Chemogenetic Manipulation of Amygdala Kappa Opioid Receptor Neurons Modulates Amygdala Neuronal Activity and Neuropathic Pain Behaviors.

Neuroplasticity in the central nucleus of the amygdala (CeA) plays a key role in the modulation of pain and its aversive component. The dynorphin/kappa opioid receptor (KOR) system in the amygdala is critical for averse-affective behaviors in pain conditions, but its mechanisms are not well understood. Here, we used chemogenetic manipulations of amygdala KOR-expressing neurons to analyze the behavioral consequences in a chronic neuropathic pain model. For the chemogenetic inhibition or activation of KOR neurons in the CeA, a Cre-inducible viral vector encoding Gi-DREADD (hM4Di) or Gq-DREADD (hM3Dq) was injected stereotaxically into the right CeA of transgenic KOR-Cre mice. The chemogenetic inhibition of KOR neurons expressing hM4Di with a selective DREADD actuator (deschloroclozapine, DCZ) in sham control mice significantly decreased inhibitory transmission, resulting in a shift of inhibition/excitation balance to promote excitation and induced pain behaviors. The chemogenetic activation of KOR neurons expressing hM3Dq with DCZ in neuropathic mice significantly increased inhibitory transmission, decreased excitability, and decreased neuropathic pain behaviors. These data suggest that amygdala KOR neurons modulate pain behaviors by exerting an inhibitory tone on downstream CeA neurons. Therefore, activation of these interneurons or blockade of inhibitory KOR signaling in these neurons could restore control of amygdala output and mitigate pain.

Turmeric Bioactive Compounds Alleviate Spinal Nerve Ligation-Induced Neuropathic Pain by Suppressing Glial Activation and Improving Mitochondrial Function in Spinal Cord and Amygdala.

This study examined the effects of turmeric bioactive compounds, curcumin C3 complex® (CUR) and bisdemethoxycurcumin (BDMC), on mechanical hypersensitivity and the gene expression of markers for glial activation, mitochondrial function, and oxidative stress in the spinal cord and amygdala of rats with neuropathic pain (NP). Twenty-four animals were randomly assigned to four groups: sham, spinal nerve ligation (SNL, an NP model), SNL+100 mg CUR/kg BW p.o., and SNL+50 mg BDMC/kg BW p.o. for 4 weeks. Mechanical hypersensitivity was assessed by the von Frey test (VFT) weekly. The lumbosacral section of the spinal cord and the right amygdala (central nucleus) were collected to determine the mRNA expression of genes (IBA-1, CD11b, GFAP, MFN1, DRP1, FIS1, PGC1α, PINK, Complex I, TLR4, and SOD1) utilizing qRT-PCR. Increased mechanical hypersensitivity and increased gene expression of markers for microglial activation (IBA-1 in the amygdala and CD11b in the spinal cord), astrocyte activation (GFAP in the spinal cord), mitochondrial dysfunction (PGC1α in the amygdala), and oxidative stress (TLR4 in the spinal cord and amygdala) were found in untreated SNL rats. Oral administration of CUR and BDMC significantly decreased mechanical hypersensitivity. CUR decreased CD11b and GFAP gene expression in the spinal cord. BDMC decreased IBA-1 in the spinal cord and amygdala as well as CD11b and GFAP in the spinal cord. Both CUR and BDMC reduced PGC1α gene expression in the amygdala, PINK1 gene expression in the spinal cord, and TLR4 in the spinal cord and amygdala, while they increased Complex I and SOD1 gene expression in the spinal cord. CUR and BDMC administration decreased mechanical hypersensitivity in NP by mitigating glial activation, oxidative stress, and mitochondrial dysfunction.

Gingerol-Enriched Ginger Supplementation Mitigates Neuropathic Pain via Mitigating Intestinal Permeability and Neuroinflammation: Gut-Brain Connection.

Objectives: Emerging evidence suggests an important role of the gut-brain axis in the development of neuropathic pain (NP). We investigated the effects of gingerol-enriched ginger (GEG) on pain behaviors, as well as mRNA expressions of inflammation via tight junction proteins in GI tissues (colon) and brain tissues (amygdala, both left and right) in animals with NP. Methods: Seventeen male rats were randomly divided into three groups: Sham, spinal nerve ligation (SNL, pain model), and SNL+0.375% GEG (wt/wt in diet) for 4 weeks. Mechanosensitivity was assessed by von Frey filament tests and hindpaw compression tests. Emotional responsiveness was measured from evoked audible and ultrasonic vocalizations. Ongoing spontaneous pain was measured in rodent grimace tests. Intestinal permeability was assessed by the lactulose/D-mannitol ratio in urine. The mRNA expression levels of neuroinflammation (NF-κB, TNF-α) in the colon and amygdala (right and left) were determined by qRT-PCR. Data was analyzed statistically. Results: Compared to the sham group, the SNL group had significantly greater mechanosensitivity (von Frey and compression tests), emotional responsiveness (audible and ultrasonic vocalizations to innocuous and noxious mechanical stimuli), and spontaneous pain (rodent grimace tests). GEG supplementation significantly reduced mechanosensitivity, emotional responses, and spontaneous pain measures in SNL rats. GEG supplementation also tended to decrease SNL-induced intestinal permeability markers. The SNL group had increased mRNA expression of NF-κB and TNF-α in the right amygdala and colon; GEG supplementation mitigated these changes in SNL-treated rats. Conclusion: This study suggests GEG supplementation palliated a variety of pain spectrum behaviors in a preclinical NP animal model. GEG also decreased SNL-induced intestinal permeability and neuroinflammation, which may explain the behavioral effects of GEG.

Dysfunction of Glutamate Delta-1 Receptor-Cerebellin 1 Trans-Synaptic Signaling in the Central Amygdala in Chronic Pain.

Chronic pain is a debilitating condition involving neuronal dysfunction, but the synaptic mechanisms underlying the persistence of pain are still poorly understood. We found that the synaptic organizer glutamate delta 1 receptor (GluD1) is expressed postsynaptically at parabrachio-central laterocapsular amygdala (PB-CeLC) glutamatergic synapses at axo-somatic and punctate locations on protein kinase C δ -positive (PKCδ+) neurons. Deletion of GluD1 impairs excitatory neurotransmission at the PB-CeLC synapses. In inflammatory and neuropathic pain models, GluD1 and its partner cerebellin 1 (Cbln1) are downregulated while AMPA receptor is upregulated. A single infusion of recombinant Cbln1 into the central amygdala led to sustained mitigation of behavioral pain parameters and normalized hyperexcitability of central amygdala neurons. Cbln2 was ineffective under these conditions and the effect of Cbln1 was antagonized by GluD1 ligand D-serine. The behavioral effect of Cbln1 was GluD1-dependent and showed lateralization to the right central amygdala. Selective ablation of GluD1 from the central amygdala or injection of Cbln1 into the central amygdala in normal animals led to changes in averse and fear-learning behaviors. Thus, GluD1-Cbln1 signaling in the central amygdala is a teaching signal for aversive behavior but its sustained dysregulation underlies persistence of pain. Significance statement: Chronic pain is a debilitating condition which involves synaptic dysfunction, but the underlying mechanisms are not fully understood. Our studies identify a novel mechanism involving structural synaptic changes in the amygdala caused by impaired GluD1-Cbln1 signaling in inflammatory and neuropathic pain behaviors. We also identify a novel means to mitigate pain in these conditions using protein therapeutics.

Kappa opioid receptor activation in the amygdala disinhibits CRF neurons to generate pain-like behaviors.

Recent evidence suggests that kappa opioid receptors (KOR) in limbic brain regions such as the amygdala contribute to pain conditions, but underlying mechanisms remain to be determined. The amygdala is an important player in averse-affective aspects of pain and pain modulation. The central nucleus (CeA) serves output functions through projection neurons that include corticotropin releasing factor (CRF) expressing neurons. The CeA is also rich in KOR. Here we tested the novel hypothesis that KOR activation in the CeA generates pain-like behaviors through a mechanism that involves inhibition of synaptic inhibition (disinhibition) of CRF neurons. Intra-CeA administration of a KOR agonist (U-69,593) increased vocalizations of naïve rats to noxious stimuli, and induced anxiety-like behaviors in the open field test (OFT) and avoidance in the conditioned place preference test, without affecting mechanosensory thresholds. Optogenetic silencing of CeA-CRF neurons blocked the facilitatory effects of systemically applied U-69,593 in naïve rats. Patch-clamp recordings of CRF neurons in rat brain slices found that U-69,593 decreased feedforward inhibitory transmission evoked by optogenetic stimulation of parabrachial afferents, but had no effect on monosynaptic excitatory transmission. U-69,593 decreased frequency, but not amplitude, of inhibitory synaptic currents, suggesting a presynaptic action. Multiphoton imaging of CeA-CRF neurons in rat brain slices showed that U-69,593 increased calcium signals evoked by electrical stimulation of presumed parabrachial input. This study shows for the first time that KOR activation increases activity of amygdala CRF neurons through synaptic disinhibition, resulting in averse-affective pain-like behaviors. Blocking KOR receptors may therefore represent a novel therapeutic strategy.

Fear extinction learning ability predicts neuropathic pain behaviors and amygdala activity in male rats.

Background The amygdala plays a key role in fear learning and extinction and has emerged as an important node of emotional-affective aspects of pain and pain modulation. Impaired fear extinction learning, which involves prefrontal cortical control of amygdala processing, has been linked to neuropsychiatric disorders. Here, we tested the hypothesis that fear extinction learning ability can predict the magnitude of neuropathic pain. Results We correlated fear extinction learning in naive adult male rats with sensory and affective behavioral outcome measures (mechanical thresholds, vocalizations, and anxiety- and depression-like behaviors) before and after the induction of the spinal nerve ligation model of neuropathic pain compared to sham controls. Auditory fear conditioning, extinction learning, and extinction retention tests were conducted after baseline testing. All rats showed increased freezing responses after fear conditioning. During extinction training, the majority (75%) of rats showed a decline in freezing level to 50% in 5 min (fear extinction+), whereas 25% of the rats maintained a high freezing level (>50%, fear extinction-). Fear extinction- rats showed decreased open-arm preference in the elevated plus maze, reflecting anxiety-like behavior, but there were no significant differences in sensory thresholds, vocalizations, or depression-like behavior (forced swim test) between fear extinction+ and fear extinction- types. In the neuropathic pain model (four weeks after spinal nerve ligation), fear extinction- rats showed a greater increase in vocalizations and anxiety-like behavior than fear extinction+ rats. Fear extinction- rats, but not fear extinction+ rats, also developed depression-like behavior. Extracellular single unit recordings of amygdala (central nucleus) neurons in behaviorally tested rats (anesthetized with isoflurane) found greater increases in background activity, bursting, and evoked activity in fear extinction- rats than fear extinction+ rats in the spinal nerve ligation model compared to sham controls. Conclusion The data may suggest that fear extinction learning ability predicts the magnitude of neuropathic pain-related affective rather than sensory behaviors, which correlates with differences in amygdala activity changes.

Pathway-Specific Alterations of Cortico-Amygdala Transmission in an Arthritis Pain Model.

Medial prefrontal cortex (mPFC) and amygdala are closely interconnected brain areas that play a key role in cognitive-affective aspects of pain through their reciprocal interactions. Clinical and preclinical evidence suggests that dysfunctions in the mPFC-amygdala circuitry underlie pain-related cognitive-affective deficits. However, synaptic mechanisms of pain-related changes in these long-range pathways are largely unknown. Here we used optogenetics and brain slice physiology to analyze synaptic transmission in different types of amygdala neurons driven by inputs from infralimbic (IL) and prelimbic (PL) subdivisions of the mPFC. We found that IL inputs evoked stronger synaptic inhibition of neurons in the latero-capsular division of the central nucleus (CeLC) of the amygdala than PL inputs, and this inhibition was impaired in an arthritis pain model. Furthermore, inhibition-excitation ratio in basolateral amygdala neurons was increased in the pain model in the IL pathway but not in the PL pathway. These results suggest that IL rather than PL controls CeLC activity, and that changes in this acute pain model occur predominantly in the IL-amygdala pathway.

5-HT2C Receptor Knockdown in the Amygdala Inhibits Neuropathic-Pain-Related Plasticity and Behaviors.

Neuroplasticity in the amygdala drives pain-related behaviors. The central nucleus (CeA) serves major amygdala output functions and can generate emotional-affective behaviors and modulate nocifensive responses. The CeA receives excitatory and inhibitory inputs from the basolateral nucleus (BLA) and serotonin receptor subtype 5-HT2CR in the BLA, but not CeA, has been implicated anxiogenic behaviors and anxiety disorders. Here, we tested the hypothesis that 5-HT2CR in the BLA plays a critical role in CeA plasticity and neuropathic pain behaviors in the rat spinal nerve ligation (SNL) model. Local 5-HT2CR knockdown in the BLA with stereotaxic injection of 5-HT2CR shRNA AAV vector decreased vocalizations and anxiety- and depression-like behaviors and increased sensory thresholds of SNL rats, but had no effect in sham controls. Extracellular single-unit recordings of CeA neurons in anesthetized rats showed that 5-HT2CR knockdown blocked the increase in neuronal activity (increased responsiveness, irregular spike firing, and increased burst activity) in SNL rats. At the synaptic level, 5-HT2CR knockdown blocked the increase in excitatory transmission from BLA to CeA recorded in brain slices from SNL rats using whole-cell patch-clamp conditions. Inhibitory transmission was decreased by 5-HT2CR knockdown in control and SNL conditions to a similar degree. The findings can be explained by immunohistochemical data showing increased expression of 5-HT2CR in non-GABAergic BLA cells in SNL rats. The results suggest that increased 5-HT2CR in the BLA contributes to neuropathic-pain-related amygdala plasticity by driving synaptic excitation of CeA neurons. As a rescue strategy, 5-HT2CR knockdown in the BLA inhibits neuropathic-pain-related behaviors.SIGNIFICANCE STATEMENT Neuroplasticity in the amygdala has emerged as an important pain mechanism. This study identifies a novel target and rescue strategy to control abnormally enhanced amygdala activity in an animal model of neuropathic pain. Specifically, an integrative approach of gene transfer, systems and brain slice electrophysiology, behavior, and immunohistochemistry was used to advance the novel concept that serotonin receptor subtype 5-HT2C contributes critically to the imbalance between excitatory and inhibitory drive of amygdala output neurons. Local viral vector-mediated 5-HT2CR knockdown in the amygdala normalizes the imbalance, decreases neuronal activity, and inhibits neuropathic-pain-related behaviors. The study provides valuable insight into serotonin receptor (dys)function in a limbic brain area.

Rescue of Impaired mGluR5-Driven Endocannabinoid Signaling Restores Prefrontal Cortical Output to Inhibit Pain in Arthritic Rats.

The medial prefrontal cortex (mPFC) serves executive functions that are impaired in neuropsychiatric disorders and pain. Underlying mechanisms remain to be determined. Here we advance the novel concept that metabotropic glutamate receptor 5 (mGluR5) fails to engage endocannabinoid (2-AG) signaling to overcome abnormal synaptic inhibition in pain, but restoring endocannabinoid signaling allows mGluR5 to increase mPFC output hence inhibit pain behaviors and mitigate cognitive deficits. Whole-cell patch-clamp recordings were made from layer V pyramidal cells in the infralimbic mPFC in rat brain slices. Electrical and optogenetic stimulations were used to analyze amygdala-driven mPFC activity. A selective mGluR5 activator (VU0360172) increased pyramidal output through an endocannabinoid-dependent mechanism because intracellular inhibition of the major 2-AG synthesizing enzyme diacylglycerol lipase or blockade of CB1 receptors abolished the facilitatory effect of VU0360172. In an arthritis pain model mGluR5 activation failed to overcome abnormal synaptic inhibition and increase pyramidal output. mGluR5 function was rescued by restoring 2-AG-CB1 signaling with a CB1 agonist (ACEA) or inhibitors of postsynaptic 2-AG hydrolyzing enzyme ABHD6 (intracellular WWL70) and monoacylglycerol lipase MGL (JZL184) or by blocking GABAergic inhibition with intracellular picrotoxin. CB1-mediated depolarization-induced suppression of synaptic inhibition (DSI) was also impaired in the pain model but could be restored by coapplication of VU0360172 and ACEA. Stereotaxic coadministration of VU0360172 and ACEA into the infralimbic, but not anterior cingulate, cortex mitigated decision-making deficits and pain behaviors of arthritic animals. The results suggest that rescue of impaired endocannabinoid-dependent mGluR5 function in the mPFC can restore mPFC output and cognitive functions and inhibit pain. Significance statement: Dysfunctions in prefrontal cortical interactions with subcortical brain regions, such as the amygdala, are emerging as important players in neuropsychiatric disorders and pain. This study identifies a novel mechanism and rescue strategy for impaired medial prefrontal cortical function in an animal model of arthritis pain. Specifically, an integrative approach of optogenetics, pharmacology, electrophysiology, and behavior is used to advance the novel concept that a breakdown of metabotropic glutamate receptor subtype mGluR5 and endocannabinoid signaling in infralimbic pyramidal cells fails to control abnormal amygdala-driven synaptic inhibition in the arthritis pain model. Restoring endocannabinoid signaling allows mGluR5 activation to increase infralimbic output hence inhibit pain behaviors and mitigate pain-related cognitive deficits.

Group II mGluRs modulate baseline and arthritis pain-related synaptic transmission in the rat medial prefrontal cortex.

The medial prefrontal cortex (mPFC) serves executive control functions that are impaired in neuropsychiatric disorders and pain. Therefore, restoring normal synaptic transmission and output is a desirable goal. Group II metabotropic glutamate receptors mGluR2 and mGluR3 are highly expressed in the mPFC, modulate synaptic transmission, and have been targeted for neuropsychiatric disorders. Their pain-related modulatory effects in the mPFC remain to be determined. Here we evaluated their ability to restore pyramidal output in an arthritis pain model. Whole-cell patch-clamp recordings of layer V mPFC pyramidal cells show that a selective group II mGluR agonist (LY379268) decreased synaptically evoked spiking in brain slices from normal and arthritic rats. Effects were concentration-dependent and reversed by a selective antagonist (LY341495). LY379268 decreased monosynaptic excitatory postsynaptic currents (EPSCs) and glutamate-driven inhibitory postsynaptic currents (IPSCs) in the pain model. Effects on EPSCs preceded those on IPSCs and could explain the overall inhibitory effect on pyramidal output. LY379268 decreased frequency, but not amplitude, of miniature EPSCs without affecting miniature IPSCs. LY341495 alone increased synaptically evoked spiking under normal conditions and in the pain model. In conclusion, group II mGluRs act on glutamatergic synapses to inhibit direct excitatory transmission and feedforward inhibition onto pyramidal cells. Their net effect is decreased pyramidal cell output. Facilitatory effects of a group II antagonist suggest the system may be tonically active to control pyramidal output. Failure to release the inhibitory tone and enhance mPFC output could be a mechanism for the development or persistence of a disease state such as pain.

Neuropeptide S: a novel regulator of pain-related amygdala plasticity and behaviors.

Amygdala plasticity is an important contributor to the emotional-affective dimension of pain. Recently discovered neuropeptide S (NPS) has anxiolytic properties through actions in the amygdala. Behavioral data also suggest antinociceptive effects of centrally acting NPS, but site and mechanism of action remain to be determined. This is the first electrophysiological analysis of pain-related NPS effects in the brain. We combined whole cell patch-clamp recordings in brain slices and behavioral assays to test the hypothesis that NPS activates synaptic inhibition of amygdala output to suppress pain behavior in an arthritis pain model. Recordings of neurons in the laterocapsular division of the central nucleus (CeLC), which serves pain-related amygdala output functions, show that NPS inhibited the enhanced excitatory drive [monosynaptic excitatory postsynaptic currents (EPSCs)] from the basolateral amygdala (BLA) in the pain state. As shown by miniature EPSC analysis, the inhibitory effect of NPS did not involve direct postsynaptic action on CeLC neurons but rather a presynaptic, action potential-dependent network mechanism. Indeed, NPS increased external capsule (EC)-driven synaptic inhibition of CeLC neurons through PKA-dependent facilitatory postsynaptic action on a cluster of inhibitory intercalated (ITC) cells. NPS had no effect on BLA neurons. High-frequency stimulation (HFS) of excitatory EC inputs to ITC cells also inhibited synaptic activation of CeLC neurons, providing further evidence that ITC activation can control amygdala output. The cellular mechanisms by which EC-driven synaptic inhibition controls CeLC output remain to be determined. Administration of NPS into ITC, but not CeLC, also inhibited vocalizations and anxiety-like behavior in arthritic rats. A selective NPS receptor antagonist ([d-Cys(tBu)(5)]NPS) blocked electrophysiological and behavioral effects of NPS. Thus NPS is a novel tool to control amygdala output and pain-related affective behaviors through a direct action on inhibitory ITC cells.

Modulation of pyramidal cell output in the medial prefrontal cortex by mGluR5 interacting with CB1.

The medial prefrontal cortex (mPFC) serves executive cognitive functions such as decision-making that are impaired in neuropsychiatric disorders and pain. We showed previously that amygdala-driven abnormal inhibition and decreased output of mPFC pyramidal cells contribute to pain-related impaired decision-making (Ji et al., 2010). Therefore, modulating pyramidal output is desirable therapeutic goal. Targeting metabotropic glutamate receptor subtype mGluR5 has emerged as a cognitive-enhancing strategy in neuropsychiatric disorders, but synaptic and cellular actions of mGluR5 in the mPFC remain to be determined. The present study determined synaptic and cellular actions of mGluR5 to test the hypothesis that increasing mGluR5 function can enhance pyramidal cell output. Whole-cell voltage- and current-clamp recordings were made from visually identified pyramidal neurons in layer V of the mPFC in rat brain slices. Both the prototypical mGluR5 agonist CHPG and a positive allosteric modulator (PAM) for mGluR5 (VU0360172) increased synaptically evoked spiking (E-S coupling) in mPFC pyramidal cells. The facilitatory effects of CHPG and VU0360172 were inhibited by an mGluR5 antagonist (MTEP). CHPG, but not VU0360172, increased neuronal excitability (frequency-current [F-I] function). VU0360172, but not CHPG, increased evoked excitatory synaptic currents (EPSCs) and amplitude, but not frequency, of miniature EPSCs, indicating a postsynaptic action. VU0360172, but not CHPG, decreased evoked inhibitory synaptic currents (IPSCs) through an action that involved cannabinoid receptor CB1, because a CB1 receptor antagonist (AM281) blocked the inhibitory effect of VU0360172 on synaptic inhibition. VU0360172 also increased and prolonged CB1-mediated depolarization-induced suppression of synaptic inhibition (DSI). Activation of CB1 with ACEA decreased inhibitory transmission through a presynaptic mechanism. The results show that increasing mGluR5 function enhances mPFC output. This effect can be accomplished by increasing excitability with an orthosteric agonist (CHPG) or by increasing excitatory synaptic drive and CB1-mediated presynaptic suppression of synaptic inhibition ("dis-inhibition") with a PAM (VU0360172). Therefore, mGluR5 may be a useful target in conditions of impaired mPFC output. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Alzheimer brain-derived tau oligomers propagate pathology from endogenous tau.

Intracerebral injection of brain extracts containing amyloid or tau aggregates in transgenic animals can induce cerebral amyloidosis and tau pathology. We extracted pure populations of tau oligomers directly from the cerebral cortex of Alzheimer disease (AD) brain. These oligomers are potent inhibitors of long term potentiation (LTP) in hippocampal brain slices and disrupt memory in wild type mice. We observed for the first time that these authentic brain-derived tau oligomers propagate abnormal tau conformation of endogenous murine tau after prolonged incubation. The conformation and hydrophobicity of tau oligomers play a critical role in the initiation and spread of tau pathology in the naïve host in a manner reminiscent of sporadic AD.

Contribution of microglia and astrocytes to the central sensitization, inflammatory and neuropathic pain in the juvenile rat.

BACKGROUND: The development of pain after peripheral nerve and tissue injury involves not only neuronal pathways but also immune cells and glia. Central sensitization is thought to be a mechanism for such persistent pain, and ATP involves in the process. We examined the contribution of glia to neuronal excitation in the juvenile rat spinal dorsal horn which is subjected to neuropathic and inflammatory pain. RESULTS: In rats subjected to neuropathic pain, immunoreactivity for the microglial marker OX42 was markedly increased. In contrast, in rats subjected to inflammatory pain, immunoreactivity for the astrocyte marker glial fibrillary acidic protein was increased slightly. Optically-recorded neuronal excitation induced by single-pulse stimulation to the dorsal root was augmented in rats subjected to neuropathic and inflammatory pain compared to control rats. The bath application of a glial inhibitor minocycline and a p38 mitogen-activated protein kinase inhibitor SB203580 inhibited the neuronal excitation in rats subjected to neuropathic pain. A specific P2X1,2,3,4 antagonist TNP-ATP largely inhibited the neuronal excitation only in rats subjected to neuropathic pain rats. In contrast, an astroglial toxin L-alpha-aminoadipate, a gap junction blocker carbenoxolone and c-Jun N-terminal kinase inhibitor SP600125 inhibited the neuronal excitation only in rats subjected to inflammatory pain. A greater number of cells in spinal cord slices from rats subjected to neuropathic pain showed Ca2+ signaling in response to puff application of ATP. This Ca2+ signaling was inhibited by minocycline and TNP-ATP. CONCLUSIONS: These results directly support the notion that microglia is more involved in neuropathic pain and astrocyte in inflammatory pain.

Long-term potentiation of neuronal excitation in the central nucleus of the rat amygdala revealed by imaging with a voltage-sensitive dye.

We examined the propagation pattern of neuronal excitation in slices from the central nucleus of the rat amygdala (CeA) using optical imaging with a voltage-sensitive dye. We analyzed the mechanisms for the long-term potentiation (LTP) of neuronal excitation induced by conditioning stimulation. High-frequency conditioning stimulation induced NMDA receptor-, cAMP-, and PKA-dependent LTP of neuronal excitation in the lateral part of the CeA. The bath application of forskolin also induced LTP that was PKA-dependent. These results suggest that the potentiation of neuronal excitation in the lateral part of the CeA is induced by the activation of intracellular cAMP/PKA signaling, which is triggered by the influx of Ca(2+) via NMDA receptors.

Synaptic plasticity in the spinal dorsal horn.

Long-term potentiation (LTP) at synapses in the spinal dorsal horn is thought to be a cellular mechanism for abnormal pain sensitivity. In this article, we review LTP in spinal projection neurons and presynaptic mechanisms of LTP in the spinal dorsal horn revealed by patch-clamp recording and optical imaging with voltage-sensitive dye.

Effect of excitatory and inhibitory agents and a glial inhibitor on optically-recorded primary-afferent excitation.

The effects of GABA, excitatory amino-acid receptors antagonists and a glial metabolism inhibitor on primary-afferent excitation in the spinal dorsal horn were studied by imaging the presynaptic excitation of high-threshold afferents in cord slices from young rats with a voltage-sensitive dye. Primary afferent fibers and terminals were anterogradely labeled with a voltage-sensitive dye from the dorsal root attached to the spinal cord slice. Single-pulse stimulation of C fiber-activating strength to the dorsal root elicited compound action potential-like optical responses in the superficial dorsal horn. The evoked presynaptic excitation was increased by the GABAA receptor antagonists picrotoxin and bicuculline, by glutamate receptor antagonists D-AP5 and CNQX, and by the glial metabolism inhibitor mono-fluoroacetic acid (MFA). The increase in presynaptic excitation by picrotoxin was inhibited in the presence of D-AP5, CNQX and MFA. Presynaptic modulation in the central terminal of fine primary afferents by excitatory and inhibitory amino acids may represent a mechanism that regulates the transmission of pain.