Fusao Kato

NMDA receptor-independent synaptic plasticity in the central amygdala in the rat model of neuropathic pain

Abstract Neurons in the latero‐capsular part of the central nucleus of the amygdala (CeA), a region now called the “nociceptive amygdala”, receive predominantly nociceptive information from the dorsal horn through afferent pathways relayed at the nucleus parabrachialis (PB). Excitatory synaptic transmission between the PB afferents and these neurons is reported to become potentiated within a few hours of the establishment of arthritic or visceral pain, making it a possible mechanism linking chronic pain and unpleasant negative emotional experiences. However, it remains unknown whether such synaptic potentiation is consolidated or becomes adaptively extinct in the longer‐lasting form of chronic pain, such as neuropathic pain, an as yet serious and frequent type of pain of important clinical concern. To address this issue, we recorded postsynaptic currents in CeA neurons evoked by PB tract stimulation in acute brain slices from young rats with neuropathic pain, as evaluated by tactile allodynic responses, following unilateral spinal nerve ligature made 1 week earlier. CeA neurons contralateral to the nerve ligation showed significantly larger‐amplitude postsynaptic currents than those in the ipsilateral CeA and sham‐ and non‐operated groups. The degree of synaptic potentiation, as compared between two sides, was positively correlated to that of tactile allodynia responses. In addition, blockade of NMDA receptors did not affect this potentiation. We conclude that potentiation of the PB‐CeA synapse is consolidated in long‐lasting neuropathic pain but that this potentiation results from a molecular mechanism distinct from that in arthritic and visceral pain.

Intragastric administration of capsiate, a transient receptor potential channel agonist, triggers thermogenic sympathetic responses

The sympathetic thermoregulatory system controls the magnitude of adaptive thermogenesis in correspondence with the environmental temperature or the state of energy intake and plays a key role in determining the resultant energy storage. However, the nature of the trigger initiating this reflex arc remains to be determined. Here, using capsiate, a digestion-vulnerable capsaicin analog, we examined the involvement of specific activation of transient receptor potential (TRP) channels within the gastrointestinal tract in the thermogenic sympathetic system by measuring the efferent activity of the postganglionic sympathetic nerve innervating brown adipose tissue (BAT) in anesthetized rats. Intragastric administration of capsiate resulted in a time- and dose-dependent increase in integrated BAT sympathetic nerve activity (SNA) over 180 min, which was characterized by an emergence of sporadic high-activity phases composed of low-frequency bursts. This increase in BAT SNA was abolished by blockade of TRP channels as well as of sympathetic ganglionic transmission and was inhibited by ablation of the gastrointestinal vagus nerve. The activation of SNA was delimited to BAT and did not occur in the heart or pancreas. These results point to a neural pathway enabling the selective activation of the central network regulating the BAT SNA in response to a specific stimulation of gastrointestinal TRP channels and offer important implications for understanding the dietary-dependent regulation of energy metabolism and control of obesity.

On-Site Energy Supply at Synapses through Monocarboxylate Transporters Maintains Excitatory Synaptic Transmission

ATP production through oxidative phosphorylation in the mitochondria is the most efficient way to provide energy to various energy-consuming activities of the neurons. These processes require a large amount of ATP molecules to be maintained. Of these, synaptic transmission is most energy consuming. Here we report that lactate transported through monocarboxylate transporters (MCTs) at excitatory synapses constitutively supports synaptic transmission, even under conditions in which a sufficient supply of glucose and intracellular ATP are present. We analyzed the effects of MCT inhibition on neuronal activities using whole-cell recordings in brain slices of rats in the nucleus of the solitary tract. MCT inhibitors (α-cyano-4-hydroxycinnamic acid (4-CIN), phloretin, and d-lactate) significantly decreased the amplitude of EPSCs without reducing release probability. Although 4-CIN significantly reduced currents mediated by heterologously expressed AMPA-Rs in oocytes (a novel finding in this study), the IC50 of the inhibitory effect on EPSC in brain slices was ∼3.8 times smaller than that on AMPA-R currents in oocytes. Removal of intracellular ATP significantly potentiated the inhibition of EPSC with 4-CIN in a manner that was counteracted by intracellular lactate addition. In addition, extracellular lactate rescued aglycemic suppression of EPSC, in a manner that was prevented by 4-CIN. Inhibition of MCTs also reduced NMDA-R-mediated EPSCs and, to a lesser extent, the IPSC. The reduction in EPSC amplitude by γ-d-glutamylglycine was enhanced by 4-CIN, suggesting also a decreased quantal content. We conclude that “on-site” astrocyte-neuron lactate transport to presynaptic and postsynaptic elements is necessary for the integrity of excitatory synaptic transmission.

The lateral parabrachial nucleus is actively involved in the acquisition of fear memory in mice

BackgroundPavlovian fear conditioning is a form of learning accomplished by associating a conditioned stimulus (CS) and an unconditioned stimulus (US). While CS–US associations are generally thought to occur in the amygdala, the pathway mediating US signal processing has only been partially identified. The external part of the pontine lateral parabrachial nucleus (elPB) is well situated for providing US nociceptive information to the central amygdala (CeA), which was recently revealed to play a primary role in fear acquisition. Therefore, we manipulated the elPB activity to examine its role in the regulation of fear learning.ResultsFirst, we transiently inactivate the elPB during the acquisition of fear memory. Mice received bilateral elPB injections of the GABAA agonist muscimol (MUS) or phosphate-buffered saline (drug control), with bilateral misplacement of MUS defined as a placement control group. After the injection, mice were conditioned with a pure tone and foot-shock. On a memory retrieval test on day 2, the freezing ratio was significantly lower in the MUS group compared with that in the drug control or placement control groups. A second retrieval test using a pip tone on day 4 following de novo training on day 3, resulted in significant freezing with no group differences, indicating integrity of fear learning and a temporary limited effect of MUS. Next, we examined whether selectively activating the elPB-CeC pathway is sufficient to induce fear learning when paired with CS. Mice with channelrhodopsin2 (ChR2) expressed in the elPB received a pure tone (CS) in association with optical stimulation in the CeA (CS-LED paired group). On the retrieval test, CS-LED paired mice exhibited significantly higher freezing ratios evoked by CS presentation compared with both control mice receiving optical stimulation immediately after being placed in the shock chamber and exposed to the CS much later (immediate shock group) and those expressing only GFP (GFP control group). These results suggest that selective stimulation of the elPB-CeC pathway substitutes for the US to induce fear learning.ConclusionsThe elPB activity is necessary and sufficient to trigger fear learning, likely as a part of the pathway transmitting aversive signals to the CeA.

Synaptic potentiation in the nociceptive amygdala following fear learning in mice

BackgroundPavlovian fear conditioning is a classical form of associative learning, which depends on associative synaptic plasticity in the amygdala. Recent findings suggest that the central amygdala (CeA) plays an active role in the acquisition of fear learning. However, little is known about the synaptic properties of the CeA in fear learning. The capsular part of the central amygdala (CeC) receives direct nociceptive information from the external part of the lateral parabrachial nucleus (lPB), as well as highly processed polymodal signals from the basolateral nucleus of the amygdala (BLA). Therefore, we focused on CeC as a convergence point for polymodal BLA signals and nociceptive lPB signals, and explored the synaptic regulation of these pathways in fear conditioning.ResultsIn this study, we show that fear conditioning results in synaptic potentiation in both lPB-CeC and BLA-CeC synapses. This potentiation is dependent on associative fear learning, rather than on nociceptive or sensory experience, or fear memory retrieval. The synaptic weight of the lPB-CeC and BLA-CeC pathways is correlated in fear-conditioned mice, suggesting that fear learning may induce activity-dependent heterosynaptic interactions between lPB-CeC and BLA-CeC pathways. This synaptic potentiation is associated with both postsynaptic and presynaptic changes in the lPB-CeC and BLA-CeC synapses.ConclusionsThese results indicate that the CeC may provide an important locus of Pavlovian association, integrating direct nociceptive signals with polymodal sensory signals. In addition to the well-established plasticity of the lateral amygdala, the multi-step nature of this association system contributes to the highly orchestrated tuning of fear learning.

Spatiotemporal and anatomical analyses of P2X receptor-mediated neuronal and glial processing of sensory signals in the rat dorsal horn

Summary Adenosine triphosphate in the dorsal horn acts on neuronal pre‐ and postsynaptic P2X receptors, especially in the deep layer, and astrocytes optimize the excitability, especially in the superficial layer. ABSTRACT Extracellularly released adenosine triphosphate (ATP) modulates sensory signaling in the spinal cord. We analyzed the spatiotemporal profiles of P2X receptor‐mediated neuronal and glial processing of sensory signals and the distribution of P2X receptor subunits in the rat dorsal horn. Voltage imaging of spinal cord slices revealed that extracellularly applied ATP (5–500 μM), which was degraded to adenosine and acting on P1 receptors, inhibited depolarizing signals and that it also enhanced long‐lasting slow depolarization, which was potentiated after ATP was washed out. This post‐ATP rebound potentiation was mediated by P2X receptors and was more prominent in the deep than in the superficial layer. Patch clamp recording of neurons in the superficial layer revealed long‐lasting enhancement of depolarization by ATP through P2X receptors during the slow repolarization phase at a single neuron level. This depolarization pattern was different from that in voltage imaging, which reflects both neuronal and glial activities. By immunohistochemistry, P2X1 and P2X3 subunits were detected in neuropils in the superficial layer. The P2X5 subunit was found in neuronal somata. The P2X6 subunit was widely expressed in neuropils in the whole gray matter except for the dorsal superficial layer. Astrocytes expressed the P2X7 subunit. These findings indicate that extracellular ATP is degraded into adenosine and prevents overexcitation of the sensory system, and that ATP acts on pre‐ and partly on postsynaptic neuronal P2X receptors and enhances synaptic transmission, predominantly in the deep layer. Astrocytes are involved in sensitization of sensory network activity more importantly in the superficial than in the deep layer.

Overlapping memory trace indispensable for linking, but not recalling, individual memories

Unrelated memories get blurred together If one retrieves two memories around the same time, a small number of neurons will become involved in both memories. Yokose et al. investigated the cellular ensemble mechanisms underlying the association between two such memories. In mice, a small population of neurons mediates the association. Memory traces for two independent emotional memories in the brain partially overlapped when the two memories were retrieved synchronously to create a linkage. Suppressing the activity of the overlapping memory trace interrupted the linkage without damaging the original memories. Science, this issue p. 398 In mice, repeated simultaneous reactivation of two initially separated memory traces links them together. Memories are not stored in isolation from other memories but are integrated into associative networks. However, the mechanisms underlying memory association remain elusive. Using two amygdala-dependent behavioral paradigms—conditioned taste aversion (CTA) and auditory-cued fear conditioning (AFC)—in mice, we found that presenting the conditioned stimulus used for the CTA task triggered the conditioned response of the AFC task after natural coreactivation of the memories. This was accompanied through an increase in the overlapping neuronal ensemble in the basolateral amygdala. Silencing of the overlapping ensemble suppressed CTA retrieval-induced freezing. However, retrieval of the original CTA or AFC memory was not affected. A small population of coshared neurons thus mediates the link between memories. They are not necessary for recalling individual memories.

Cellular tagging as a neural network mechanism for behavioural tagging

Behavioural tagging is the transformation of a short-term memory, induced by a weak experience, into a long-term memory (LTM) due to the temporal association with a novel experience. The mechanism by which neuronal ensembles, each carrying a memory engram of one of the experiences, interact to achieve behavioural tagging is unknown. Here we show that retrieval of a LTM formed by behavioural tagging of a weak experience depends on the degree of overlap with the neuronal ensemble corresponding to a novel experience. The numbers of neurons activated by weak training in a novel object recognition (NOR) task and by a novel context exploration (NCE) task, denoted as overlapping neurons, increases in the hippocampal CA1 when behavioural tagging is successfully achieved. Optical silencing of an NCE-related ensemble suppresses NOR–LTM retrieval. Thus, a population of cells recruited by NOR is tagged and then preferentially incorporated into the memory trace for NCE to achieve behavioural tagging.

Codeine presynaptically inhibits the glutamatergic synaptic transmission in the nucleus tractus solitarius of the guinea pig

Although codeine is the most prominent and centrally acting antitussive agent, the precise sites and mode of its action have not been fully understood yet. In the present study, we examined the effects of codeine on synaptic transmission in second-order neurons of the nucleus tractus solitarius (NTS), which is the first central relay site receiving tussigenic afferent fibers, by using whole-cell patch-clamp recordings in guinea-pig brainstem slices. Codeine (0.3-3 mM) significantly decreased the amplitude of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of the tractus solitarius in a naloxone-reversible and concentration-dependent manner, but it had no effect on the decay time of evoked EPSCs (eEPSCs). The inhibition of eEPSCs was accompanied by an increased paired-pulse ratio of two consecutive eEPSCs. The inward current induced by application of AMPA remained unchanged after codeine application. A voltage-sensitive K+ channel blocker, 4-aminopyridine (4-AP) attenuated the inhibitory effect of codeine on eEPSCs. These results suggest that codeine inhibits excitatory transmission from the primary afferent fibers to the second-order NTS neurons through the opioid receptors that activate the 4-AP sensitive K+ channels located at presynaptic terminals.

Facilitation of spontaneous glycine release by anoxia potentiates NMDA receptor current in the hypoglossal motor neurons of the rat

Deficiency in energy supply, such as occurs during hypoxia, anoxia, metabolic stress and mitochondrial failure, strongly affects the excitability of central neurons. Such lowered energy supply evokes various changes in spontaneous synaptic input to the hippocampal and cortical neurons. However, how this energy deprivation affects synaptic input to motor neurons, which are also vulnerable to energy deprivation, has never been addressed. Here we report for the first time the effect of metabolic stress on synaptic input to motor neurons by recording postsynaptic currents in the hypoglossal nucleus. Chemical anoxia with NaCN (1 mm) and anoxia with 95% N2 induced a persistent inward current and a marked and robust increase in action potential‐independent synaptic input. This increase was abolished by strychnine, but not by picrotoxin, CNQX or MK‐801, indicating glycine release facilitation. Blockade of voltage‐dependent Ca2+ channels and extracellular Ca2+ deprivation strongly attenuated this facilitation. The amplitude of inward currents evoked by local application of NMDA to the motor neurons in the presence of strychnine was significantly increased during NaCN application. A saturating concentration of d‐serine occluded this potentiation, suggesting that released glycine activated the glycine‐binding sites of NMDA receptors. By contrast, neurons in the dorsal motor nucleus of the vagus showed no detectable change in synaptic input in response to NaCN. These data suggest that increase in synaptically released glycine in response to metabolic stress may play an exacerbating role in NMDA receptor‐mediated excitotoxicity in motor neurons.