Abstract Background Carbon dioxide (CO2 ) inhalation, a biological challenge and pathologic marker in panic disorder, evokes intense fear and panic attacks in susceptible individuals. The molecular ...identity and anatomic location of CO2 -sensing systems that translate CO2 -evoked fear remain unclear. We investigated contributions of microglial acid sensor T cell death–associated gene-8 (TDAG8) and microglial proinflammatory responses in CO2 -evoked behavioral and physiological responses. Methods CO2 -evoked freezing, autonomic, and respiratory responses were assessed in TDAG8-deficient (–/– ) and wild-type (+/+ ) mice. Involvement of TDAG8-dependent microglial activation and proinflammatory cytokine interleukin (IL)-1β with CO2 -evoked responses was investigated using microglial blocker, minocycline, and IL-1β antagonist IL-1RA. CO2 -chemosensitive firing responses using single-cell patch clamping were measured in TDAG8–/– and TDAG8+/+ mice to gain functional insights. Results TDAG8 expression was localized in microglia enriched within the sensory circumventricular organs. TDAG8–/– mice displayed attenuated CO2 -evoked freezing and sympathetic responses. TDAG8 deficiency was associated with reduced microglial activation and proinflammatory cytokine IL-1β within the subfornical organ. Central infusion of microglial activation blocker minocycline and IL-1β antagonist IL-1RA attenuated CO2 -evoked freezing. Finally, CO2 -evoked neuronal firing in patch-clamped subfornical organ neurons was dependent on acid sensor TDAG8 and IL-1β. Conclusions Our data identify TDAG8-dependent microglial acid sensing as a unique chemosensor for detecting and translating hypercapnia to fear-associated behavioral and physiological responses, providing a novel mechanism for homeostatic threat detection of relevance to psychiatric conditions such as panic disorder.
•Mice deficient in CX3CR1 receptor show increased fear acquisition and reinstatement.•Unconditioned acoustic startle responses are not impacted by CX3CR1 deficiency.•CX3CR1−/− mice elicit reduced ...anxiety-like behaviors.•CX3CR1−/− mice have altered microglial morphology and elevated cfos in PVN and PFC.•CX3CR1 receptor regulates stress and fear circuits impacting behaviors pertinent to PTSD.
Mounting evidence supports immune dysfunction in psychiatric conditions such as post-traumatic stress disorder (PTSD). The association of immunomodulatory mechanisms with PTSD-relevant behavior and physiology is not well understood. Communication between neurons and microglia, resident immune cells of the central nervous system, is crucial for optimal regulation of behavior and physiology. In this regard, the fractalkine CX3CL1, secreted from neurons and its target, the microglial CX3CR1 receptor represent a primary neuron-microglia inter-regulatory system important for synaptic plasticity and function. The current study investigated the impact of CX3CR1 deficiency on behaviors relevant to PTSD, such as fear acquisition and memory, acoustic startle response and anxiety-like behavior. Morphological analysis of microglia and neuronal activation within PTSD-relevant forebrain nuclei regulating stress and fear behaviors was also conducted. CX3CR1-deficient (CX3CR1−/−) mice elicited increased fear acquisition as well as reinstatement of fear as compared to wild type (CX3CR1+/+) mice. Conditioned fear and extinction were not significantly different between genotypes. No significant differences were observed in unconditioned acoustic startle response between genotypes. CX3CR1−/− mice showed reduced anxiety-like behaviors as compared with CX3CR1+/+ mice. Morphological assessment of microglia showed region-selective effects of CX3CR1 deficiency, primarily within hypothalamic and cortical areas. Lastly, CX3CR1−/− mice elicited elevated neuronal activity in the PVN and the ventral tegmental-interpeduncular area following reinstatement of fear. Collectively, our data suggest that impaired CX3CR1 function may evoke region-selective alterations in forebrain circuits regulating stress, anxiety and fear, impacting behaviors relevant to disorders such as PTSD.
Difficulty in appropriately responding to threats is a key feature of psychiatric disorders, especially fear-related conditions such as panic disorder (PD) and posttraumatic stress disorder (PTSD). ...Most prior work on threat and fear regulation involves exposure to external threatful cues. However, fear can also be triggered by aversive, within-the-body, sensations. This interoceptive signaling of fear is highly relevant to PD and PTSD but is not well understood, especially in the context of sex. Using female and male mice, the current study investigated fear-associated spontaneous and conditioned behaviors to carbon dioxide (CO2) inhalation, a potent interoceptive threat that induces fear and panic. We also investigated whether behavioral sensitivity to CO2 is associated with delayed PTSD-relevant behaviors. CO2 evoked heterogenous freezing behaviors in both male and female animals. However, active, rearing behavior was significantly reduced in CO2-exposed male but not female mice. Interestingly, behavioral sensitivity to CO2 was associated with compromised fear extinction, independent of sex. However, in comparison to CO2-exposed males, females elicited less freezing and higher rearing during extinction suggesting an engagement of active versus passive defensive coping. Persistent neuronal activation marker ΔFosB immuno-mapping revealed attenuated engagement of infralimbic-prefrontal areas in both sexes but higher activation of brain stem locus coeruleus (LC) area in females. Inter-regional co-activation mapping revealed sex-independent disruptions in the infralimbic-amygdala associations but altered LC associations only in CO2-exposed female mice. Lastly, dopamine β hydroxylase positive (DβH + ve) noradrenergic neuronal cell counts in the LC correlated with freezing and rearing behaviors during CO2 inhalation and extinction only in female but not male mice. Collectively, these data provide evidence for higher active defensive responding to interoceptive threat CO2-associated fear in females that may stem from increased recruitment of the brainstem noradrenergic system. Our findings reveal distinct contributory mechanisms that may promote sex differences in fear and panic associated pathologies.
Abstract Activation of the maternal innate immune system, termed “maternal immune activation” (MIA), represents a common environmental risk factor for schizophrenia. Whereas evidence suggests ...dysregulation of GABA systems may underlie the pathophysiology of schizophrenia, a role for MIA in alteration of GABAergic systems is less clear. Here, pregnant rats received either the viral mimetic polyriboinosinic–polyribocytidilic acid or vehicle injection on gestational day 14. Glutamic acid decarboxylase-67 (GAD67 ) mRNA expression was examined in male offspring at postnatal day (P)14, P30 and P60. At P60, GAD67 mRNA was elevated in hippocampus and thalamus and decreased in prefrontal cortex of MIA offspring. MIA-induced alterations in GAD expression could contribute to the pathophysiology of schizophrenia.
► Prenatal immune activation blunts MK-801-induced increase in extracellular glutamate. ► Prefrontal cortex basal glutamate is elevated by prenatal immune activation. ► Paliperidone treatment ...normalized PFC glutamate in poly I:C offspring. ► Palliperidone and risperidone prevent MK-801-induced glutamate increase.
The NMDA glutamate hypofunction model of schizophrenia is based in part upon acute effects of NMDA receptor blockade in humans and rodents. Several laboratories have reported glutamate system abnormalities following prenatal exposure to immune challenge, a known environmental risk factor for schizophrenia. Here we report indices of NMDA glutamate receptor hypofunction following prenatal immune activation, as well as the effects of treatment during periadolescence with the atypical antipsychotic medications risperidone and paliperidone. Pregnant Sprague-Dawley rats were injected with polyinosinic:polycytidylic acid (poly I:C) or saline on gestational day 14. Male offspring were treated orally via drinking water with vehicle, risperidone (0.01
mg/kg/day), or paliperidone (0.01
mg/kg/day) between postnatal days 35 and 56 (periadolescence) and extracellular glutamate levels in the prefrontal cortex were determined by microdialysis at PD 56. Consistent with decreased NMDA receptor function, MK-801-induced increases in extracellular glutamate concentration were markedly blunted following prenatal immune activation. Further suggesting NMDA receptor hypofunction, prefrontal cortex basal extracellular glutamate was significantly elevated (
p
<
0.05) in offspring of poly I:C treated dams. Pretreatment with low dose paliperidone or risperidone (0.01
mg/kg/day postnatal days 35–56) normalized prefrontal cortical basal extracellular glutamate (
p
<
0.05 vs. poly I:C vehicle-treatment). Pretreatment with paliperidone and risperidone also prevented the acute MK-801-induced increase in extracellular glutamate. These observations demonstrate decreased NMDA receptor function and elevated extracellular glutamate, two key features of the NMDA glutamate receptor hypofunction model of schizophrenia, during periadolescence following prenatal immune activation. Treatment with the atypical antipsychotic medications paliperidone and risperidone normalized basal extracellular glutamate. Demonstration of glutamatergic abnormalities consistent with the NMDA glutamate receptor hypofunction model of schizophrenia as an early developmental consequence of prenatal immune action provides a model to identify novel early interventions targeting glutamatergic systems which play an important role in both positive and negative symptoms of schizophrenia.
Abstract Aim Studies characterizing treatment interventions in a naturalistic setting suggest that antidepressant and antipsychotic medications may be equally effective in improving clinical outcome ...in individuals at high risk for first-episode psychosis. Of interest, both beneficial as well as potentially adverse effects have been observed following fluoxetine treatment in a mouse prenatal immune activation model of relevance to psychosis prevention. We sought to extend those findings by examining the effects of fluoxetine, as well as the antipsychotic medication aripiprazole, in a rat prenatal immune activation model. Methods Pregnant Sprague–Dawley rats were injected with poly I:C or saline on gestational day 14. Offspring of poly I:C and saline-treated dams received fluoxetine (10.0 mg/kg/d), aripiprazole (0.66 mg/kg/d), or vehicle from postnatal days 35 to 70. Locomotor responses to novelty, saline injection, and amphetamine (1 and 5 mg/kg) were determined at three months, i.e., 21 days following drug discontinuation. Results Both fluoxetine and aripiprazole had beneficial effects on behavioral response to amphetamine (1 mg/kg) at 3 months, ameliorating the impact of prenatal immune activation on offspring of poly I:C-treated dams. Significantly, both drugs also exerted effects in offspring of control (saline-treated) dams on locomotor response to injection. Conclusions Fluoxetine and aripiprazole pretreatment of poly I:C offspring from postnatal days 35 to 70 stabilized response to amphetamine exposure persisting through 3 months of age, similar to earlier findings in mice that fluoxetine treatment following prenatal immune activation prevented altered locomotor response to amphetamine. The current data also confirm earlier findings of potential adverse behavioral effects in offspring of control dams following treatment with fluoxetine and antipsychotic medications, highlighting the potential for both therapeutic as well as safety concerns with exposure to preventive pharmacological treatments over the course of adolescent development. Further study is needed to determine clinical and epidemiological consequences of these pre-clinical findings.
Individuals with fear-associated conditions such as panic disorder (PD) and posttraumatic stress disorder (PTSD) display increased emotional responses to interoceptive triggers, such as CO2 ...inhalation, that signal a threat to physiological homeostasis. Currently, effector systems and mechanisms underlying homeostatic modulation of fear memory are not well understood. In this regard, the renin angiotensin system (RAS), particularly the angiotensin receptor type 1 (AT1R), a primary homeostatic regulatory target, has gained attention. RAS polymorphisms have been reported in PD and PTSD, and recent studies report AT1R-mediated modulation of fear extinction. However, contribution of AT1Rs in fear evoked by the interoceptive threat of CO2 has not been investigated. Using pharmacological, behavioral, and AT1R/ACE gene transcription analyses, we assessed central AT1R recruitment in CO2-associated fear. CO2 inhalation led to significant AT1R and ACE mRNA upregulation in homeostatic regulatory regions, subfornical organ (SFO) and paraventricular nucleus (PVN), in a temporal manner. Intracerebroventricular infusion of selective AT1R antagonist, losartan, significantly attenuated freezing during CO2 inhalation, and during re-exposure to CO2 context, suggestive of AT1R modulation of contextual fear. Regional Fos mapping in losartan-treated mice post-behavior revealed significantly attenuated labeling in areas regulating defensive behavior, contextual fear, and threat responding; such as, the bed nucleus of stria terminalis, dorsal periaqueductal gray, hypothalamic nuclei, hippocampus, and prefrontal areas such as the prelimbic, infralimbic, and anterior cingulate cortices. Sub-regions of the amygdala did not show CO2-associated AT1R regulation or altered Fos labeling. Collectively, our data suggests central AT1R recruitment in modulation of fear behaviors associated with CO2 inhalation via engagement of neurocircuits regulating homeostasis and defensive behaviors. Our data provides mechanistic insights into the interoceptive regulation of fear, relevant to fear related disorders such as PD and PTSD.
•CO2, an interoceptive threat, increases AT1 receptor and ACE mRNA in SFO and PVN.•AT1 receptor antagonist, losartan, attenuates CO2 evoked freezing and contextual fear.•Losartan reduced cFos in defensive behavior & threat response regulatory areas.•Brain AT1R circuits in CO2-evoked fear provide insights into panic disorder and PTSD.
Highlights • CO2 inhalation, a biological challenge for panic and fear elicits differential behavioral sensitivity in rat strains. • Long Evans and Wistar-Kyoto rats show significantly higher CO2 ...-evoked immobility than Wistar and Sprague–Dawley rats. • CO2 -sensitive strains have decreased TPH2-positive serotonergic neurons in panic regulatory raphe subnuclei, DRVL-VLPAG. • CO2 -sensitive strains have increased DβH-positive noradrenergic neurons in the locus coeruleus, a CO2 -chemosensitive site. • Rodent models of CO2 -sensitivity will facilitate understanding of CO2 -hypersensitivity in panic and anxiety disorders.
An important role of pH homeostasis has been suggested in the physiology of panic disorder, with acidosis as an interoceptive trigger leading to fear and panic. Identification of novel mechanisms ...that can translate acidosis into fear will promote a better understanding of panic physiology. The current study explores a role of the subfornical organ (SFO), a blood–brain barrier compromised brain area, in translating acidosis to fear‐relevant behaviors. We performed SFO‐targeted acidification in male, wild‐type mice and mice lacking microglial acid‐sensing G protein–coupled receptor—T‐cell death‐associated gene 8 (TDAG8). Localized SFO acidification evoked significant freezing and reduced exploration that was dependent on the presence of acid‐sensor TDAG8. Acidosis promoted the activation of SFO microglia and neurons that were absent in TDAG8‐deficient mice. The assessment of regional neuronal activation in wild‐type and TDAG8‐deficient mice following SFO acidification revealed significant acidosis and genotype‐dependent alterations in the hypothalamus, amygdala, prefrontal cortex, and periaqueductal gray nuclei. Furthermore, mapping of interregional co‐activation patterns revealed that SFO acidosis promoted positive hypothalamic‐cortex associations and desynchronized SFO‐cortex and amygdala‐cortex associations, suggesting an interplay of homeostatic and fear regulatory areas. Importantly, these alterations were not evident in TDAG8‐deficient mice. Overall, our data support a regulatory role of subfornical organ microglial acid sensing in acidosis‐evoked fear, highlighting a centralized role of blood–brain barrier compromised nodes in interoceptive sensing and behavioral regulation. Identification of pathways by which humoral information can modulate fear behavior is relevant to panic disorder, where aberrant interoceptive signaling has been reported.
Homeostatic pH disturbance, specifically acidosis, is a trigger for panic attacks relevant to panic disorder; however, contributory mechanisms are still unclear. Here we report a role of microglial acid chemosensory G protein–coupled receptor, T‐cell death‐associated gene 8, within the subfornical organ in orchestrating acidosis‐evoked, fear‐associated behavior of relevance to panic.
•Subfornical organ (SFO) infusion of IL-1R1 antagonist attenuates CO2-evoked fear.•SFO IL-1RA prevents enhanced conditioned fear and delayed fear extinction by CO2.•SFO IL-1RA regulation of fear ...engages cortico-amygdala-PAG subregions.•CO2 regulates IL-1β in monocytes and SFO; IL-1R1 localizes to SFO endothelium.•SFO IL-1R1 is a “bottom-up” neuroimmune mediator of fear relevant to panic and PTSD.
Impaired threat responding and fear regulation is a hallmark of psychiatric conditions such as post-traumatic stress disorder (PTSD) and Panic Disorder (PD). Most studies have focused on external psychogenic threats to study fear, however, accumulating evidence suggests a primary role of homeostatic perturbations and interoception in regulating emotional behaviors. Heightened reactivity to interoceptive threat carbon dioxide (CO2) inhalation associates with increased risk for developing PD and PTSD, however, contributory mechanisms and molecular targets are not well understood. Previous studies from our group suggested a potential role of interleukin 1 receptor (IL-1R1) signaling within BBB-devoid sensory circumventricular organ, the subfornical organ (SFO) in CO2-evoked fear. However, the necessity of SFO-IL-1R1 in regulating CO2-associated spontaneous fear as well as, long-term fear potentiation relevant to PD/PTSD has not been investigated. The current study tested male mice with SFO-targeted microinfusion of the IL-1R1 antagonist (IL-1RA) or vehicle in a recently developed CO2-startle-fear conditioning-extinction paradigm. Consistent with our hypothesis, SFO IL-1RA treatment elicited significant attenuation of freezing and increased rearing during CO2 inhalation suggesting SFO-IL1R1 regulation of spontaneous fear to CO2. Intriguingly, SFO IL-1RA treatment normalized CO2-associated potentiation of conditioned fear and impaired extinction a week later suggesting modulation of long-term fear by SFO-IL-1R1 signaling. Post behavior FosB mapping revealed recruitment of prefrontal cortex-amygdala-periaqueductal gray (PAG) areas in SFO-IL-1RA mediated effects. Additionally, we localized cellular IL-1R1 expression within the SFO to blood vessel endothelial cells and observed CO2-induced alterations in IL-1β/IL-1R1 expression in peripheral mononuclear cells and SFO. Lastly, CO2-evoked microglial activation was attenuated in SFO-IL-1RA treated mice. These observations suggest a peripheral monocyte-endothelial-microglia interplay in SFO-IL-1R1 modulation of CO2-associated spontaneous fear and delayed fear memory. Collectively, our data highlight a novel, “bottom-up” neuroimmune mechanism that integrates interoceptive and exteroceptive threat processing of relevance to fear-related pathologies.
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