The rostral ventromedial medulla modulates pain and depression-related behaviors caused by social stress.

ABSTRACT: The rostral ventromedial medulla (RVM) is a crucial structure in the descending pain modulatory system, playing a key role as a relay for both the facilitation and inhibition of pain. The chronic social defeat stress (CSDS) model has been widely used to study stress-induced behavioral impairments associated with depression in rodents. Several studies suggest that CSDS also causes changes related to chronic pain. In this study, we aimed to investigate the involvement of the RVM in CSDS-induced behavioral impairments, including those associated with chronic pain. We used chemogenetics to activate or inhibit the RVM during stress. The results indicated that the RVM is a vital hub influencing stress outcomes. Rostral ventromedial medulla activation during CSDS ameliorates all the stress outcomes, including social avoidance, allodynia, hyperalgesia, anhedonia, and behavioral despair. In addition, RVM inhibition in animals exposed to a subthreshold social defeat stress protocol induces a susceptible phenotype, facilitating all stress outcomes. Finally, chronic RVM inhibition-without any social stress stimulus-induces chronic pain but not depressive-like behaviors. Our findings provide insights into the comorbidity between chronic pain and depression by indicating the involvement of the RVM in establishing social stress-induced behavioral responses associated with both chronic pain and depression.

Involvement of brain cell phenotypes in stress-vulnerability and resilience.

Stress-related disorders' prevalence is epidemically increasing in modern society, leading to a severe impact on individuals' well-being and a great economic burden on public resources. Based on this, it is critical to understand the mechanisms by which stress induces these disorders. The study of stress made great progress in the past decades, from deeper into the hypothalamic-pituitary-adrenal axis to the understanding of the involvement of a single cell subtype on stress outcomes. In fact, many studies have used state-of-the-art tools such as chemogenetic, optogenetic, genetic manipulation, electrophysiology, pharmacology, and immunohistochemistry to investigate the role of specific cell subtypes in the stress response. In this review, we aim to gather studies addressing the involvement of specific brain cell subtypes in stress-related responses, exploring possible mechanisms associated with stress vulnerability versus resilience in preclinical models. We particularly focus on the involvement of the astrocytes, microglia, medium spiny neurons, parvalbumin neurons, pyramidal neurons, serotonergic neurons, and interneurons of different brain areas in stress-induced outcomes, resilience, and vulnerability to stress. We believe that this review can shed light on how diverse molecular mechanisms, involving specific receptors, neurotrophic factors, epigenetic enzymes, and miRNAs, among others, within these brain cell subtypes, are associated with the expression of a stress-susceptible or resilient phenotype, advancing the understanding/knowledge on the specific machinery implicate in those events.

The Role of The Rostral Ventromedial Medulla in Stress Responses.

The rostral ventromedial medulla (RVM) is a brainstem structure critical for the descending pain modulation system involved in both pain facilitation and inhibition through its projection to the spinal cord. Since the RVM is well connected with pain- and stress-engaged brain structures, such as the anterior cingulate cortex, nucleus accumbens, and amygdala, its involvement in stress responses has become a matter of great interest. While chronic stress has been proposed as a trigger of pain chronification and related psychiatric comorbidities due to maladaptive stress responses, acute stress triggers analgesia and other adaptative responses. Here we reviewed and highlighted the critical role of the RVM in stress responses, mainly in acute stress-induced analgesia (SIA) and chronic stress-induced hyperalgesia (SIH), providing insights into pain chronification processes and comorbidity between chronic pain and psychiatric disorders.

Molecular, Circuit, and Stress Response Characterization of Ventral Pallidum Npas1-Neurons.

Altered activity of the ventral pallidum (VP) underlies disrupted motivation in stress and drug exposure. The VP is a very heterogeneous structure composed of many neuron types with distinct physiological properties and projections. Neuronal PAS 1-positive (Npas1+) VP neurons are thought to send projections to brain regions critical for motivational behavior. While Npas1+ neurons have been characterized in the globus pallidus external, there is limited information on these neurons in the VP. To address this limitation, we evaluated the projection targets of the VP Npas1+ neurons and performed RNA-sequencing on ribosome-associated mRNA from VP Npas1+ neurons to determine their molecular identity. Finally, we used a chemogenetic approach to manipulate VP Npas1+ neurons during social defeat stress (SDS) and behavioral tasks related to anxiety and motivation in Npas1-Cre mice. We used a similar approach in females using the chronic witness defeat stress (CWDS). We identified VP Npas1+ projections to the nucleus accumbens, ventral tegmental area, medial and lateral habenula, lateral hypothalamus, thalamus, medial and lateral septum, and periaqueductal gray area. VP Npas1+ neurons displayed distinct translatome representing distinct biological processes. Chemogenetic activation of hM3D(Gq) receptors in VP Npas1+ neurons increased susceptibility to a subthreshold SDS and anxiety-like behavior in the elevated plus maze and open field while the activation of hM4D(Gi) receptors in VP Npas1+ neurons enhanced resilience to chronic SDS and CWDS. Thus, the activity of VP Npas1+ neurons modulates susceptibility to social stressors and anxiety-like behavior. Our studies provide new information on VP Npas1+ neuron circuitry, molecular identity, and their role in stress response.SIGNIFICANCE STATEMENT The ventral pallidum (VP) is a structure connected to both reward-related and aversive brain centers. It is a key brain area that signals the hedonic value of natural rewards. Disruption in the VP underlies altered motivation in stress and substance use disorder. However, VP is a very heterogeneous area with multiple neuron subtypes. This study characterized the projection pattern and molecular signatures of VP Neuronal PAS 1-positive (Npas1+) neurons. We further used tools to alter receptor signaling in VP Npas1+ neurons in stress to demonstrate a role for these neurons in stress behavioral outcomes. Our studies have implications for understanding brain cell type identities and their role in brain disorders, such as depression, a serious disorder that is precipitated by stressful events.

Come out and play.

We all know that we should exercise so that we can reach a healthier physiological state. However, this knowledge is often not enough to prompt us to regularly engage in exercise. Here, I discuss why and what we could do to try to overcome this circumstance.

Neutrophil-Derived COX-2 has a Key Role during Inflammatory Hyperalgesia.

Inflammation is a vital process for the injured tissue restoration and one of its hallmarks is inflammatory hyperalgesia. The cyclooxygenase (COX) pathway is strongly related to the inflammatory and painful process. Usually, the COX-1 isoform is described as homeostatic, while COX-2 is characterized as inducible in inflammatory conditions. Although it is well known that neutrophil cells are the first to arrive at the inflamed site and the major source of COX-2 is still unknown, the specific role of neutrophil-derived COX-2 in the pain process is. Thus, in the present study, we demonstrate for the first time that neutrophil-derived COX-2 plays a key role in peripheral inflammatory hyperalgesia. Conditional knockout mice for COX-2 in neutrophils (COX-2 fl/fl: Mrp8cre±) exhibited higher pain sensitivity after carrageenan (CG) injection and long-lasting IL-1ß-induced hyperalgesia compared with the control group (COX-2 fl/fl). Also, CG-induced inflammation in COX-2 fl/fl: Mrp8cre± mice showed COX-1 overexpression, and increased neutrophil migration and pro-inflammatory cytokines (e.g., IL-1ß and CXCL1). These findings revealed that neutrophil COX-2 has an important role in the regulation of inflammatory hyperalgesia.

The BDNF-TrkB Pathway Acts Through Nucleus Accumbens D2 Expressing Neurons to Mediate Stress Susceptible Outcomes.

Brain-derived neurotrophic factor (BDNF) has a critical role in stress response including neuropsychiatric disorders that are precipitated by stress, such as major depressive disorder (MDD). BDNF acts through its full-length BDNF receptor tyrosine kinase B (TrkB) to trigger a pro-plasticity effect. In contrast, the truncated isoform of the BDNF receptor (TrkB.t1) triggers an anti-plasticity effect. In stress outcomes, BDNF acting in the hippocampus has a stress resilience effect, and, inversely, in the nucleus accumbens (NAc), BDNF acts as a stress susceptible molecule. It is unknown if BDNF-TrkB acts on a specific NAc projection neuron, i.e., medium spiny neuron (MSN or spiny projection neuron), a subtype in stress outcomes. To determine this, we performed chronic social or vicarious witness defeat stress (CSDS or CWDS) in mice expressing TrkB.t1 in dopamine receptor 1 or 2 containing MSNs (D1- or D2-MSNs). Our results showed that TrkB.t1 overexpression in NAc D2-MSNs prevented the CSDS-induced social avoidance or other stress susceptible behaviors in male and female mice. We further showed that this overexpression in D2-MSNs blocked stress susceptible behavior induced by intra-NAc BDNF infusion. In contrast, our results demonstrate that overexpression of TrkB.t1 on NAc D1-MSNs facilitates the SDS susceptible behaviors. Our study provides enhanced details into the NAc cell subtype role of BDNF-TrkB signaling in stress outcomes.

Running wheel exercise induces therapeutic and preventive effects on inflammatory stimulus-induced persistent hyperalgesia in mice.

Chronic pain affects significant portion of the world's population and physical exercise has been extensively indicated as non-pharmacological clinical intervention to relieve symptoms in chronic pain conditions. In general, studies on pain chronification and physical exercise intervention have focused on neuropathic pain, although chronic pain commonly results from an original inflammatory episode. Based on this, the objective of the present study was to investigate the therapeutic and preventive effect of the running wheel exercise on the persistent hyperalgesia induced by repetitive inflammatory stimulus, a rodent model that simulates clinical conditions of chronic pain that persist even with no more inflammatory stimulus present. To evaluate the therapeutic effect of physical exercise, we first induced persistent hyperalgesia through 14 days of PGE2 hind paw injections and, after that, mice have access to the regular voluntary running wheel. To evaluate the preventive effect of physical exercise, we first left the mice with access to the regular voluntary running wheel and, after that, we performed 14 days of PGE2 hind paw injection. Our results showed that voluntary running wheel exercise reduced persistent mechanical and chemical hyperalgesia intensity induced by repetitive inflammatory stimulus. In addition, we showed that this therapeutic effect is long-lasting and is observed even if started belatedly, i.e. two weeks after the development of hyperalgesia. Also, our results showed that voluntary running wheel exercise absolutely prevented persistent mechanical and chemical hyperalgesia induction. We can conclude that physical exercise has therapeutic and preventive effect on inflammatory stimulus-induced persistent hyperalgesia. Our data from animal experiments bypass placebo effects bias of the human studies and reinforce physical exercise clinical recommendations to treat and prevent chronic pain.

Peripheral Inflammatory Hyperalgesia Depends on P2X7 Receptors in Satellite Glial Cells.

Peripheral inflammatory hyperalgesia depends on the sensitization of primary nociceptive neurons. Inflammation drives molecular alterations not only locally but also in the dorsal root ganglion (DRG) where interleukin-1 beta (IL-1ß) and purinoceptors are upregulated. Activation of the P2X7 purinoceptors by ATP is essential for IL-1ß maturation and release. At the DRG, P2X7R are expressed by satellite glial cells (SGCs) surrounding sensory neurons soma. Although SGCs have no projections outside the sensory ganglia these cells affect pain signaling through intercellular communication. Therefore, here we investigated whether activation of P2X7R by ATP and the subsequent release of IL-1ß in DRG participate in peripheral inflammatory hyperalgesia. Immunofluorescent images confirmed the expression of P2X7R and IL-1ß in SGCs of the DRG. The function of P2X7R was then verified using a selective antagonist, A-740003, or antisense for P2X7R administered in the L5-DRG. Inflammation was induced by CFA, carrageenan, IL-1ß, or PGE2 administered in rat's hind paw. Blockage of P2X7R at the DRG reduced the mechanical hyperalgesia induced by CFA, and prevented the mechanical hyperalgesia induced by carrageenan or IL-1ß, but not PGE2. It was also found an increase in P2X7 mRNA expression at the DRG after peripheral inflammation. IL-1ß production was also increased by inflammatory stimuli in vivo and in vitro, using SGC-enriched cultures stimulated with LPS. In LPS-stimulated cultures, activation of P2X7R by BzATP induced the release of IL-1ß, which was blocked by A-740003. In summary, our data suggest that peripheral inflammation leads to the activation of P2X7R expressed by SGCs at the DRG. Then, ATP-induced activation of P2X7R mediates the release of IL-1ß from SGC. This evidence places the SGC as an active player in the establishment of peripheral inflammatory hyperalgesia and highlights the importance of the events in DRG for the treatment of inflammatory diseases.

Social defeat stress-induced hyperalgesia is mediated by nav 1.8+ nociceptive fibers.

Recently the voltage-gated sodium (Nav) channels began to be studied as possible targets for analgesic drugs. In addition, specific Nav 1.8 blockers are currently being used to treat some types of chronic pain pathologies such as neuropathies and fibromyalgia. Nav 1.8+ fibers convey nociceptive information to brain structures belonging to the limbic system, which is involved in the pathophysiology of major depressive disorders. From this, using a model of chronic social defeat stress (SDS) and intrathecal injections of Nav 1.8 antisense, this study investigated the possible involvement of Nav 1.8+ nociceptive fibers in SDS- induced hyperalgesia in C57/BL mice. Our results showed that SDS induced a depressive-like behavior of social avoidance and increased the sensitivity to mechanical (electronic von Frey test) and chemical (capsaicin test) nociceptive stimuli. We also showed that intrathecal injection of Nav 1.8 antisense reversed the SDS-induced hyperalgesia as demonstrated by both, mechanical and chemical nociceptive tests. We confirmed the antisense efficacy and specificity in a separate no-defeated cohort through real-time PCR, which showed a significant reduction of Nav 1.8 mRNA and no reduction of Nav 1.7 and Nav 1.9 in the L4, L5 and L6 dorsal root ganglia (DRG). The present study advances the understanding of SDS-induced hyperalgesia, which seems to be dependent on Nav 1.8+ nociceptive fibers.

Using the Parafilm-assisted Microdissection (PAM) Method to Sample Rodent Nucleus Accumbens.

Microdissection techniques are very important for anatomical and functional studies focused on neuroscience, where it is often necessary microdissect specific brain areas to perform molecular or anatomical analyses. The parafilm®-assisted microdissection (PAM) was previously described and involves the microdissection of tissue sections mounted on parafilm-covered glass slides. In this work, we describe the use of the PAM method to microdissect rodent nucleus accumbens (NAc). (1) We first describe the best way to perform the mouse euthanasia and how to remove the brain. (2) Next, we describe how to prepare the slides with parafilm® that will be used to receive the brain slices. (3) Following, we describe how to handle the brain in the cryostat, how to align the hemispheres and how to identify the NAc antero-posterior limits. (4) We also describe how to perform the staining and dehydration of the slices, a critical step to facilitate the microdissection and preserve macromolecules. (5) In the final step, we describe how to identify the dorso-ventral and latero-medial limits of the NAc and, finally, how to perform the manual microdissection of the area. This is a low-cost technique that allows the researcher to specifically microdissect any brain region, from which intact RNA and proteins can be extracted to perform several molecular analyses (e.g., real-time PCR, Western blot, and RNA-seq).

Social Defeat Stress (SDS) in Mice: Using Swiss Mice as Resident.

[Abstract] Due to the high prevalence and great economic impact of depression, studies with animal models have been increasingly used to identify neurobiological mechanisms associated with this disorder. However, many animal models use stressful conditions that are not consistent with what we observe in the modern human world. Examples are the chronic unpredictable stress and the electric shock model used in rodents. It's well established the social stress as the major cause of depressive disorder in human, in this way a social defeat stress model was recently standardized and can induce depressive-like behavior of social avoidance, a typical human depressive behavior. In this model, mice are exposed on consecutive days to an aggressor mouse, suffering brief periods of physical aggression followed by longer periods of visual and olfactory (sensory) contact and, as a consequence, a relationship of social submission is characterized. Thus, the objective of this work is to describe a social defeat stress protocol using swiss mice as resident, also describing valuable procedural suggestions that will help researchers to reproduce the model easily.

Physical Activity Induces Nucleus Accumbens Genes Expression Changes Preventing Chronic Pain Susceptibility Promoted by High-Fat Diet and Sedentary Behavior in Mice.

Recent findings from rodent studies suggest that high-fat diet (HFD) increases hyperalgesia independent of obesity status. Furthermore, weight loss interventions such as voluntary physical activity (PA) for adults with obesity or overweight was reported to promote pain reduction in humans with chronic pain. However, regardless of obesity status, it is not known whether HFD intake and sedentary (SED) behavior is underlies chronic pain susceptibility. Moreover, differential gene expression in the nucleus accumbens (NAc) plays a crucial role in chronic pain susceptibility. Thus, the present study used an adapted model of the inflammatory prostaglandin E2 (PGE2)-induced persistent hyperalgesia short-term (PH-ST) protocol for mice, an HFD, and a voluntary PA paradigm to test these hypotheses. Therefore, we performed an analysis of differential gene expression using a transcriptome approach of the NAc. We also applied a gene ontology enrichment tools to identify biological processes associated with chronic pain susceptibility and to investigate the interaction between the factors studied: diet (standard diet vs. HFD), physical activity behavior (SED vs. PA) and PH-ST (PGE vs. saline). Our results demonstrated that HFD intake and sedentary behavior promoted chronic pain susceptibility, which in turn was prevented by voluntary physical activity, even when the animals were fed an HFD. The transcriptome of the NAc found 2,204 differential expression genes and gene ontology enrichment analysis revealed 41 biologic processes implicated in chronic pain susceptibility. Taking these biological processes together, our results suggest that genes related to metabolic and mitochondria stress were up-regulated in the chronic pain susceptibility group (SED-HFD-PGE), whereas genes related to neuroplasticity were up-regulated in the non-chronic pain susceptibility group (PA-HFD-PGE). These findings provide pieces of evidence that HFD intake and sedentary behavior provoked gene expression changes in the NAc related to promotion of chronic pain susceptibility, whereas voluntary physical activity provoked gene expression changes in the NAc related to prevention of chronic pain susceptibility. Finally, our findings confirmed previous literature supporting the crucial role of voluntary physical activity to prevent chronic pain and suggest that low levels of voluntary physical activity would be helpful and highly recommended as a complementary treatment for those with chronic pain.

Social defeat stress induces hyperalgesia and increases truncated BDNF isoforms in the nucleus accumbens regardless of the depressive-like behavior induction in mice.

Epidemiological studies have shown a close association between pain and depression. There is evidence showing this association as patients with depression show a high chronic pain prevalence and vice versa. Considering that social stress is critical for the development of depression in humans, we used a social defeat stress (SDS) model which induces depressive-like behavior in mice. In this model, mice are exposed to an aggressor mouse for ten days, suffering brief periods of agonistic contact and long periods of sensory contact. Some mice display social avoidance, a depressive-like behavior, and are considered susceptible, while some mice do not, and are considered resilient. Thus, we investigated the nociceptive behavior of mice submitted to SDS and the neuroplastic changes in dopaminergic mesolimbic system. Our results showed that the stressed mice (resilient and susceptible) presented a higher sensitivity to pain than the control mice in chemical and mechanical tests. We also verified that susceptible mice have higher Bdnf mRNA in the VTA compared to the resilient and control mice. The stressed mice had less mature BDNF and more truncated BDNF protein in the NAc compared with control mice. Although social stress may trigger the development of depression and hyperalgesia, these two conditions may manifest independently as social stress induced hyperalgesia even in mice that did not display depressive-like behavior. Also, increased Bdnf in the VTA seems to be associated with depressive-like behavior, whereas high levels of truncated BDNF and low mature BDNF appear to be associated with hyperalgesia induced by social defeat stress.

Nucleus accumbens dopaminergic neurotransmission switches its modulatory action in chronification of inflammatory hyperalgesia.

Dopaminergic neurotransmission in the nucleus accumbens, a central component of the mesolimbic system, has been associated with acute pain modulation. As there is a transition from acute to chronic pain ('chronification'), modulatory structures may be involved in chronic pain development. Thus, this study aimed to elucidate the role of nucleus accumbens dopaminergic neurotransmission in chronification of pain. We used a rat model in which daily subcutaneous injection of prostaglandin E2 in the hindpaw for 14 days induces a long-lasting state of nociceptor sensitization that lasts for at least 30 days following the end of the treatment. Our findings demonstrated that the increase of dopamine in the nucleus accumbens by local administration of GBR12909 (0.5 nmol/0.25 µL), a dopamine reuptake inhibitor, blocked prostaglandin E2 -induced acute hyperalgesia. This blockade was prevented by a dopamine D2 receptor antagonist (raclopride, 10 nmol/0.25 µL) but not changed by a D1 receptor antagonist (SCH23390, 0.5, 3 or 10 nmol/0.25 µL), both co-administered with GBR12909 in the nucleus accumbens. In contrast, the induction of persistent hyperalgesia was facilitated by continuous infusion of GBR12909 in the nucleus accumbens (0.021 nmol/0.5 µL/h) over 7 days of prostaglandin E2 treatment. The development of persistent hyperalgesia was impaired by SCH23390 (0.125 nmol/0.5 µL/h) and raclopride (0.416 nmol/0.5 µL/h), both administered continuously in the nucleus accumbens over 7 days. Taken together, our data suggest that the chronification of pain involves the plasticity of dopaminergic neurotransmission in the nucleus accumbens, which switches its modulatory role from antinociceptive to pronociceptive.