Agent-based modeling of the central amygdala and pain using cell-type specific physiological parameters.

The amygdala is a brain area involved in emotional regulation and pain. Over the course of the last 20 years, multiple researchers have studied sensory and motor connections within the amygdala in trying to understand the ultimate role of this structure in pain perception and descending control of pain. A number of investigators have been using cell-type specific manipulations to probe the underlying circuitry of the amygdala. As data have accumulated in this research space, we recognized a critical need for a single framework to integrate these data and evaluate emergent system-level responses. In this manuscript, we present an agent-based computational model of two distinct inhibitory neuron populations in the amygdala, those that express protein kinase C delta (PKCδ) and those that express somatostatin (SOM). We utilized a network of neural links to simulate connectivity and the transmission of inhibitory signals between neurons. Type-specific parameters describing the response of these neurons to noxious stimuli were estimated from published physiological and immunological data as well as our own wet-lab experiments. The model outputs an abstract measure of pain, which is calculated in terms of the cumulative pro-nociceptive and anti-nociceptive activity across neurons in both hemispheres of the amygdala. Results demonstrate the ability of the model to produce changes in pain that are consistent with published studies and highlight the importance of several model parameters. In particular, we found that the relative proportion of PKCδ and SOM neurons within each hemisphere is a key parameter in predicting pain and we explored model predictions for three possible values of this parameter. We compared model predictions of pain to data from our earlier behavioral studies and found areas of similarity as well as distinctions between the data sets. These differences, in particular, suggest a number of wet-lab experiments that could be done in the future.

Intracellular FGF14 (iFGF14) Is Required for Spontaneous and Evoked Firing in Cerebellar Purkinje Neurons and for Motor Coordination and Balance.

Mutations in FGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia (SCA27). In addition, mice lacking Fgf14 (Fgf14(-/-)) exhibit an ataxia phenotype resembling SCA27, accompanied by marked changes in the excitability of cerebellar granule and Purkinje neurons. It is not known, however, whether these phenotypes result from defects in neuronal development or if they reflect a physiological requirement for iFGF14 in the adult cerebellum. Here, we demonstrate that the acute and selective Fgf14-targeted short hairpin RNA (shRNA)-mediated in vivo "knock-down" of iFGF14 in adult Purkinje neurons attenuates spontaneous and evoked action potential firing without measurably affecting the expression or localization of voltage-gated Na(+) (Nav) channels at Purkinje neuron axon initial segments. The selective shRNA-mediated in vivo "knock-down" of iFGF14 in adult Purkinje neurons also impairs motor coordination and balance. Repetitive firing can be restored in Fgf14-targeted shRNA-expressing Purkinje neurons, as well as in Fgf14(-/-) Purkinje neurons, by prior membrane hyperpolarization, suggesting that the iFGF14-mediated regulation of the excitability of mature Purkinje neurons depends on membrane potential. Further experiments revealed that the loss of iFGF14 results in a marked hyperpolarizing shift in the voltage dependence of steady-state inactivation of the Nav currents in adult Purkinje neurons. We also show here that expressing iFGF14 selectively in adult Fgf14(-/-) Purkinje neurons rescues spontaneous firing and improves motor performance. Together, these results demonstrate that iFGF14 is required for spontaneous and evoked action potential firing in adult Purkinje neurons, thereby controlling the output of these cells and the regulation of motor coordination and balance.

Distinct balance of excitation and inhibition in an interareal feedforward and feedback circuit of mouse visual cortex.

Mouse visual cortex is subdivided into multiple distinct, hierarchically organized areas that are interconnected through feedforward (FF) and feedback (FB) pathways. The principal synaptic targets of FF and FB axons that reciprocally interconnect primary visual cortex (V1) with the higher lateromedial extrastriate area (LM) are pyramidal cells (Pyr) and parvalbumin (PV)-expressing GABAergic interneurons. Recordings in slices of mouse visual cortex have shown that layer 2/3 Pyr cells receive excitatory monosynaptic FF and FB inputs, which are opposed by disynaptic inhibition. Most notably, inhibition is stronger in the FF than FB pathway, suggesting pathway-specific organization of feedforward inhibition (FFI). To explore the hypothesis that this difference is due to diverse pathway-specific strengths of the inputs to PV neurons we have performed subcellular Channelrhodopsin-2-assisted circuit mapping in slices of mouse visual cortex. Whole-cell patch-clamp recordings were obtained from retrobead-labeled FF(V1→LM)- and FB(LM→V1)-projecting Pyr cells, as well as from tdTomato-expressing PV neurons. The results show that the FF(V1→LM) pathway provides on average 3.7-fold stronger depolarizing input to layer 2/3 inhibitory PV neurons than to neighboring excitatory Pyr cells. In the FB(LM→V1) pathway, depolarizing inputs to layer 2/3 PV neurons and Pyr cells were balanced. Balanced inputs were also found in the FF(V1→LM) pathway to layer 5 PV neurons and Pyr cells, whereas FB(LM→V1) inputs to layer 5 were biased toward Pyr cells. The findings indicate that FFI in FF(V1→LM) and FB(LM→V1) circuits are organized in a pathway- and lamina-specific fashion.

Divergent changes in PBN excitability in a mouse model of neuropathic pain.

The transition from acute to chronic pain involves maladaptive plasticity in central nociceptive pathways. Growing evidence suggests that changes within the parabrachial nucleus (PBN), an important component of the spino-parabrachio-amygdaloid pain pathway, are key contributors to the development and maintenance of chronic pain. In animal models of chronic pain, PBN neurons become sensitive to normally innocuous stimuli and responses to noxious stimuli become amplified and more often produce after-discharges that outlast the stimulus. Using ex vivo slice electrophysiology and two mouse models of neuropathic pain, sciatic cuff and chronic constriction of the infraorbital nerve (CCI-ION), we find that changes in the firing properties of PBN neurons and a shift in inhibitory synaptic transmission may underlie this phenomenon. Compared to PBN neurons from shams, a larger proportion of PBN neurons from mice with a sciatic cuff were spontaneously active at rest, and these same neurons showed increased excitability relative to shams. In contrast, quiescent PBN neurons from cuff mice were less excitable than those from shams. Despite an increase in excitability in a subset of PBN neurons, the presence of after-discharges frequently observed in vivo were largely absent ex vivo in both injury models. However, GABAB-mediated presynaptic inhibition of GABAergic terminals is enhanced in PBN neurons after CCI-ION. These data suggest that the amplified activity of PBN neurons observed in rodent models of chronic pain arise through a combination of changes in firing properties and network excitability.Significance Statement Hyperactivity of neurons in the parabrachial nucleus (PBN) is causally linked to exaggerated pain behaviors in rodent models of chronic pain but the underlying mechanisms remain unknown. Using two mouse models of neuropathic pain, we show the intrinsic properties of PBN neurons are largely unaltered following injury. However, subsets of PBN neurons become more excitable and GABAB receptor mediated suppression of inhibitory terminals is enhanced after injury. Thus, shifts in network excitability may be a contributing factor in injury induced potentiation of PBN activity.

Loss of Intracellular Fibroblast Growth Factor 14 (iFGF14) Increases the Excitability of Mature Hippocampal and Cortical Pyramidal Neurons.

Mutations in FGF14 , which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia type 27 (SCA27), a multisystem disorder associated with progressive deficits in motor coordination and cognitive function. Mice ( Fgf14 -/- ) lacking iFGF14 display similar phenotypes, and we have previously shown that the deficits in motor coordination reflect reduced excitability of cerebellar Purkinje neurons, owing to the loss of iFGF14-mediated regulation of the voltage-dependence of inactivation of the fast transient component of the voltage-gated Na + (Nav) current, I NaT . Here, we present the results of experiments designed to test the hypothesis that loss of iFGF14 also attenuates the intrinsic excitability of mature hippocampal and cortical pyramidal neurons. Current-clamp recordings from adult mouse hippocampal CA1 pyramidal neurons in acute in vitro slices, however, revealed that repetitive firing rates were higher in Fgf14 -/- , than in wild type (WT), cells. In addition, the waveforms of individual action potentials were altered in Fgf14 -/- hippocampal CA1 pyramidal neurons, and the loss of iFGF14 reduced the time delay between the initiation of axonal and somal action potentials. Voltage-clamp recordings revealed that the loss of iFGF14 altered the voltage-dependence of activation, but not inactivation, of I NaT in CA1 pyramidal neurons. Similar effects of the loss of iFGF14 on firing properties were evident in current-clamp recordings from layer 5 visual cortical pyramidal neurons. Additional experiments demonstrated that the loss of iFGF14 does not alter the distribution of anti-Nav1.6 or anti-ankyrin G immunofluorescence labeling intensity along the axon initial segments (AIS) of mature hippocampal CA1 or layer 5 visual cortical pyramidal neurons in situ . Taken together, the results demonstrate that, in contrast with results reported for neonatal (rat) hippocampal pyramidal neurons in dissociated cell culture, the loss of iFGF14 does not disrupt AIS architecture or Nav1.6 localization/distribution along the AIS of mature hippocampal (or cortical) pyramidal neurons in situ .

Cells and circuits for amygdala neuroplasticity in the transition to chronic pain.

Maladaptive plasticity is linked to the chronification of diseases such as pain, but the transition from acute to chronic pain is not well understood mechanistically. Neuroplasticity in the central nucleus of the amygdala (CeA) has emerged as a mechanism for sensory and emotional-affective aspects of injury-induced pain, although evidence comes from studies conducted almost exclusively in acute pain conditions and agnostic to cell type specificity. Here, we report time-dependent changes in genetically distinct and projection-specific CeA neurons in neuropathic pain. Hyperexcitability of CRF projection neurons and synaptic plasticity of parabrachial (PB) input at the acute stage shifted to hyperexcitability without synaptic plasticity in non-CRF neurons at the chronic phase. Accordingly, chemogenetic inhibition of the PB→CeA pathway mitigated pain-related behaviors in acute, but not chronic, neuropathic pain. Cell-type-specific temporal changes in neuroplasticity provide neurobiological evidence for the clinical observation that chronic pain is not simply the prolonged persistence of acute pain.

The parabrachial to central amygdala pathway is critical to injury-induced pain sensitization in mice.

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PBN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. However, the functional significance of this pathway in the modulation of the somatosensory component of pain was recently challenged by studies showing that spinal nociceptive neurons do not target CeA-projecting PBN cells and that manipulations of this pathway have no effect on reflexive-defensive somatosensory responses to peripheral noxious stimulation. Here, we showed that activation of CeA-projecting PBN neurons is critical to increase both stimulus-evoked and spontaneous nociceptive responses following an injury in male and female mice. Using optogenetic-assisted circuit mapping, we confirmed a functional excitatory projection from PBN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increased the expression of the neuronal activity marker Fos in CeA-projecting PBN neurons and that chemogenetic inactivation of these cells decreased behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we showed that chemogenetic activation of CeA-projecting PBN neurons is sufficient to induced bilateral hypersensitivity without injury. Together, our results indicate that the PBN→CeA pathway is a key modulator of pain-related behaviors that can increase reflexive-defensive and affective-motivational responses to somatosensory stimulation in injured states without affecting nociception under normal physiological conditions.

Anti-hyperalgesic and anti-inflammatory effects of 4R-tobacco cembranoid in a mouse model of inflammatory pain.

4R is a tobacco cembranoid that binds to and modulates cholinergic receptors and exhibits neuroprotective and anti-inflammatory activity. Given the established function of the cholinergic system in pain and inflammation, we propose that 4R is also analgesic. Here, we tested the hypothesis that systemic 4R treatment decreases pain-related behaviors and peripheral inflammation via modulation of the alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) in a mouse model of inflammatory pain. We elicited inflammation by injecting Complete Freund's Adjuvant (CFA) into the hind paw of male and female mice. We then assessed inflammation-induced hypersensitivity to cold, heat, and tactile stimulation using the Acetone, Hargreaves, and von Frey tests, respectively, before and at different time points (2.5 h - 8d) after a single systemic 4R (or vehicle) administration. We evaluated the contribution of α7 nAChRs 4R-mediated analgesia by pre-treating mice with a selective antagonist of α7 nAChRs followed by 4R (or vehicle) administration prior to behavioral tests. We assessed CFA-induced paw edema and inflammation by measuring paw thickness and quantifying immune cell infiltration in the injected hind paw using hematoxylin and eosin staining. Lastly, we performed immunohistochemical and flow cytometric analyses of paw skin in α7 nAChR-cre::Ai9 mice to measure the expression of α7 nAChRs on immune subsets. Our experiments show that systemic administration of 4R decreases inflammation-induced peripheral hypersensitivity in male and female mice and inflammation-induced paw edema in male but not female mice. Notably, 4R-mediated analgesia and anti-inflammatory effects lasted up to 8d after a single systemic administration on day 1. Pretreatment with an α7 nAChR-selective antagonist prevented 4R-mediated analgesia and anti-inflammatory effects, demonstrating that 4R effects are via modulation of α7 nAChRs. We further show that a subset of immune cells in the hind paw expresses α7 nAChRs. However, the number of α7 nAChR-expressing immune cells is unaltered by CFA or 4R treatment, suggesting that 4R effects are independent of α7 nAChR-expressing immune cells. Together, our findings identify a novel function of the 4R tobacco cembranoid as an analgesic agent in both male and female mice that reduces peripheral inflammation in a sex-dependent manner, further supporting the pharmacological targeting of the cholinergic system for pain treatment.

I(A) channels encoded by Kv1.4 and Kv4.2 regulate neuronal firing in the suprachiasmatic nucleus and circadian rhythms in locomotor activity.

Neurons in the suprachiasmatic nucleus (SCN) display coordinated circadian changes in electrical activity that are critical for daily rhythms in physiology, metabolism, and behavior. SCN neurons depolarize spontaneously and fire repetitively during the day and hyperpolarize, drastically reducing firing rates, at night. To explore the hypothesis that rapidly activating and inactivating A-type (I(A)) voltage-gated K(+) (Kv) channels, which are also active at subthreshold membrane potentials, are critical regulators of the excitability of SCN neurons, we examined locomotor activity and SCN firing in mice lacking Kv1.4 (Kv1.4(-/-)), Kv4.2 (Kv4.2(-/-)), or Kv4.3 (Kv4.3(-/-)), the pore-forming (α) subunits of I(A) channels. Mice lacking either Kv1.4 or Kv4.2 α subunits have markedly shorter (0.5 h) periods of locomotor activity than wild-type (WT) mice. In vitro extracellular multi-electrode recordings revealed that Kv1.4(-/-) and Kv4.2(-/-) SCN neurons display circadian rhythms in repetitive firing, but with shorter periods (0.5 h) than WT cells. In contrast, the periods of wheel-running activity in Kv4.3(-/-) mice and firing in Kv4.3(-/-) SCN neurons were indistinguishable from WT animals and neurons. Quantitative real-time PCR revealed that the transcripts encoding all three Kv channel α subunits, Kv1.4, Kv4.2, and Kv4.3, are expressed constitutively throughout the day and night in the SCN. Together, these results demonstrate that Kv1.4- and Kv4.2-encoded I(A) channels regulate the intrinsic excitability of SCN neurons during the day and night and determine the period and amplitude of circadian rhythms in SCN neuron firing and locomotor behavior.

The sodium channel accessory subunit Navß1 regulates neuronal excitability through modulation of repolarizing voltage-gated K⁺ channels.

The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (I(A)) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na⁺ channel accessory subunit Navß1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navß1 decreases I(A) densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navß1. Biochemical and voltage-clamp experiments further demonstrated that Navß1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navß1 as a component of native neuronal Kv4.2-encoded I(A) channel complexes and a novel regulator of I(A) channel densities and neuronal excitability.

Divergent changes in PBN excitability in a mouse model of neuropathic pain.

The transition from acute to chronic pain involves maladaptive plasticity in central nociceptive pathways. Growing evidence suggests that changes within the parabrachial nucleus (PBN), an important component of the spino-parabrachio-amygdaloid pain pathway, are key contributors to the development and maintenance of chronic pain. In animal models of chronic pain, PBN neurons become sensitive to normally innocuous stimuli and responses to noxious stimuli become amplified and more often produce after-discharges that outlast the stimulus. Using ex vivo slice electrophysiology and two mouse models of neuropathic pain, sciatic cuff and chronic constriction of the infraorbital nerve (CCI-ION), we find that changes in the firing properties of PBN neurons and a shift in inhibitory synaptic transmission may underlie this phenomenon. Compared to PBN neurons from shams, a larger proportion of PBN neurons from mice with a sciatic cuff were spontaneously active at rest, and these same neurons showed increased excitability relative to shams. In contrast, quiescent PBN neurons from cuff mice were less excitable than those from shams. Despite an increase in excitability in a subset of PBN neurons, the presence of after-discharges frequently observed in vivo were largely absent ex vivo in both injury models. However, GABAB-mediated presynaptic inhibition of GABAergic terminals is enhanced in PBN neurons after CCIION. These data suggest that the amplified activity of PBN neurons observed in rodent models of chronic pain arise through a combination of changes in firing properties and network excitability.

A Parabrachial-to-Amygdala Circuit That Determines Hemispheric Lateralization of Somatosensory Processing.

BACKGROUND: The central amygdala (CeA) is a bilateral hub of pain and emotional processing with well-established functional lateralization. We reported that optogenetic manipulation of neural activity in the left and right CeA has opposing effects on bladder pain. METHODS: To determine the influence of calcitonin gene-related peptide (CGRP) signaling from the parabrachial nucleus on this diametrically opposed lateralization, we administered CGRP and evaluated the activity of CeA neurons in acute brain slices as well as the behavioral signs of bladder pain in the mouse. RESULTS: We found that CGRP increased firing in both the right and left CeA neurons. Furthermore, we found that CGRP administration in the right CeA increased behavioral signs of bladder pain and decreased bladder pain-like behavior when administered in the left CeA. CONCLUSIONS: These studies reveal a parabrachial-to-amygdala circuit driven by opposing actions of CGRP that determines hemispheric lateralization of visceral pain.

Sex differences in pain-related behaviors and clinical progression of disease in mouse models of colonic pain.

ABSTRACT: Previous studies have reported sex differences in patients with irritable bowel syndrome and inflammatory bowel disease, including differences in visceral pain perception. Despite this, sex differences in behavioral manifestations of visceral pain and underlying pathology of the gastrointestinal tract have been largely understudied in preclinical research. In this study, we evaluated potential sex differences in spontaneous nociceptive responses, referred abdominal hypersensitivity, disease progression, and bowel pathology in mouse models of acute and persistent colon inflammation. Our experiments show that females exhibit more nociceptive responses and referred abdominal hypersensitivity than males in the context of acute but not persistent colon inflammation. We further demonstrate that, after acute and persistent colon inflammation, pain-related behavioral responses in females and males are distinct, with increases in licking of the abdomen only observed in females and increases in abdominal contractions only seen in males. During persistent colon inflammation, males exhibit worse disease progression than females, which is manifested as worse physical appearance and higher weight loss. However, no measurable sex differences were observed in persistent inflammation-induced bowel pathology, stool consistency, or fecal blood. Overall, our findings demonstrate sex differences in pain-related behaviors and disease progression in the context of acute and persistent colon inflammation, highlighting the importance of considering sex as a biological variable in future mechanistic studies of visceral pain as well as in the development of diagnostics and therapeutic options for chronic gastrointestinal diseases.

The parabrachial to central amygdala circuit is a key mediator of injury-induced pain sensitization.

The spino-ponto-amygdaloid pathway is a major ascending circuit relaying nociceptive information from the spinal cord to the brain. Potentiation of excitatory synaptic transmission in the parabrachial nucleus (PbN) to central amygdala (CeA) pathway has been reported in rodent models of persistent pain. At the behavioral level, the PbN→CeA pathway has been proposed to serve as a general alarm system to potential threats that modulates pain-related escape behaviors, threat memory, aversion, and affective-motivational (but not somatosensory) responses to painful stimuli. Increased sensitivity to previously innocuous somatosensory stimulation is a hallmark of chronic pain. Whether the PbN→CeA circuit contributes to heightened peripheral sensitivity following an injury, however, remains unknown. Here, we demonstrate that activation of CeA-projecting PbN neurons contributes to injury-induced behavioral hypersensitivity but not baseline nociception in male and female mice. Using optogenetic assisted circuit mapping, we confirmed a functional excitatory projection from PbN→CeA that is independent of the genetic or firing identity of CeA cells. We then showed that peripheral noxious stimulation increases the expression of the neuronal activity marker c-Fos in CeA-projecting PbN neurons and chemogenetic inactivation of these cells reduces behavioral hypersensitivity in models of neuropathic and inflammatory pain without affecting baseline nociception. Lastly, we show that chemogenetic activation of CeA-projecting PbN neurons is sufficient to induce bilateral hypersensitivity without injury. Together, our results demonstrate that the PbN→CeA pathway is a key modulator of pain-related behaviors that can amplify responses to somatosensory stimulation in pathological states without affecting nociception under normal physiological conditions. Significance Statement: Early studies identified the spino-ponto-amygdaloid pathway as a major ascending circuit conveying nociceptive inputs from the spinal cord to the brain. The functional significance of this circuit to injury-induced hypersensitivity, however, remains unknown. Here, we addressed this gap in knowledge using viral-mediated anatomical tracers, ex-vivo electrophysiology and chemogenetic intersectional approaches in rodent models of persistent pain. We found that activation of this pathway contributes to injury-induced hypersensitivity, directly demonstrating a critical function of the PbN→CeA circuit in pain modulation.

A-type K+ channels encoded by Kv4.2, Kv4.3 and Kv1.4 differentially regulate intrinsic excitability of cortical pyramidal neurons.

Rapidly activating and rapidly inactivating voltage-gated A-type K+ currents, IA, are key determinants of neuronal excitability and several studies suggest a critical role for the Kv4.2 pore-forming α subunit in the generation of IA channels in hippocampal and cortical pyramidal neurons. The experiments here demonstrate that Kv4.2, Kv4.3 and Kv1.4 all contribute to the generation of IA channels in mature cortical pyramidal (CP) neurons and that Kv4.2-, Kv4.3- and Kv1.4-encoded IA channels play distinct roles in regulating the intrinsic excitability and the firing properties of mature CP neurons. In vivo loss of Kv4.2, for example, alters the input resistances, current thresholds for action potential generation and action potential repolarization of mature CP neurons. Elimination of Kv4.3 also prolongs action potential duration, whereas the input resistances and the current thresholds for action potential generation in Kv4.3−/− and WT CP neurons are indistinguishable. In addition, although increased repetitive firing was observed in both Kv4.2−/− and Kv4.3−/− CP neurons, the increases in Kv4.2−/− CP neurons were observed in response to small, but not large, amplitude depolarizing current injections, whereas firing rates were higher in Kv4.3−/− CP neurons only with large amplitude current injections. In vivo loss of Kv1.4, in contrast, had minimal effects on the intrinsic excitability and the firing properties of mature CP neurons. Comparison of the effects of pharmacological blockade of Kv4-encoded currents in Kv1.4−/− and WT CP neurons, however, revealed that Kv1.4-encoded IA channels do contribute to controlling resting membrane potentials, the regulation of current thresholds for action potential generation and repetitive firing rates in mature CP neurons.

An inhibitory circuit from central amygdala to zona incerta drives pain-related behaviors in mice.

Central amygdala neurons expressing protein kinase C-delta (CeA-PKCδ) are sensitized following nerve injury and promote pain-related responses in mice. The neural circuits underlying modulation of pain-related behaviors by CeA-PKCδ neurons, however, remain unknown. In this study, we identified a neural circuit that originates in CeA-PKCδ neurons and terminates in the ventral region of the zona incerta (ZI), a subthalamic structure previously linked to pain processing. Behavioral experiments show that chemogenetic inhibition of GABAergic ZI neurons induced bilateral hypersensitivity in uninjured mice and contralateral hypersensitivity after nerve injury. In contrast, chemogenetic activation of GABAergic ZI neurons reversed nerve injury-induced hypersensitivity. Optogenetic manipulations of CeA-PKCδ axonal terminals in the ZI further showed that inhibition of this pathway reduces nerve injury-induced hypersensitivity whereas activation of the pathway produces hypersensitivity in the uninjured paws. Altogether, our results identify a novel nociceptive inhibitory efferent pathway from CeA-PKCδ neurons to the ZI that bidirectionally modulates pain-related behaviors in mice.

Activation of metabotropic glutamate receptor 5 in the amygdala modulates pain-like behavior.

The central nucleus of the amygdala (CeA) has been identified as a site of nociceptive processing important for sensitization induced by peripheral injury. However, the cellular signaling components underlying this function remain unknown. Here, we identify metabotropic glutamate receptor 5 (mGluR5) as an integral component of nociceptive processing in the CeA. Pharmacological activation of mGluRs with (R,S)-3,5-dihydroxyphenylglycine (DHPG) in the CeA of mice is sufficient to induce peripheral hypersensitivity in the absence of injury. DHPG-induced peripheral hypersensitivity is reduced via pharmacological blockade of mGluR5 or genetic disruption of mGluR5. Furthermore, pharmacological blockade or conditional deletion of mGluR5 in the CeA abrogates inflammation-induced hypersensitivity, demonstrating the necessity of mGluR5 in CeA-mediated pain modulation. Moreover, we demonstrate that phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2) is downstream of mGluR5 activation in the CeA and is necessary for the full expression of peripheral inflammation-induced behavioral sensitization. Finally, we present evidence of right hemispheric lateralization of mGluR5 modulation of amygdalar nociceptive processing. We demonstrate that unilateral pharmacological activation of mGluR5 in the CeA produces distinct behavioral responses depending on whether the right or left amygdala is injected. We also demonstrate significantly higher levels of mGluR5 expression in the right amygdala compared with the left under baseline conditions, suggesting a potential mechanism for right hemispheric lateralization of amygdala function in pain processing. Together, these results establish an integral role for mGluR5 and ERK1/2 in nociceptive processing in the CeA.

Cell-Type Specificity of Neuronal Excitability and Morphology in the Central Amygdala.

Central amygdala (CeA) neurons expressing protein kinase Cδ (PKCδ+) or somatostatin (Som+) differentially modulate diverse behaviors. The underlying features supporting cell-type-specific function in the CeA, however, remain unknown. Using whole-cell patch-clamp electrophysiology in acute mouse brain slices and biocytin-based neuronal reconstructions, we demonstrate that neuronal morphology and relative excitability are two distinguishing features between Som+ and PKCδ+ neurons in the laterocapsular subdivision of the CeA (CeLC). Som+ neurons, for example, are more excitable, compact, and with more complex dendritic arborizations than PKCδ+ neurons. Cell size, intrinsic membrane properties, and anatomic localization were further shown to correlate with cell-type-specific differences in excitability. Lastly, in the context of neuropathic pain, we show a shift in the excitability equilibrium between PKCδ+ and Som+ neurons, suggesting that imbalances in the relative output of these cells underlie maladaptive changes in behaviors. Together, our results identify fundamentally important distinguishing features of PKCδ+ and Som+ cells that support cell-type-specific function in the CeA.

The kv4.2 potassium channel subunit is required for pain plasticity.

A-type potassium currents are important determinants of neuronal excitability. In spinal cord dorsal horn neurons, A-type currents are modulated by extracellular signal-regulated kinases (ERKs), which mediate central sensitization during inflammatory pain. Here, we report that Kv4.2 mediates the majority of A-type current in dorsal horn neurons and is a critical site for modulation of neuronal excitability and nociceptive behaviors. Genetic elimination of Kv4.2 reduces A-type currents and increases excitability of dorsal horn neurons, resulting in enhanced sensitivity to tactile and thermal stimuli. Furthermore, ERK-mediated modulation of excitability in dorsal horn neurons and ERK-dependent forms of pain hypersensitivity are absent in Kv4.2(-/-) mice compared to wild-type littermates. Finally, mutational analysis of Kv4.2 indicates that S616 is the functionally relevant ERK phosphorylation site for modulation of Kv4.2-mediated currents in neurons. These results show that Kv4.2 is a downstream target of ERK in spinal cord and plays a crucial role in pain plasticity.