via translaminar dendritic extensions and, more generally, that local connectivity is governed by rules that are specific to the laminar position and morphology of the postsynaptic neuron.

Application of in vivo patch-clamp technique to pharmacological analysis of synaptic transmission in the CNS

Slice preparations as well as acutely dissociated and cultured neurons from various regions in the CNS have been widely used to analyze pharmacological properties of synaptic responses and receptors expressed at the pre- and post-synaptic sites. However, the essential properties are not obviously different from neuron to neuron. These characteristics of neurons in the CNS make it difficult to elucidate their functional significances. It is, therefore, preferable that the pharmacological analysis should be made from identified neurons by stimulation of identified inputs. The in vivo patch-clamp recording technique allows us to clarify the synaptic responses evoked by the various known natural stimuli applied to the skin or other parts and makes it possible to interpret with more certainty the behavioral changes by synaptic plasticity observed at the single cell level. Although the in vivo technique has obvious advantages in analysis of physiological responses, this method is, however, confined so far to neurons located at the near surface of the CNS for pharmacological analysis, because of the diffusion problem of the chemical to deeper neurons. Thus, combinatorial studies with dissociated or cultured neurons or with slice preparations are clearly required for further understanding of pharmacological properties of neurons in the CNS.

The effect of B-HT 920, a dopamine D2 agonist, on bar-press feeding in the monkey

Although the dopamine (DA) system has been shown to regulate food intake, the function of the DA receptor subtypes on behavior still remains to be elucidated. In the present study, we examined the effect of B-HT 920, a selective agonist of DA D2 receptors that preferentially affect presynaptic autoreceptors, on both food consumption and execution of a high fixed-ratio bar-press task for food reward in monkeys. Two kinds of bar-press task were used: 1) a cue-triggered bar-press task during the first 40 trials, and 2) a self-paced bar-press task in which the monkeys freely performed bar-press trials until they were satiated. A SC injection of B-HT 920 (25 micrograms/kg) increased food consumption in the home cage. The same facilitatory effect on food consumption was also observed in the operant task condition. During the cue-triggered bar-press task, however, both the latency of the bar-press responses to a cue light and the time required to complete the bar-press trials were prolonged after the injection of B-HT 920. The results suggest that the activation of D2 autoreceptors suppresses the operant food acquisition behavior and increases food consumption through an inhibition of the satiety mechanism rather than an activation of any hunger-related drive.

In vivo patch clamp analysis of synaptic responses evoked in primary somatosensory cortex in inflamed rats

Previous studies on frequency modulation (FM) reported that the primary auditory cortex (A1) neurons in anesthetized cats transiently responded to instantaneous FM-frequency (Fi) only at the boundary of cell’s tonal frequency-receptive-field (FRF), and preferred fast FM-speeds. In the present study on awake cats, we found a group of A1 neurons that show sustained FM-responses and prefer slow FM-speeds. These cells showed sustained responses to pure tone stimuli. When the sweep-speed of FM stimuli slowly traversed a cell’s FRF, the cell continuously responded to the FM tone until it passed FRF. The peak response was triggered by Fi corresponding to the frequency, which evoked the best response in pure tone stimuli. Cells preferring upward FM direction tended to have dominant high frequency inhibitory-FRF (defined by two tone stimuli), suggesting contribution of inhibitory-FRF to the direction selectivity. Thus, the sustained response cells may play a role in continuously encoding FM-sweep trajectories of cat’s vocalizations.

156 Modification of bursting activities in somatosensory cortex in inflamed-rats in vivo

Aim. The aim was to differentiate the effects of sucrose loading on thermal pain sensitivity in diabetic and non-diabetic rats. Materials and methods. Five months of age male diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, and their age-matched non-diabetic genetic controls Long Evans Tokusima Otsuka (LETO) rats, were used. The animals were fed with standard laboratory chow and divided into the following four groups: Group O-sucrose (n = 20) – OLETF rats were loaded for 2 months with 30% sucrose in the drinking water (sucrose period). Then the rats were put again on pure tap water (washout period) until the end of the experiment. Group O-control (n = 9) – on tap water in the course of the whole experiment. Group L-sucrose (n = 16) and group L-control (n = 9) – age-matched LETO rats had the same schedule like the groups O-sucrose and O-control, respectively. Sensitivity to noxious thermal stimuli was assessed by the latency time during tail-flick test. The beam was focused on the ventral surface, 5 cm from the tip of the tail. Results. At baseline, at the end of the sucrose period, and 2 moths later (during the washout period), respectively, the groups presented with following latencies (means ± SD): O-sucrose = 6.9 ± 1.4; 7.5 ± 0.9 and 8.4 ± 0.9 (p < 0.5 vs.baseline and vs. O-control); O-control = 7.6 ± 0.6; 7.1 ± 0.7 and 7.2 ± 1.0 (p < 0.5 vs. Lcontrol); L-sucrose = 7.6 ± 1.0; 6.2 ± 1.0 and 5.8 ± 1.4 (p < 0.5 vs. baseline); L-control = 8.4 ± 1.4; 6.0 ± 1.4 and 6.0 ± 0.8 (p < 0.5 vs. baseline). Conclusion. Sucrose loading decreases pain sensitivity, measured with tail-flick test in diabetic, but not in non-diabetic rats.