Guangchen Ji

Cognitive impairment in pain through amygdala-driven prefrontal cortical deactivation.

Cognitive deficits such as impaired decision-making can be a consequence of persistent pain. Normal functions of the intact amygdala and prefrontal cortex are required for emotion-based decision-making that relies on the ability to assess risk, attribute value, and identify advantageous strategies. We tested the hypothesis that pain-related cognitive deficits result from amygdala-driven impairment of medial prefrontal cortical (mPFC) function. To do this, we used electrophysiological single-unit recordings in vivo, patch clamp in brain slices, and various behavioral assays to show that increased neuronal activity in the amygdala in an animal model of arthritis pain was accompanied by decreased mPFC activation and impaired decision-making. Furthermore, pharmacologic inhibition (with a corticotropin-releasing factor 1 receptor antagonist) of pain-related hyperactivity in the basolateral amygdala (BLA), but not central amygdala (CeA), reversed deactivation of mPFC pyramidal cells and improved decision-making deficits. Pain-related cortical deactivation resulted from a shift of balance between inhibitory and excitatory synaptic transmission. Direct excitatory transmission to mPFC pyramidal cells did not change in the pain model, whereas polysynaptic inhibitory transmission increased. GABAergic transmission was reduced by non-NMDA receptor antagonists, suggesting that synaptic inhibition was glutamate driven. The results are consistent with a model of BLA-driven feedforward inhibition of mPFC neurons. In contrast to the differential effects of BLA versus CeA hyperactivity on cortical-cognitive functions, both amygdala nuclei modulate emotional-affective pain behavior. Thus, this study shows that the amygdala contributes not only to emotional-affective but also cognitive effects of pain. The novel amygdalo-cortical pain mechanism has important implications for our understanding of amygdala functions and amygdalo-cortical interactions.

Techniques for assessing knee joint pain in arthritis

The assessment of pain is of critical importance for mechanistic studies as well as for the validation of drug targets. This review will focus on knee joint pain associated with arthritis. Different animal models have been developed for the study of knee joint arthritis. Behavioral tests in animal models of knee joint arthritis typically measure knee joint pain rather indirectly. In recent years, however, progress has been made in the development of tests that actually evaluate the sensitivity of the knee joint in arthritis models. They include measurements of the knee extension angle struggle threshold, hind limb withdrawal reflex threshold of knee compression force, and vocalizations in response to stimulation of the knee. A discussion of pain assessment in humans with arthritis pain conditions concludes this review.

Hemispheric Lateralization of Pain Processing by Amygdala Neurons

Recent biochemical and behavioral data suggest right-hemispheric lateralization of amygdala functions in pain. Our previous electrophysiological studies showed pain-related neuroplasticity in the latero-capsular division of the central nucleus of the amygdala (CeLC) in the right brain hemisphere. Here we determined differences in the processing of pain-related signals in right versus left CeLC neurons. Individual CeLC neurons were recorded extracellularly before and after induction of an arthritis pain state in anesthetized rats. Brief innocuous and noxious test stimuli were applied to peripheral tissues ipsi- and contralateral to the recording site. A monoarthritis was induced in the ipsi- or contralateral knee by intraarticular injections of kaolin and carrageenan. Under normal conditions, CeLC neurons in the left amygdala had smaller receptive fields than those in the right, but the magnitude of background and evoked activity was not significantly different. After arthritis induction, neurons in the right, but not left, CeLC developed increased background activity and evoked responses, irrespective of the location of the arthritis (ipsi- or contralateral to the recording site). A protein kinase A (PKA) inhibitor decreased the activity of right CeLC neurons after arthritis induction but had no effect in the left amygdala. Forskolin, however, increased the activity of left and right CeLC neurons under normal conditions. The results show for the first time laterality of pain-related electrophysiological activity changes in individual amygdala neurons. Whereas both left and right amygdala neurons receive nociceptive inputs and can become sensitized in principle, a yet unknown mechanism prevents PKA activation and pain-related changes in the left amygdala.

Mitochondrial Reactive Oxygen Species Are Activated by mGluR5 through IP3 and Activate ERK and PKA to Increase Excitability of Amygdala Neurons and Pain Behavior

Reactive oxygen species (ROS) such as superoxide are emerging as important signaling molecules in physiological plasticity but also in peripheral and spinal cord pain pathology. Underlying mechanisms and pain-related ROS signaling in the brain remain to be determined. Neuroplasticity in the amygdala plays a key role in emotional-affective pain responses and depends on group I metabotropic glutamate receptors (mGluRs) and protein kinases. Using patch-clamp, live-cell imaging, and behavioral assays, we tested the hypothesis that mitochondrial ROS links group I mGluRs to protein kinase activation to increase neuronal excitability and pain behavior. Agonists for mGluR1/5 (DHPG) or mGluR5 (CHPG) increased neuronal excitability of neurons in the laterocapsular division of the central nucleus of the amygdala (CeLC). DHPG effects were inhibited by an mGluR5 antagonist (MTEP), IP3 receptor blocker (xestospongin C), or ROS scavengers (PBN, tempol), but not by an mGluR1 antagonist (LY367385) or NO synthase inhibitor (l-NAME). Tempol inhibited the effects of IP3 but not those of a PKC activator, indicating that ROS activation was IP3 mediated. Live-cell imaging in CeLC-containing brain slices directly showed DHPG-induced and synaptically evoked mitochondrial superoxide production. DHPG also increased pain-related vocalizations and spinal reflexes through a mechanism that required mGluR5, IP3, and ROS. Combined application of inhibitors of ERK (U0126) and PKA (KT5720) was necessary to block completely the excitatory effects of a ROS donor (tBOOH). A PKC inhibitor (GF109203X) had no effect. Antagonists and inhibitors alone did not affect neuronal excitability. The results suggest an important role for the novel mGluR5- IP3-ROS-ERK/PKA signaling pathway in amygdala pain mechanisms.

Pain-related deactivation of medial prefrontal cortical neurons involves mGluR1 and GABAA receptors

Pain-related hyperactivity in the amygdala leads to deactivation of the medial prefrontal cortex (mPFC) and decision-making deficits. The mechanisms of pain-related inhibition of the mPFC are not yet known. Here, we used extracellular single-unit recordings of prelimbic mPFC neurons to determine the role of GABA(A) receptors and metabotropic glutamate receptor (mGluR) subtypes, mGluR1 and mGluR5, in pain-related activity changes of mPFC neurons. Background and evoked activity of mPFC neurons decreased after arthritis induction. To determine pain-related changes, the same neuron was recorded continuously before and after induction of arthritis in one knee joint by intra-articular injection of kaolin/carrageenan. Stereotaxic administration of a GABA(A) receptor antagonist {[R-(R*,S*)]-5-(6,8-dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl)-5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dioxolo[4,5-g]isoquinolinium iodide (bicuculline)} into the mPFC by microdialysis reversed pain-related inhibition, whereas offsite injections into the adjacent anterior cingulate cortex had no or opposite effects on prelimbic mPFC neurons. A selective mGluR1/5 agonist [(S)-3,5-dihydroxyphenylglycine (DHPG)] inhibited background and evoked activity under normal conditions through a GABAergic mechanism, because the inhibitory effect was blocked with bicuculline. In the arthritis pain state, DHPG, alone or in the presence of bicuculline, had no effect. Consistent with the involvement of mGluR1 in pain-related inhibition of the mPFC, a selective mGluR1 antagonist [(S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid] reversed the pain-related decrease of background and evoked activity of mPFC neurons in arthritis, whereas a selective mGluR5 antagonist [2-methyl-6-(phenylethynyl)pyridine hydrochloride] had no effect. The mGluR antagonists had no effect under normal conditions. We interpret our data to suggest that pain-related inhibition of mPFC neurons in the arthritis model depends on mGluR1-mediated endogenous activation of GABA(A) receptors. Exogenous activation of mGluR1/5 produces GABAergic inhibition under normal conditions. Restoring normal activity in the mPFC may be a therapeutic strategy to improve cognitive deficits associated with persistent pain.

Pro- and Anti-Nociceptive Effects of Corticotropin-Releasing Factor (CRF) in Central Amygdala Neurons Are Mediated Through Different Receptors

Corticotropin-releasing factor (CRF) is not only a stress hormone but also acts as a neuromodulator outside the hypothalamic-pituitary-adrenocortical axis, playing an important role in anxiety, depression, and pain modulation. The underlying mechanisms remain to be determined. A major site of extra-hypothalamic expression of CRF and its receptors is the amygdala, a key player in affect-related disorders such as anxiety. The latero-capsular division of the central nucleus of the amygdala (CeLC) is also important for pain modulation and pain affect. This study analyzed the effects of CRF on nociceptive processing in CeLC neurons and the contribution of CRF1 and CRF2 receptors and protein kinases A and C. Extracellular single-unit recordings were made from CeLC neurons in anesthetized adult rats. All neurons responded more strongly to noxious than innocuous mechanical stimulation of the knee. Evoked responses and background activity were measured before and during administration of CRF into the CeLC by microdialysis. CRF was administered alone or together with receptor antagonists or protein kinase inhibitors. CRF (0.01-1 microM; concentrations in microdialysis probe; 15 min) facilitated the evoked responses more strongly than background activity; a higher concentration (10 microM) had inhibitory effects. Facilitation by CRF (0.1 microM) was reversed by a selective CRF1 receptor antagonist (NBI27914, 10 microM) but not a CRF2 receptor antagonist (astressin-2B, 100 microM) and by a protein kinase A (PKA) inhibitor (KT5720, 100 microM) but not a protein kinase C inhibitor (GF109203X, 100 microM). Inhibitory effects of CRF (10 microM) were reversed by astressin-2B. These data suggest that CRF has dual effects on amygdala neurons: CRF1 receptor-mediated PKA-dependent facilitation and CRF2 receptor-mediated inhibition.

Somatostatin modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel

&NA; Activation of peripheral somatostatin receptors (SSTRs) inhibits sensitization of nociceptors, thus having a short term or phasic effect [Pain 90 (2001) 233] as well as maintaining a tonic inhibitory control over nociceptors [J Neurosci 21 (2001) 4042]. The present study provides several lines of evidence that an important mechanism underlying SSTR modulation of nociceptors is regulation of the transient receptor potential vanilloid 1 ion channel (TRPV1, formerly the VR1 receptor). Double labeling of L5 dorsal root ganglion cells demonstrates that ∼60% of SSTR2a‐labeled cells are positive for TRPV1. Conversely, ∼33% of TRPV1‐labeled cells are positive for SSTR2a. In vivo behavioral studies demonstrate that intraplantar injection of 20.0 but not 2.0 &mgr;M octreotide (OCT, SSTR agonist) significantly reduces capsaicin (CAP, a ligand for TRPV1) ‐induced flinching and lifting/licking behaviors. This occurs through local activation of SSTRs in the injected hindpaw and is reversed following co‐application of the SSTR antagonist cyclo‐somatostatin (c‐SOM). In vitro studies using a skin‐nerve preparation demonstrate that activation of peripheral SSTRs on nociceptors with 20.0 &mgr;M OCT significantly reduces CAP‐induced activity and can prevent CAP‐induced desensitization. Furthermore, blockade of peripheral SSTRs with c‐SOM dramatically enhances CAP‐induced behaviors and nociceptor activity, demonstrating SSTR‐induced tonic inhibitory modulation of TRPV1. Finally, TRPV1 does not appear to be under tonic opioid receptor control since the opioid antagonist naloxone does not change CAP‐induced excitation and does not effect OCT‐induced inhibition of CAP responses. These data strongly suggest that SSTRs modulate nociceptors through phasic and tonic regulation of peripheral TRPV1 receptors.

Modulation of medial prefrontal cortical activity using in vivo recordings and optogenetics

BackgroundThe medial prefrontal cortex (mPFC) serves major executive functions. mPFC output to subcortical brain areas such as the amygdala controls emotional processing and plays an important role in fear extinction. Impaired mPFC function correlates with extinction deficits in anxiety disorders such as PTSD and with cognitive decision-making deficits in neuropsychiatric disorders and persistent pain. Controlling mPFC output is a desirable therapeutic goal in neuropsychiatric disorders but functional differences of cell types (pyramidal cells and interneurons) and regions (infralimbic and prelimbic) represent a challenge. This electrophysiological study used optogenetics for the cell- and region-specific modulation of mPFC pyramidal output in the intact anesthetized animal.ResultsExtracellular single-unit recordings were made from infralimbic (IL) pyramidal cells, IL interneurons and prelimbic (PL) pyramidal cells 2–3 weeks after intra-IL injection of a viral vector encoding channel rhodopsin 2 (ChR2) under the control of the CaMKII promoter (rAAV5/CaMKIIa-ChR2(H134R)-EYFP) or a control vector that lacked the ChR2 sequence (rAAV5/CaMKIIa-EYFP). Optical stimulation with laser-generated blue light pulses delivered through an optical fiber to the IL increased spontaneous and evoked action potential firing of ChR2 expressing IL pyramidal cells but had no effect on IL interneurons that were distinguished from pyramidal cells based on their higher firing rate and shorter spike duration. Optical activation of IL pyramidal cells also inhibited PL pyramidal cells, suggesting that IL output controls PL output. The effects were light intensity-dependent and reversible. Confocal microscopy confirmed ChR2-EYFP or control vector expression in mPFC pyramidal cells but not in GABAergic cells.ConclusionsThe novelty of our study is the analysis of optogenetic effects on background and evoked activity of defined cell types in different mPFC regions. The electrophysiological in vivo results directly demonstrate the optogenetic modulation of mPFC activity in a region- and cell type-specific manner, which is significant in conditions of impaired mPFC output.

Facilitation of synaptic transmission and pain responses by CGRP in the amygdala of normal rats

Calcitonin gene-related peptide (CGRP) plays an important role in peripheral and central sensitization. CGRP also is a key molecule in the spino-parabrachial-amygdaloid pain pathway. Blockade of CGRP1 receptors in the spinal cord or in the amygdala has antinociceptive effects in different pain models. Here we studied the electrophysiological mechanisms of behavioral effects of CGRP in the amygdala in normal animals without tissue injury.Whole-cell patch-clamp recordings of neurons in the latero-capsular division of the central nucleus of the amygdala (CeLC) in rat brain slices showed that CGRP (100 nM) increased excitatory postsynaptic currents (EPSCs) at the parabrachio-amygdaloid (PB-CeLC) synapse, the exclusive source of CGRP in the amygdala. Consistent with a postsynaptic mechanism of action, CGRP increased amplitude, but not frequency, of miniature EPSCs and did not affect paired-pulse facilitation. CGRP also increased neuronal excitability. CGRP-induced synaptic facilitation was reversed by an NMDA receptor antagonist (AP5, 50 μM) or a PKA inhibitor (KT5720, 1 μM), but not by a PKC inhibitor (GF109203X, 1 μM). Stereotaxic administration of CGRP (10 μM, concentration in microdialysis probe) into the CeLC by microdialysis in awake rats increased audible and ultrasonic vocalizations and decreased hindlimb withdrawal thresholds. Behavioral effects of CGRP were largely blocked by KT5720 (100 μM) but not by GF109203X (100 μM).The results show that CGRP in the amygdala exacerbates nocifensive and affective behavioral responses in normal animals through PKA- and NMDA receptor-dependent postsynaptic facilitation. Thus, increased CGRP levels in the amygdala might trigger pain in the absence of tissue injury.

Intact Aδ-fibers up-regulate transient receptor potential A1 and contribute to cold hypersensitivity in neuropathic rats

Mechanisms underlying cold hypersensitivity in neuropathic states are unclear. Recent data indicate both transient receptor potential (TRP) M8 and TRPA1 play a role. In relation to TRPA1, there are reported increases in mRNA. However, it is unknown whether TRPA1 mRNA is translated into functional receptors, whether these receptors are found on peripheral nociceptors and what population of primary afferents expresses the receptors. The present study provides several lines of evidence that TRPA1 receptors are expressed on intact primary sensory neurons and contribute to cold hypersensitivity following spinal nerve ligation (SNL). Immunohistochemical studies show that expression of TRPA1 is significantly increased in the ipsilateral compared with the contralateral L4 dorsal root ganglion (DRG). Using mustard oil (MO, selective TRPA1 agonist), Ca(2+) imaging demonstrates an increase in the percentage of MO-sensitive L4 DRG cells in SNL compared with sham and naive rats. The magnitude of the Ca(2+) response evoked by MO is also significantly larger in SNL compared with sham and naive rats. Behavioral studies demonstrate that SNL results in increased nocifensive behaviors to mechanical and cold stimulation that is not seen in sham or naive rats. Behavioral responses in sham rats are no different from naive rats. In vitro single fiber recordings demonstrate Adelta-fibers (intact L4 axons) in the nerve-injured hind paw have conduction velocities no different from naive rats. In contrast, compared with naive rats, mechanical thresholds of the Adelta-fibers in SNL rats are significantly decreased, the proportion of cold-sensitive and MO-sensitive Adelta-fibers is significantly increased and the response magnitude of Adelta-fibers to MO is significantly increased. MO-induced activity in Adelta-fibers is significantly reduced by Ruthenium Red (TRPA1 receptor antagonist). These results demonstrate that TRPA1 is expressed on peripheral nociceptors, and they are up-regulated on intact Adelta-fibers following nerve injury, contributing to cold hypersensitivity.