SUMMARY Pain perception is essential for survival and can be amplified or suppressed by expectations, experiences, and context. The neural mechanisms underlying bidirectional modulation of pain remain largely unknown. Here, we demonstrate that the central nucleus of the amygdala (CeA) functions as a pain rheostat, decreasing or increasing pain-related behaviors in mice. This dual and opposing function of the CeA is encoded by opposing changes in the excitability of two distinct subpopulations of GABAergic neurons that receive excitatory inputs from the parabrachial nucleus (PB). Thus, cells expressing protein kinase C-delta (CeA-PKCδ) are sensitized by nerve injury and increase pain-related responses. In contrast, cells expressing somatostatin (CeA-Som) are inhibited by nerve injury and their activity drives antinociception. Together, these results demonstrate that the CeA can amplify or suppress pain in a cell-type-specific manner, uncovering a previously unknown mechanism underlying bidirectional control of pain in the brain.

Summary Neuronal synaptic connections are either chemical or electrical, and these two types of synapses work together to dynamically define neural circuit function[1]. While we know a great deal about the molecules that support chemical synapse formation and function, we know little about the macromolecular complexes that regulate electrical synapses. Electrical synapses are created by gap junction channels (GJs) that provide direct ionic communication between neurons[2]. While often molecularly and functionally symmetric, recent work has found that pre- and postsynaptic neurons can contribute different GJ-forming proteins, creating molecularly asymmetric channels that are correlated with functional asymmetry at the synapse[3,4]. Associated with the GJs are structures observed by electron microscopy termed the Electrical Synapse Density (ESD)[5]. The ESD has been suggested to be critical for the formation and function of the electrical synapse, yet the biochemical makeup of these structures is poorly understood. Here we find that electrical synapse formation in vivo requires an intracellular scaffold called Tight Junction Protein 1b (Tjp1b). Tjp1b is localized to the electrical synapse where it is required for the stabilization of the GJs and for electrical synapse function. Strikingly, we find that Tjp1b protein localizes and functions asymmetrically, exclusively on the postsynaptic side of the synapse. Our findings support a novel model of electrical synapse molecular asymmetry at the level of an intracellular scaffold that is required for building the electrical synapse. We propose that such ESD asymmetries could be used by all nervous systems to support molecular and functional asymmetries at electrical synapses.

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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.

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.

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.

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, affective-motivational (but not somatosensory) responses to painful stimuli, aversion, and threat memory. Increased sensitivity to previously innocuous somatosensory stimulation is a hallmark of chronic pain. Whether the PbN→CeA circuit contributes to heightened sensitivity to somatosensory stimulation 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 pain-related behaviors that can amplify responses to somatosensory stimulation in pathological states without affecting nociception under normal physiological conditions.