Chemogenetic activation of the ventral subiculum-BNST pathway reduces context fear expression.

An inability to reduce fear in nonthreatening environments characterizes many anxiety disorders. The pathway from the ventral subiculum (vSUB) to the bed nucleus of the stria terminalis (BNST) is more active in safe contexts than in aversive ones, as indexed by FOS expression. Here, we used chemogenetic techniques to specifically activate the vSUB-BNST pathway during both context and cued fear expression by expressing a Cre-dependent hM3D(Gq) receptor in BNST-projecting vSUB neurons. Activation of the vSUB-BNST pathway reduced context but not cued fear expression. These data suggest that the vSUB-BNST pathway contributes to behavioral responses to nonaversive contexts.

Aversive Contexts Reduce Activity in the Ventral Subiculum- BNST Pathway.

Many anxiety disorders can be characterized by abnormalities in detecting and learning about threats, and the inability to reduce fear responses in non-threatening environments. PTSD may be the most representative of context processing pathology, as intrusive memories are experienced in "safe" contexts. The ventral subiculum (vSUB), the main output of the ventral hippocampus, encodes environmental cues and is critical for context processing. The bed nucleus of the stria terminalis (BNST) contributes to anxiety-like behaviors as well as context fear conditioning. Given the important roles of the BNST and the vSUB in these anxiety and fear-related behaviors, and the anatomical connections between the two brain regions, the major aims of this study were to characterize the anatomy and function of the vSUB-BNST pathway. First, using the retrograde tracer cholera toxin, we mapped the topographical arrangement of the vSUB-BNST pathway. Dual retrograde tracing experiments revealed neurons projecting to the BNST and those projecting to the basolateral amygdala are distinct populations. Second, we assessed whether activity in this pathway, as indexed by FOS immunohistochemistry, was modulated by context fear conditioning. Our data reveal less activation of the vSUB-BNST pathway in both males and females in aversive contexts and the greatest activation when animals explored a neutral familiar context. In addition, the vSUB of females contained fewer GABAergic neurons compared to males. These findings suggest that the vSUB-BNST pathway is involved in eliciting appropriate responses to contexts.

Sex Differences in BNST and Amygdala Activation by Contextual, Cued, and Unpredictable Threats.

Fear and anxiety can be described as emotional and physical responses to predictable and unpredictable threats. While the amygdala is necessary for context and cued fear conditioning, the bed nucleus of the stria terminalis (BNST) is important for anxiety-like behavior and conditioned responses to diffuse and/or unpredictable threats. However, we still lack knowledge about how the BNST and amygdala nuclei act in coordination. Moreover, the incidence of anxiety disorders and posttraumatic stress disorder (PTSD) is substantially higher in women than in men, but most studies of fear conditioning are conducted in male rodents. Here, we asked whether the BNST and the lateral, basal, and central nuclei of the amygdala are active during the expression of fear conditioning in male and female rats using FOS immunohistochemistry. We first show that the BNST is indeed involved in context fear expression in males, but not in females. The lateral amygdala was active in both sexes during context fear expression. We next trained animals using tone cues paired with an unconditioned stimulus (US), or tone cues which were unpaired with the US, and thus nonpredictive. Females displayed greater fear expression to these unpaired tones than males. FOS was upregulated in both the BNST and the basal amygdala during fear expression to unpaired tones in both sexes. The differential processing of fear responses by males and females highlights the need to acknowledge sex differences in conditioned fear memory.

Extended amygdala circuits are differentially activated by context fear conditioning in male and female rats.

As the incidence of anxiety disorders is more prevalent in females, comparing the neural underpinnings of anxiety in males and females is imperative. The bed nucleus of the stria terminalis (BNST) contributes to long-lasting, anxiety-like states including the expression of context fear conditioning. Currently, there is conflicting evidence as to which nuclei of the BNST contribute to these behaviors. The anterolateral portion of the BNST (BNST-AL) located dorsal to the anterior commissure and lateral to the stria terminalis sends robust projections to the central nucleus of the amygdala (CE). Here we asked whether the BNST-AL is active during the expression of context fear conditioning in both male and female rats. At the cellular level, the expression of context fear produced upregulation of the immediate-early gene ARC in the BNST-AL as well as an upregulation of ARC specifically in neurons projecting to the CE, as labeled by the retrograde tracer Fluorogold infused into the CE. However, this pattern of ARC expression was observed in male rats only. Excitotoxic lesions of the BNST reduced context fear expression in both sexes, suggesting that a different set of BNST subnuclei may be recruited by the expression of fear and anxiety-like behaviors in females. Overall, our data highlight the involvement of the BNST-AL in fear expression in males, and suggest that subnuclei of the BNST may be functionally different in male and female rats.

Top-Down Cortical Control of Acute and Chronic Pain.

Acute pain has an evolutionary role in the detection of physical harm and the response to it. In some cases, however, acute pain can impair function and lead to other morbidities. Chronic pain, meanwhile, can present as a psychopathological condition that significantly interferes with daily living. Most basic and translational pain research has focused on the molecular and cellular mechanisms in the spinal and peripheral nervous systems. In contrast, the brain plays a key role in the affective manifestation and cognitive control of pain. In particular, several cortical regions, such as the somatosensory cortex, prefrontal cortex, insular, and anterior cingulate cortex, are well known to be activated by acute pain signals, and neurons in these regions have been demonstrated to undergo changes in response to chronic pain. Furthermore, these cortical regions can project to a number of forebrain and limbic structures to exert powerful top-down control of not only sensory pain transmission but also affective pain expression, and such cortical regulatory mechanisms are particularly relevant in chronic pain states. Newer techniques have emerged that allow for detailed studies of central pain circuits in animal models, as well as how such circuits are modified by the presence of chronic pain and other predisposing psychosomatic factors. These mechanistic approaches can complement imaging in human studies. At the therapeutic level, a number of pharmacological and nonpharmacological interventions have recently been shown to engage these top-down control systems to provide analgesia. In this review, we will discuss how pain signals reach important cortical regions and how these regions in turn project to subcortical areas of the brain to exert profound modulation of the pain experience. In addition, we will discuss the clinical relevance of such top-down pain regulation mechanisms.

Rate and Temporal Coding Mechanisms in the Anterior Cingulate Cortex for Pain Anticipation.

Pain is a complex sensory and affective experience. Through its anticipation, animals can learn to avoid pain. Much is known about passive avoidance during a painful event; however, less is known about active pain avoidance. The anterior cingulate cortex (ACC) is a critical hub for affective pain processing. However, there is currently no mechanism that links ACC activities at the cellular level with behavioral anticipation or avoidance. Here we asked whether distinct populations of neurons in the ACC can encode information for pain anticipation. We used tetrodes to record from ACC neurons during a conditioning assay to train rats to avoid pain. We found that in rats that successfully avoid acute pain episodes, neurons that responded to pain shifted their firing rates to an earlier time, whereas neurons that responded to the anticipation of pain increased their firing rates prior to noxious stimulation. Furthermore, we found a selected group of neurons that shifted their firing from a pain-tuned response to an anticipatory response. Unsupervised learning analysis of ensemble spike activity indicates that temporal spiking patterns of ACC neurons can indeed predict the onset of pain avoidance. These results suggest rate and temporal coding schemes in the ACC for pain avoidance.

Scaling Up Cortical Control Inhibits Pain.

Acute pain evokes protective neural and behavioral responses. Chronic pain, however, disrupts normal nociceptive processing. The prefrontal cortex (PFC) is known to exert top-down regulation of sensory inputs; unfortunately, how individual PFC neurons respond to an acute pain signal is not well characterized. We found that neurons in the prelimbic region of the PFC increased firing rates of the neurons after noxious stimulations in free-moving rats. Chronic pain, however, suppressed both basal spontaneous and pain-evoked firing rates. Furthermore, we identified a linear correlation between basal and evoked firing rates of PFC neurons, whereby a decrease in basal firing leads to a nearly 2-fold reduction in pain-evoked response in chronic pain states. In contrast, enhancing basal PFC activity with low-frequency optogenetic stimulation scaled up prefrontal outputs to inhibit pain. These results demonstrate a cortical gain control system for nociceptive regulation and establish scaling up prefrontal outputs as an effective neuromodulation strategy to inhibit pain.

Assessment of Aversion of Acute Pain Stimulus through Conditioned Place Aversion.

Pain is a complex experience. The aversive component of pain has been assessed through conditioned place aversion in rodents. However, this behavioral test does not allow the evaluation of the aversion of an acute pain stimulus. In Zhang et al. (2017), we provide an updated version of a Conditioned Place Aversion paradigm to address this challenge. In this protocol, a detailed version of this method is described.

Chronic pain induces generalized enhancement of aversion.

A hallmark feature of chronic pain is its ability to impact other sensory and affective experiences. It is notably associated with hypersensitivity at the site of tissue injury. It is less clear, however, if chronic pain can also induce a generalized site-nonspecific enhancement in the aversive response to nociceptive inputs. Here, we showed that chronic pain in one limb in rats increased the aversive response to acute pain stimuli in the opposite limb, as assessed by conditioned place aversion. Interestingly, neural activities in the anterior cingulate cortex (ACC) correlated with noxious intensities, and optogenetic modulation of ACC neurons showed bidirectional control of the aversive response to acute pain. Chronic pain, however, altered acute pain intensity representation in the ACC to increase the aversive response to noxious stimuli at anatomically unrelated sites. Thus, chronic pain can disrupt cortical circuitry to enhance the aversive experience in a generalized anatomically nonspecific manner.

Genetic ablation of GINIP-expressing primary sensory neurons strongly impairs Formalin-evoked pain.

Primary sensory neurons are heterogeneous by myriad of molecular criteria. However, the functional significance of this remarkable heterogeneity is just emerging. We precedently described the GINIP+ neurons as a new subpopulation of non peptidergic C-fibers encompassing the free nerve ending cutaneous MRGPRD+ neurons and C-LTMRs. Using our recently generated ginip mouse model, we have been able to selectively ablate the GINIP+ neurons and assess their functional role in the somatosensation. We found that ablation of GINIP+ neurons affected neither the molecular contents nor the central projections of the spared neurons. GINIP-DTR mice exhibited impaired sensation to gentle mechanical stimuli applied to their hairy skin and had normal responses to noxious mechanical stimuli applied to their glabrous skin, under acute and injury-induced conditions. Importantly, loss of GINIP+ neurons significantly altered formalin-evoked first pain and drastically suppressed the second pain response. Given that MRGPRD+ neurons have been shown to be dispensable for formalin-evoked pain, our study suggest that C-LTMRs play a critical role in the modulation of formalin-evoked pain.

GINIP, a Gαi-interacting protein, functions as a key modulator of peripheral GABAB receptor-mediated analgesia.

One feature of neuropathic pain is a reduced GABAergic inhibitory function. Nociceptors have been suggested to play a key role in this process. However, the mechanisms behind nociceptor-mediated modulation of GABA signaling remain to be elucidated. Here we describe the identification of GINIP, a Gαi-interacting protein expressed in two distinct subsets of nonpeptidergic nociceptors. GINIP null mice develop a selective and prolonged mechanical hypersensitivity in models of inflammation and neuropathy. GINIP null mice show impaired responsiveness to GABAB, but not to delta or mu opioid receptor agonist-mediated analgesia specifically in the spared nerve injury (SNI) model. Consistently, GINIP-deficient dorsal root ganglia neurons had lower baclofen-evoked inhibition of high-voltage-activated calcium channels and a defective presynaptic inhibition of lamina IIi interneurons. These results further support the role of unmyelinated C fibers in injury-induced modulation of spinal GABAergic inhibition and identify GINIP as a key modulator of peripherally evoked GABAB-receptors signaling.

Uncoupling of molecular maturation from peripheral target innervation in nociceptors expressing a chimeric TrkA/TrkC receptor.

Neurotrophins and their receptors control a number of cellular processes, such as survival, gene expression and axonal growth, by activating multiple signalling pathways in peripheral neurons. Whether each of these pathways controls a distinct developmental process remains unknown. Here we describe a novel knock-in mouse model expressing a chimeric TrkA/TrkC (TrkAC) receptor from TrkA locus. In these mice, prospective nociceptors survived, segregated into appropriate peptidergic and nonpeptidergic subsets, projected normally to distinct laminae of the dorsal spinal cord, but displayed aberrant peripheral target innervation. This study provides the first in vivo evidence that intracellular parts of different Trk receptors are interchangeable to promote survival and maturation of nociceptors and shows that these developmental processes can be uncoupled from peripheral target innervation. Moreover, adult homozygous TrkAC knock-in mice displayed severe deficits in acute and tissue injury-induced pain, representing the first viable adult Trk mouse mutant with a pain phenotype.