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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 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 StatementEarly 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-vivoelectrophysiology 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.

BackgroundThe 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.MethodsTo determine the influence of calcitonin gene-related peptide (CGRP) signaling from the parabrachial nucleus (PBN) 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.ResultsWe 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.ConclusionsThese studies reveal a parabrachial-to-amygdala circuit driven by opposing actions of CGRP that determines hemispheric lateralization of visceral pain.

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are activated in response to inflammation and modulate pain in humans and rodent models. The use of α7nAChRs agonists as a therapeutic option for inflammation and pain is challenged by unwanted effects resulting from constant activation and/or desensitization of α7nAChRs. Positive allosteric modulators (PAMs) represent a compelling alternative as they increase endogenous nicotinic transmission but do not result in progressive desensitization or loss of receptor function. In the present study, we evaluated the function of the 4R tobacco cembranoid (4R) as a PAM of α7nAChR that reduces inflammation and pain-related behaviors in mouse models of inflammatory pain. Our electrophysiological experiments show that 4R potentiates choline-evoked currents in SH-SY5Y cells overexpressing α7nAChRs in a dose-dependent manner. At the behavioral level, we show that subcutaneous administration of 4R decreases inflammation-induced thermal but not tactile hypersensitivity or formalin-induced spontaneous nociceptive responses in both male and female mice. We further show reduced inflammation-induced paw edema in 4R-treated males, with no measurable effect observed in female mice. Altogether, the results from the experiments in this study identify 4R as a PAM of α7nAChRs that reduces thermal hypersensitivity in male and female mice and inflammation in a sex-specific manner. These findings highlight the use of 4R as a potential novel treatment strategy for pain and inflammation.

Previous studies have reported sex differences in irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) patients, 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 visceral 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 visceral nociceptive responses and referred abdominal hypersensitivity than males in the context of acute but not persistent colon inflammation. We further demonstrate that, following acute and persistent colon inflammation, visceral 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 that visceral pain-related behaviors and disease progression in the context of acute and persistent colon inflammation are sex-dependent, 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.

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.

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 functional monosynaptic inhibitory 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 further show that chemogenetic inhibition of GABAergic ZI neurons is sufficient to induce bilateral hypersensitivity in uninjured mice as well as contralateral hypersensitivity after nerve injury. In contrast, chemogenetic activation of GABAergic ZI neurons reverses nerve injury-induced hypersensitivity, demonstrating that silencing of the ZI is required for injury-induced behavioral hypersensitivity. Our results identify a previously unrecognized inhibitory efferent pathway from CeA-PKCδ neurons to the ZI and demonstrate that ZI-GABAergic neurons can bidirectionally modulate pain-related behaviors in mice.

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., Author summary In this manuscript, we present a computational modeling approach to understand and predict pain output from a part of the brain, the amygdala, involved in stress adaptation, emotional regulation, and pain. Over the last several years, a variety of groups have begun to dissect the specific cells that are responsible for the impact of amygdala activation on pain, which can include both increases and decreases in pain and pain-like output in animal models. It is helpful and necessary to use computational models to develop a framework to understand the amygdala as these wet lab techniques add to the complexity of our understanding of the brain structure. The model presented here was based on our recent published physiology experiments along with multiple examples of expression data. This model can be used to design future wet-lab experiments and can continue to be refined to help us evaluate the impact of the amygdala and similar limbic system structures in pain and other disease. Here we present the first computational model for amygdala signaling that includes physiological and histological properties of neurons and allows dynamic simulation of nociceptive signal propagation through the network.

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. Significance Statement Genetically distinct neurons in the central nucleus of the amygdala (CeA) are functionally opposed in the modulation of many behavioral outputs, including fear and pain-related behaviors. In this study, we use whole-cell patch-clamp electrophysiology in acute mouse brain slices in combination with molecular genetics and biocytin-based neuronal reconstructions to characterize protein kinase Cδ-expressing (PKCδ+) and somatostatin-expressing (Som+) neurons in the CeA. Our experiments revealed striking differences in both electrophysiological and morphologic properties of these two subpopulations of CeA neurons, Som+ neurons are more excitable, compact, and have more complex dendritic arborizations compared with PKCδ+ neurons. These results support the notion that genetically distinct CeA neurons have differing functions and properties, advancing our understanding of the mechanisms underlying cellular and functional heterogeneity in the CeA.

Women are more sensitive to pain and make up most of the patients suffering from chronic pathological pain conditions, particularly pelvic and gastrointestinal disorders. Despite this prevalence, most preclinical studies in rodents have focused on males due to the potential variability that the estrous cycle stage might add when studying female subjects. The failure to account for sex differences and the estrous cycle effects in pain leaves unanswered important questions regarding mechanisms driving pain-related behaviors and limits our ability to translate preclinical findings to clinical applications. Therefore, a systematic evaluation of potential differences between sexes and/or across the estrous cycle in pain thresholds and in rodent models of visceral pain is warranted. In the present study, we used Von Frey filaments to measure abdominal and paw sensitivity to tactile stimulation and the intracolonic capsaicin model of visceral pain to measure visceral sensitivity. Pain-related behaviors were compared between male and female mice across the different stages of the estrous cycle. Our results demonstrate that abdominal sensitivity to tactile stimulation is strongly dependent on the phase of the estrous cycle, with mice in metestrous/diestrous (M/D) exhibiting significantly greater responses to abdominal stimulation compared to those in proestrous/estrous (P/E). Responses to abdominal stimulation in male mice were similar to females in M/D and were significantly differently from females in P/E. Interestingly, the sex and estrous cycle differences were specific to the abdomen as no significant differences in paw sensitivity were observed between males and females or across the estrous cycle. Quantification of pain-related behaviors following intracolonic injection of capsaicin further revealed that female mice display significantly higher nociceptive responses to intracolonic capsaicin, compared to males. Altogether, our results indicate that sex and the estrous cycle are important biological variables to be considered when studying abdominal and visceral sensitivity in mice.

Neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in mammals, display daily rhythms in electrical activity with more depolarized resting potentials and higher firing rates during the day than at night. Although these daily variations in the electrical properties of SCN neurons are required for circadian rhythms in physiology and behavior, the mechanisms linking changes in neuronal excitability to the molecular clock are not known. Recently, we reported that mice deficient for either Kcna4 ( Kv1.4-/-) or Kcnd2 ( Kv4.2-/-; but not Kcnd3, Kv4.3-/-), voltage-gated K+ (Kv) channel pore-forming subunits that encode subthreshold, rapidly activating, and inactivating K+ currents (IA), have shortened (0.5 h) circadian periods in SCN firing and in locomotor activity compared with wild-type (WT) mice. In the experiments here, we used a mouse ( Per2Luc) line engineered with a bioluminescent reporter construct, PERIOD2::LUCIFERASE (PER2::LUC), replacing the endogenous Per2 locus, to test the hypothesis that the loss of Kv1.4- or Kv4.2-encoded IA channels also modifies circadian rhythms in the expression of the clock protein PERIOD2 (PER2). We found that SCN explants from Kv1.4-/-Per2Luc and Kv4.2-/- Per2Luc, but not Kv4.3-/-Per2Luc, mice have significantly shorter (by approximately 0.5 h) circadian periods in PER2 rhythms, compared with explants from Per2Luc mice, revealing that the membrane properties of SCN neurons feedback to regulate clock (PER2) expression. The combined loss of both Kv1.4- and Kv4.2-encoded IA channels in Kv1.4-/-/Kv4.2-/-Per2Luc SCN explants did not result in any further alterations in PER2 rhythms. Interestingly, however, mice lacking both Kv1.4 and Kv4.2 show a striking (approximately 1.8 h) advance in their daily activity onset in a light cycle compared with WT mice, suggesting additional roles for Kv1.4- and Kv4.2-encoded IA channels in controlling the light-dependent responses of neurons within and/or outside of the SCN to regulate circadian phase of daily activity.

The central nucleus of the amygdala (CeA) encompasses the main output pathways of the amygdala, a temporal lobe structure essential in affective and cognitive dimensions of pain. A major population of neurons in the CeA send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. CeA-PAG neurons are topographically organized based on their targeted subregion within the PAG. PAG-projecting neurons in the central medial (CeM) and central lateral (CeL) regions of CeA are intrinsically distinct. CeL-PAG neurons are a homogeneous population of intrinsically distinct neurons while CeM-PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM-PAG subtypes are altered in the complete Freund's adjuvant model of inflammatory pain.A major population of neurons in the central nucleus of amygdala (CeA) send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. While the CeA-PAG pathway has proved to be a component of descending anti-nociceptive circuitry, the functional organization of CeA-PAG neurons remains unclear. We identified CeA-PAG neurons in C57BL/6 mice of both sexes using intracranial injection of a fluorescent retrograde tracer into the PAG. In acute brain slices, we investigated the topographical and intrinsic characteristics of retrogradely labelled CeA-PAG neurons using epifluorescence and whole-cell electrophysiology. We also measured changes to CeA-PAG neurons in the complete Freund's adjuvant (CFA) model of inflammatory pain. Neurons in the central lateral (CeL) and central medial (CeM) amygdala project primarily to different regions of the PAG. CeL-PAG neurons consist of a relatively homogeneous population of intrinsically distinct neurons while CeM-PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM-PAG subtypes are altered 1 day after induction of the CFA inflammatory pain model. Collectively, our results provide insight into pain-induced changes to a specific population of CeA neurons that probably play a key role in the integration of noxious input with endogenous analgesia and behavioural coping response.

The central amygdala (CeA) has key functions in modulating pain-related behaviors and exhibits experience-dependent plasticity in the context of chronic pain. Recent work from our lab has further revealed that distinct CeA cell types contribute differently to behavioral outcomes in pathological pain states in mice. Cells expressing Protein Kinase C delta (CeA-PKCδ), for example, promote hypersensitivity and display increased excitability following injury. By contrast, somatostatin-expressing CeA (CeA-Som) neurons contribute to an endogenous analgesic tone at baseline and display suppressed excitability during nerve-injury. The cellular mechanisms driving this opposing modulation of pain-related behaviors are still poorly understood. We hypothesized that the convergence of genetic identity, intrinsic membrane properties and morphological characteristics of distinct subpopulations of neurons contributes to the functional heterogeneity in the CeA. Here, we begin to test this hypothesis by characterizing the electrophysiological and morphological properties of CeA-PKCδ and CeA-Som neurons. Preliminary results from whole-cell patch-clamp electrophysiology experiments in acute amygdala slices show that the electrophysiological properties of CeA-PKCδ and CeA-Som neurons are distinct. Firing properties in CeA-PKCδ neurons, for example, are highly heterogeneous, with spontaneously active, accommodating, and non-accommodating cells found among this population. In contrast, the overwhelming majority of CeA-Som neurons are either spontaneously active or non-accommodating. Notably, non-accommodating CeA-Som neurons are more excitable than non-accommodating CeA-PKCδ neurons, displaying increased firing responses to depolarizing current injection, reduced latencies to first spike and after hyperpolarizations and changes in resting membrane potentials. Ongoing experiments are evaluating the morphological properties of these genetically distinct cells, with the end goal of correlating pain-related behaviors with morphological properties, genetic identity and firing properties. Altogether, our results demonstrate that the output gain of CeA-Som cells is significantly greater than that of CeA-PKCδ neurons, suggesting that cell-type-specific differences in neuronal excitability might contribute to functional heterogeneity in the CeA.

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.

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 amygdala has been proposed to serve as a neural center for the modulation of pain perception. Numerous anatomical and behavioral studies demonstrate that exogenous manipulations of the amygdala (i.e., lesions, drug infusions) modulate behavioral responses to acute noxious stimuli; however, little is known about the endogenous molecular changes in the amygdala that contribute to alterations in nociceptive processing during persistent noxious stimuli that resemble pathological pain conditions. In the present study, we demonstrate that endogenous molecular changes in the amygdala play a crucial role in modulating long-lasting peripheral hypersensitivity associated with persistent inflammation and we further identify the extracellular signal-regulated kinase (ERK) as a molecular substrate underlying this behavioral sensitization. Using the formalin test as a mouse model of persistent inflammatory pain, we show that activation of ERK in the amygdala is both necessary for and sufficient to induce long-lasting peripheral hypersensitivity to tactile stimulation. Thus, blockade of inflammation-induced ERK activation in the amygdala significantly reduced long-lasting peripheral hypersensitivity associated with persistent inflammation, and pharmacological activation of ERK in the amygdala induced peripheral hypersensitivity in the absence of inflammation. Importantly, blockade of ERK activation in the amygdala did not affect responses to acute noxious stimuli in the absence of inflammation, indicating that modulation of nociceptive responses by amygdala ERK activation is specific to the persistent inflammatory state. Altogether, our results demonstrate a functional role of the ERK signaling cascade in the amygdala in inflammation-induced peripheral hypersensitivity.

The suprachiasmatic nucleus (SCN) regulates daily rhythms in physiology and behavior. Previous studies suggest a critical role for neurons expressing vasoactive intestinal peptide (VIP) in coordinating rhythmicity and synchronization in the SCN. Here we examined the firing properties of VIP-expressing SCN neurons in acute brain slices. Active and passive membrane properties were measured in VIP and in non-VIP neurons during the day and at night. Current-clamp recordings revealed that both VIP and non-VIP neurons were spontaneously active, with higher firing rates during the day than at night. Average firing frequencies, however, were higher in VIP neurons (3.1 ± 0.2 Hz, day and 2.4 ± 0.2 Hz, night) than in non-VIP neurons (1.8 ± 0.2 Hz, day and 0.9 ± 0.2 Hz, night), both day and night. The waveforms of individual action potentials in VIP and non-VIP neurons were also distinct. Action potential durations (APD50) were shorter in VIP neurons (3.6 ± 0.1 ms, day and 2.9 ± 0.1 ms, night) than in non-VIP neurons (4.4 ± 0.3 ms, day and 3.5 ± 0.2 ms, night) throughout the light-dark cycle. In addition, afterhyperpolarization (AHP) amplitudes were larger in VIP neurons (21 ± 0.8 mV, day and 24.9 ± 0.9 mV, night) than in non-VIP neurons (17.2 ± 1.1 mV, day and 20.5 ± 1.2 mV, night) during the day and at night. Furthermore, significant day/night differences were observed in APD50 and AHP amplitudes in both VIP and non-VIP SCN neurons, consistent with rhythmic changes in ionic conductances that contribute to shaping the firing properties of both cell types. The higher day and night firing rates of VIP neurons likely contribute to synchronizing electrical activity in the SCN.

The recent failure of substance P receptor (NK1) antagonists in clinical trials for treatment of pain spawned additional studies asking why preclinical models implicating SP in chronic pain did not translate into a viable target for analgesic development. This review focuses on recent pain model system studies, suggesting that it is not SP or NK1 per se, but rather the cells that express NK1 that are crucial for maintaining chronic pain.

Terminal deoxynucleotidyl transferase (TdT), a template-independent DNA polymerase, contributes to antigen receptor diversity in lymphocytes. Using in situ hybridization, we found that tdt is expressed within neurons of the adult mouse brain. tdt mRNA was localized within pyramidal neurons in the hippocampus, granule and polymorphic cells in the dentate gyrus, Purkinje neurons in the cerebellum, and cortical cells. Increased levels of tdt mRNA in the hippocampus, neocortex, and cerebellum were associated with rearing C57BL/6 mice, but not DBA/2 mice, in enriched environments. Unlike wild types (WT), tdt (-/-) mice did not show improvement in spatial learning and memory as a result of rearing in enriched environments. These results suggest that tdt may be involved in learning and memory saving.

The immediate-early gene hzf-3, also known as nurr1, is a member of the inducible orphan nuclear receptor family and is one candidate in the search for genes associated with learning and memory processes. Here we report that acquisition of a spatial food search task is accompanied by elevated levels of hzf-3 mRNA in the hippocampus. Adult male Long-Evans rats were handled, food-restricted, and allowed to habituate to the maze prior to training. During acquisition, rats were given one training session per day for 5 days. Each training session consisted of five trials in which animals searched the maze for food located in 4 of 16 holes in the floor of the maze. Training resulted in spatial acquisition of the task. Northern blot analysis showed significant increases in hippocampal hzf-3 mRNA 3 h after training in the maze. Next, brains were obtained from Naive, Habituated, Day 1, Day 3, and Day 5 animals and processed for in situ hybridization. The results showed significant increases of hzf-3 mRNA in CA1 and CA3 subregions of the dorsal hippocampus during acquisition of the task. We conclude that expression of the hzf-3 gene in the brain is associated with long-term spatial memory processes. The present results are the first to implicate an orphan nuclear receptor in long-term information storage in the hippocampus.

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 FFV1→LM- and FBLM→V1-projecting Pyr cells, as well as from tdTomato-expressing PV neurons. The results show that the FFV1→LMpathway provides on average 3.7-fold stronger depolarizing input to layer 2/3 inhibitory PV neurons than to neighboring excitatory Pyr cells. In the FBLM→V1pathway, depolarizing inputs to layer 2/3 PV neurons and Pyr cells were balanced. Balanced inputs were also found in the FFV1→LMpathway to layer 5 PV neurons and Pyr cells, whereas FBLM→V1inputs to layer 5 were biased toward Pyr cells. The findings indicate that FFI in FFV1→LMand FBLM→V1circuits are organized in a pathway- and lamina-specific fashion.

In many peripheral and central neurons, A-type K+ currents, IA, have been identified and shown to be key determinants in shaping action potential waveforms and repetitive firing properties, as well as in the regulation of synaptic transmission and synaptic plasticity. The functional properties and physiological roles of native neuronal IA, however, have been shown to be quite diverse in different types of neurons. Accumulating evidence suggests that this functional diversity is generated by multiple mechanisms, including the expression and subcellular distributions of IA channels encoded by different voltage-gated K+ (Kv) channel pore-forming (α) subunits, interactions of Kv α subunits with cytosolic and/or transmembrane accessory subunits and regulatory proteins and post-translational modifications of channel subunits. Several recent reports further suggest that local protein translation in the dendrites of neurons and interactions between IA channels with other types of voltage-gated ion channels further expands the functional diversity of native neuronal IA channels. Here, we review the diverse molecular mechanisms that have been shown or proposed to underlie the functional diversity of native neuronal IA channels.

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.

Metabotropic glutamate receptors (mGluRs) play important roles in the modulation of nociception. The group I mGluRs (mGlu1 and mGlu5) modulate nociceptive plasticity via activation of extracellular signal-regulated kinase (ERK) signaling. We reported recently that the K+channel Kv4.2 subunit underlies A-type K+currents in the spinal cord dorsal horn and is modulated by the ERK signaling pathway. Kv4.2-mediated A-type currents are important determinants of dorsal horn neuronal excitability and central sensitization that underlies hypersensitivity after tissue injury. In the present study, we demonstrate that ERK-mediated phosphorylation of Kv4.2 is downstream of mGlu5 activation in spinal cord dorsal horn neurons. Activation of group I mGluRs inhibited Kv4.2-mediated A-type K+currents and increased neuronal excitability in dorsal horn neurons. These effects were mediated by activation of mGlu5, but not mGlu1, and were dependent on ERK activation. Analysis of Kv4.2 phosphorylation site mutants clearly identified S616 as the residue responsible for mGlu5–ERK-dependent modulation of A-type currents and excitability. Furthermore, nociceptive behavior induced by activation of spinal group I mGluRs was impaired in Kv4.2 knock-out mice, demonstrating that,in vivo, modulation of Kv4.2 is downstream of mGlu5 activation. Altogether, our results indicate that activation of mGlu5 leads to ERK-mediated phosphorylation and modulation of Kv4.2-containing potassium channels in dorsal horn neurons. This modulation may contribute to nociceptive plasticity and central sensitization associated with chronic inflammatory pain conditions.

SummaryA-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.

In a rat model of cocaine craving, a new study shows that activation of the protein kinase ERK in the amygdala is necessary and sufficient for the growth of craving over time in withdrawal. The combination of experimental approaches used by these authors will serve as a model for future studies examining the molecular mechanisms underlying a cognitive or behavioral process.

The extracellular signal-regulated kinase (ERK) cascade has been shown to be a key modulator of pain processing in the central nucleus of the amygdala (CeA) in mice. ERK is activated in the CeA during persistent inflammatory pain and this activation is both necessary and sufficient to induce peripheral tactile hypersensitivity. Interestingly, biochemical studies show that inflammation-induced ERK activation in the CeA only occurs in the right, but not the left hemisphere. This inflammation-induced ERK activation in the right CeA is independent of the side of peripheral inflammation, suggesting that there is a dominant role of the right hemisphere in the modulation of pain by ERK activation in the CeA. However, the functional significance of this biochemical lateralization has yet to be determined. In the present study, we tested the hypothesis that modulation of pain by ERK signaling in the CeA is functionally lateralized. We acutely blocked ERK activation in the CeA by infusing the MEK inhibitor U0126 into the right or the left hemisphere and then measured the behavioral effects on inflammation-induced mechanical hypersensitivity in mice. Our results show that blockade of ERK activation in the right, but not the left CeA, decreases inflammation-induced peripheral hypersensitivity independent of the side of peripheral injury. These findings demonstrate that modulation of pain by ERK signaling in the CeA is functionally lateralized to the right hemisphere, suggesting a dominant role of the right amygdala in pain processing.