Regulation of neuropathic pain by microglial Orai1 channels.

Microglia are important mediators of neuroinflammation, which underlies neuropathic pain. However, the molecular checkpoints controlling microglial reactivity are not well-understood. Here, we investigated the role of Orai1 channels for microglia-mediated neuroinflammation following nerve injury and find that deletion of Orai1 in microglia attenuates Ca2+ signaling and the production of inflammatory cytokines by proalgesic agonists. Conditional deletion of Orai1 attenuated microglial proliferation in the dorsal horn, spinal cytokine levels, and potentiation of excitatory neurotransmission following peripheral nerve injury. These cellular effects were accompanied by mitigation of pain hyperalgesia in microglial Orai1 knockout mice. A small-molecule Orai1 inhibitor, CM4620, similarly mitigated allodynia in male mice. Unexpectedly, these protective effects were not seen in female mice, revealing sexual dimorphism in Orai1 regulation of microglial reactivity and hyperalgesia. Together, these findings indicate that Orai1 channels are key regulators of the sexually dimorphic role of microglia for the neuroinflammation that underlies neuropathic pain.

Excitatory VTA to DH projections provide a valence signal to memory circuits.

The positive or negative value (valence) of past experiences is normally integrated into neuronal circuits that encode episodic memories and plays an important role in guiding behavior. Here, we show, using mouse behavioral models, that glutamatergic afferents from the ventral tegmental area to the dorsal hippocampus (VTA→DH) signal negative valence to memory circuits, leading to the formation of fear-inducing context memories and to context-specific reinstatement of fear. To a lesser extent, these projections also contributed to opioid-induced place preference, suggesting a role in signaling positive valence as well, and thus a lack of dedicated polarity. Manipulations of VTA terminal activity were more effective in females and paralleled by sex differences in glutamatergic signaling. By prioritizing retrieval of negative and positive over neutral memories, the VTA→DH circuit can facilitate the selection of adaptive behaviors when current and past experiences are valence congruent.

BOLD temporal variability differentiates wakefulness from anesthesia-induced unconsciousness.

Even though a number of findings, based on information content or information integration, are shown to define neural underpinnings characteristic of a conscious experience, the neurophysiological mechanism of consciousness is still poorly understood. Here, we investigated the brain activity and functional connectivity changes that occur in the isoflurane-anesthetized unconscious state in contrast to the awake state in rats (awake and/or anesthetized, n = 68 rats). We examined nine information measures previously shown to distinguish between conscious states: blood oxygen level-dependent (BOLD) variability, functional connectivity strength, modularity, weighted modularity, efficiency, clustering coefficient, small-worldness, and spatial and temporal Lempel-Ziv complexity measure. We also identified modular membership, seed-based network connectivity, and absolute and normalized power spectrums to assess the integrity of the BOLD functional networks between awake and anesthesia. fMRI BOLD variability and related absolute power were the only information measures significantly higher during the awake state compared with isoflurane anesthesia across animals, and with varying levels of anesthesia, after correcting for motion and respiration confounds. Thus, we conclude that, at least under the specific conditions examined here, global measures of information integration/sharing do not properly distinguish the anesthetized state from wakefulness, and heightened overall, global and local, BOLD variability is the most reliable determinant of conscious brain activity relative to isoflurane anesthesia. NEW & NOTEWORTHY Multiple metrics previously suggested to be able to distinguish between states of consciousness were compared, within and across rats in awake and isoflurane anesthesia-induced unconsciousness. All measures tested showed sensitivity to confounds, correcting for motion and for respiration changes due to anesthesia. Resting state local BOLD variability and the related absolute power were the only information measures that robustly differentiated wakefulness states. These results caution against the general applicability of global information measures in identifying levels of consciousness, thus challenging the popular concept that these measures reflect states of consciousness, and also pointing to local signal variability as a more reliable indicator of states of wakefulness.

Role of adult hippocampal neurogenesis in persistent pain.

The full role of adult hippocampal neurogenesis (AHN) remains to be determined, yet it is implicated in learning and emotional functions, and is disrupted in negative mood disorders. Recent evidence indicates that AHN is decreased in persistent pain consistent with the idea that chronic pain is a major stressor, associated with negative moods and abnormal memories. Yet, the role of AHN in development of persistent pain has remained unexplored. In this study, we test the influence of AHN in postinjury inflammatory and neuropathic persistent pain-like behaviors by manipulating neurogenesis: pharmacologically through intracerebroventricular infusion of the antimitotic AraC; ablation of AHN by x-irradiation; and using transgenic mice with increased or decreased AHN. Downregulating neurogenesis reversibly diminished or blocked persistent pain; oppositely, upregulating neurogenesis led to prolonged persistent pain. Moreover, we could dissociate negative mood from persistent pain. These results suggest that AHN-mediated hippocampal learning mechanisms are involved in the emergence of persistent pain.

Expression of DNA methyltransferases in adult dorsal root ganglia is cell-type specific and up regulated in a rodent model of neuropathic pain.

Neuropathic pain is associated with hyperexcitability and intrinsic firing of dorsal root ganglia (DRG) neurons. These phenotypical changes can be long lasting, potentially spanning the entire life of animal models, and depend on altered expression of numerous proteins, including many ion channels. Yet, how DRGs maintain long-term changes in protein expression in neuropathic conditions remains unclear. DNA methylation is a well-known mechanism of epigenetic control of gene expression and is achieved by the action of three enzymes: DNA methyltransferase (DNMT) 1, 3a, and 3b, which have been studied primarily during development. We first performed immunohistochemical analysis to assess whether these enzymes are expressed in adult rat DRGs (L4-5) and found that DNMT1 is expressed in both glia and neurons, DNMT3a is preferentially expressed in glia and DNMT3b is preferentially expressed in neurons. A rat model of neuropathic pain was then used to determine whether nerve injury may induce epigenetic changes in DRGs at multiple time points after pain onset. Real-time RT PCR analysis revealed robust and time-dependent changes in DNMT transcript expression in ipsilateral DRGs from spared nerve injury (SNI) but not sham rats. Interestingly, DNMT3b transcript showed a robust upregulation that appeared already 1 week after surgery and persisted at 4 weeks (our endpoint); in contrast, DNMT1 and DNMT3a transcripts showed only moderate upregulation that was transient and did not appear until the second week. We suggest that DNMT regulation in adult DRGs may be a contributor to the pain phenotype and merits further study.

Predictive dynamics of human pain perception.

While the static magnitude of thermal pain perception has been shown to follow a power-law function of the temperature, its dynamical features have been largely overlooked. Due to the slow temporal experience of pain, multiple studies now show that the time evolution of its magnitude can be captured with continuous online ratings. Here we use such ratings to model quantitatively the temporal dynamics of thermal pain perception. We show that a differential equation captures the details of the temporal evolution in pain ratings in individual subjects for different stimulus pattern complexities, and also demonstrates strong predictive power to infer pain ratings, including readouts based only on brain functional images.

Abnormalities in hippocampal functioning with persistent pain.

Chronic pain patients exhibit increased anxiety, depression, and deficits in learning and memory. Yet how persistent pain affects the key brain area regulating these behaviors, the hippocampus, has remained minimally explored. In this study we investigated the impact of spared nerve injury (SNI) neuropathic pain in mice on hippocampal-dependent behavior and underlying cellular and molecular changes. In parallel, we measured the hippocampal volume of three groups of chronic pain patients. We found that SNI animals were unable to extinguish contextual fear and showed increased anxiety-like behavior. Additionally, SNI mice compared with Sham animals exhibited hippocampal (1) reduced extracellular signal-regulated kinase expression and phosphorylation, (2) decreased neurogenesis, and (3) altered short-term synaptic plasticity. To relate the observed hippocampal abnormalities with human chronic pain, we measured the volume of human hippocampus in chronic back pain (CBP), complex regional pain syndrome (CRPS), and osteoarthritis patients (OA). Compared with controls, CBP and CRPS, but not OA, had significantly less bilateral hippocampal volume. These results indicate that hippocampus-mediated behavior, synaptic plasticity, and neurogenesis are abnormal in neuropathic rodents. The changes may be related to the reduction in hippocampal volume we see in chronic pain patients, and these abnormalities may underlie learning and emotional deficits commonly observed in such patients.

Chronic neuropathic pain-like behavior correlates with IL-1ß expression and disrupts cytokine interactions in the hippocampus.

We have proposed that neuropathic pain engages emotional learning, suggesting the involvement of the hippocampus. Because cytokines in the periphery contribute to induction and maintenance of neuropathic pain but might also participate centrally, we used 2 neuropathic pain models, chronic constriction injury (CCI) and spared nerve injury (SNI), to investigate the temporal profile of hippocampal cytokine gene expression in 2 rat strains that show different postinjury behavioral threshold sensitivities. SNI induced long-lasting allodynia in both strains, while CCI induced allodynia with time-dependent recovery in Sprague Dawley (SD) and no allodynia in Wistar Kyoto (WK) rats. In WK rats, only SNI induced sustained upregulation of hippocampal interleukin (IL)-1ß, while IL-6 expression was transiently increased and no significant changes in IL-1ra expression were detected. Conversely, in SD rats, SNI resulted in sustained and robust increased hippocampal IL-1ß expression, which was only transient in rats with CCI. In this strain, IL-6 expression was not affected in any of the 2 injury models and IL-1ra expression was significantly increased in rats with SNI or CCI at late phases. We found that the degree and development of neuropathic pain depend on the specific nerve injury model and rat strain; that hippocampal IL-1ß mRNA levels correlate with neuropathic pain behavior; that, in contrast to sham-operated animals, there are no correlations between hippocampal IL-1ß and IL-1ra or IL-6 in neuropathic rats; and that alterations in cytokine expression are restricted to the hippocampus contralateral to the injury side, again implying that the observed changes reflect nociception.

Prefrontal cortex and spinal cord mediated anti-neuropathy and analgesia induced by sarcosine, a glycine-T1 transporter inhibitor.

Sarcosine is a competitive inhibitor of glycine type 1 transporter. We hypothesized that it may have analgesic and anti-neuropathic efficacy by a dual action: affecting neurotransmission in the prefrontal cortex as well as within the spinal cord. In rats with spared nerve injury (SNI) oral sarcosine reduced mechanical sensitivity for the injured limb (anti-neuropathy or anti-allodynia) as well as for the uninjured limb (analgesia), showing better dose efficacy for the injured limb. Intrathecal administration of sarcosine was more effective in reducing mechanical sensitivity for the uninjured paw. In contrast, prefrontal cortex infusions of sarcosine acutely reduced mechanical sensitivity for the injured paw. Repeated daily oral sarcosine induced anti-neuropathy, observed only after days of repeated treatment; this long-term effect disappeared a few days after treatment cessation. The findings indicate that manipulating glycine-T1 transporter at multiple central sites can induce acute analgesia, as well as acute and long-term reduction in neuropathic pain behavior. Analgesic effects seem primarily mediated through spinal cord circuitry while anti-neuropathic effects seem mediated through prefrontal cortex circuitry, most likely through distinct molecular pathways. The results suggest that such an approach may provide a novel venue for treating clinical pain conditions.

Identifying directed links in large scale functional networks: application to brain fMRI.

BACKGROUND: Biological experiments increasingly yield data representing large ensembles of interacting variables, making the application of advanced analytical tools a forbidding task. We present a method to extract networks of correlated activity, specifically from functional MRI data, such that: (a) network nodes represent voxels, and (b) the network links can be directed or undirected, representing temporal relationships between the nodes. The method provides a snapshot of the ongoing dynamics of the brain without sacrificing resolution, as the analysis is tractable even for very large numbers of voxels. RESULTS: We find that, based on topological properties of the networks, the method provides enough information about the dynamics to discriminate between subtly different brain states. Moreover, the statistical regularities previously reported are qualitatively preserved, i.e. the resulting networks display scale-free and small-world topologies. CONCLUSION: Our method expands previous approaches to render large scale functional networks, and creates the basis for an extensive and -due to the presence of mixtures of directed and undirected links- richer motif analysis of functional relationships.

D-cycloserine reduces neuropathic pain behavior through limbic NMDA-mediated circuitry.

Human brain imaging studies suggest that chronic neuropathic pain has a strong emotional component that is mediated by medial prefrontal cortex (mPFC) activity; in rodents, the mPFC is involved in emotional and cognitive aspects of behavior, including the extinction of Pavlovian fear conditioning. Together, these findings suggest that the cortex may modulate the memory trace of pain. As D-cycloserine (DCS), a partial agonist of the NMDA receptor, can enhance learning and potentiate the extinction of acquired fear, in the present study we tested its efficacy in neuropathic pain behavior. In rats with spared nerve injury (SNI), repeated daily oral administration of DCS reduced mechanical sensitivity of the injured limb in a dose-dependent manner; this effect continued for weeks after the cessation of DCS treatment. In addition, re-exposure to DCS further enhanced antinociceptive behavior. Repeated oral DCS administration also reduced cancer chemotherapy drug-induced neuropathic pain behavior. Infusions of DCS directly into the mPFC (especially within prelimbic cortex) or the amygdala (but not into thalamus, insula, or occipital cortex) acutely induced antinociception in SNI rats. The antinociceptive effect of intra-mPFC DCS infusions was mimicked by NMDA and glycine, and blocked by HA 966. In the mPFC of SNI rats, NR2B expression was down-regulated; however, this effect was reversed with repeated oral DCS. Lastly, infusions of DCS into mPFC reversed place avoidance behavior induced by mechanical stimulation of the injured paw in SNI rats. These findings indicate that limbic NMDA-mediated circuitry is involved in long-term reduction in neuropathic pain behavior.

Inflammatory and neuropathic pain animals exhibit distinct responses to innocuous thermal and motoric challenges.

Most current methods for assessing pain in animals are based on reflexive measures and require constant interaction between the observer and the animal. Here we explore two new fully automated methods to quantify the impact of pain on the overall behavior of the organism. Both methods take advantage of the animals' natural preference for a dark environment. We used a box divided into two compartments: dark and bright. In the motoric operant task, "AngleTrack", one end of the box was raised so that the animals had to climb uphill to go from the light to the dark compartment. In the thermal operant task, "ThermalTrack", the floor of the dark compartment was heated to a given temperature, while the light compartment remained at 25 degrees C. Rats were individually placed in the light box and their crossing between chambers monitored automatically for 30 minutes. The angle of the box, or the temperature of the dark compartment, was altered to challenge the animals' natural preference. We test the hypothesis that different models of pain (inflammatory or neuropathic) can be differentiated based on performance on these devices. Three groups of rats were tested at five different challenge levels on both tasks: 1) normal, 2) neuropathic injury pain (Spared Nerve Injury), and 3) inflammatory pain (intraplantar injection of Carrageenan). We monitored the position of the animals as well as their rate of switching between compartments. We find significant differences between the three groups and between the challenge levels both in their average position with respect to time, and in their switching rates. This suggests that the angle-track and thermal-track may be useful in assessing automatically the global impact of different types of pain on behavior.