Commensal microbiota drive the functional diversification of colon macrophages.

Mononuclear phagocytes are a heterogeneous population of leukocytes essential for immune homeostasis that develop tissue-specific functions due to unique transcriptional programs driven by local microenvironmental cues. Single cell RNA sequencing (scRNA-seq) of colonic myeloid cells from specific pathogen free (SPF) and germ-free (GF) C57BL/6 mice revealed extensive heterogeneity of both colon macrophages (MPs) and dendritic cells (DCs). Modeling of developmental pathways combined with inference of gene regulatory networks indicate two major trajectories from common CCR2+ precursors resulting in colon MP populations with unique transcription factors and downstream target genes. Compared to SPF mice, GF mice had decreased numbers of total colon MPs, as well as selective proportional decreases of two major CD11c+CD206intCD121b+ and CD11c-CD206hiCD121b- colon MP populations, whereas DC numbers and proportions were not different. Importantly, these two major colon MP populations were clearly distinct from other colon MP populations regarding their gene expression profile, localization within the lamina propria (LP) and ability to phagocytose macromolecules from the blood. These data uncover the diversity of intestinal myeloid cell populations at the molecular level and highlight the importance of microbiota on the unique developmental as well as anatomical and functional fates of colon MPs.

The Behavioral Sequelae of Social Defeat Require Microglia and Are Driven by Oxidative Stress in Mice.

Chronic social defeat (CSD) in male mice can produce anxiety and aberrant socialization. Animals susceptible to CSD show activation of microglia, which have elevated levels of oxidative stress markers. We hypothesized that microglia and reactive oxygen species (ROS) production contribute to the CSD stress-induced changes in affective behavior. First, we selectively depleted microglia (99%) by administering the CSF1R (colony-stimulating factor 1 receptor) antagonist PLX5622 before and during the 14 d CSD procedure. Microglia-depleted mice in contrast to nondepleted mice were protected from the stress effects measured by light/dark and social interaction tests. ROS production, measured histochemically following dihydroethidium administration, was elevated by CSD, and the production was reduced to basal levels in mice lacking microglia. The deleterious stress effects were also blocked in nondepleted mice by continuous intracerebral administration of N-acetylcysteine (NAC), a ROS inhibitor. In a second experiment, at the end of the CSD period, PLX5622 was discontinued to allow microglial repopulation. After 14 d, the brain had a full complement of newly generated microglia. At this time, the mice that had previously been protected now showed behavioral deficits, and their brain ROS production was elevated, both in all brain cells and in repopulated microglia. NAC administration during repopulation prevented the behavioral decline in the repopulated mice, and it supported behavioral recovery in nondepleted stressed mice. The data suggest that microglia drive elevated ROS production during and after stress exposure. This elevated ROS activity generates a central state supporting dysregulated affect, and it hinders the restoration of behavioral and neurochemical homeostasis after stress cessation.SIGNIFICANCE STATEMENT Chronic psychosocial stress is associated with psychiatric disorders such as depression and anxiety. Understanding the details of CNS cellular contributions to stress effects could lead to the development of intervention strategies. Inflammation and oxidative stress are positively linked to depression severity, but the cellular nature of these processes is not clear. The chronic social defeat (CSD) paradigm in mice produces mood alterations and microglial activation characterized by elevated reactive oxygen species (ROS) production. The depletion of microglia or ROS inhibition prevented adverse stress effects. Microglial repopulation of the brain post-CSD reintroduced adverse stress effects, and ROS inhibition in this phase protected against the effects. The results suggest that stress-induced microglial ROS production drives a central state that supports dysregulated affective behavior.

Social defeat induces depressive-like states and microglial activation without involvement of peripheral macrophages.

BACKGROUND: We are interested in the causal interactions between psychological stress and activity within different compartments of the immune system. Psychosocial stress has been reported to not only alter microglia morphology but also produce anxiety-like and depressive-like effects by triggering CNS infiltration of macrophages from the periphery. We sought to test these phenomena in a somewhat different but standardized model of chronic social defeat (SD) stress. METHODS: We used a paradigm of dyadic home pairing of dominant and subordinate mice that has been validated to induce powerful anxiety-like and depressive-like effects manifested by behavior assessed in social tasks. We administered the SD stress for 3 days (acute SD) or 14 days (chronic SD) and looked for monocyte entry into the brain by three independent means, including CD45 activation states assessed by flow cytometry and tracking fluorescently tagged peripheral cells from Ccr2 (wt/rfp) and Ubc (gfp/gfp) reporter mice. We further characterized the effects of SD stress on microglia using quantitative morphometric analysis, ex vivo phagocytosis assays, flow cytometry, and immunochemistry. RESULTS: We saw no evidence of stress-induced macrophage entry after acute or chronic defeat stress. In comparison, brain infiltration of peripheral cells did occur after endotoxin administration. Furthermore, mutant mice lacking infiltrating macrophages due to CCR2 knockout developed the same degree of chronic SD-induced depressive behavior as wildtype mice. We therefore focused more closely on the intrinsic immune cells, the microglia. Using Cx3cr1 (wt/gpf) microglial reporter mice, we saw by quantitative methods that microglial morphology was not altered by stress at either time point. However, chronic SD mice had elevated numbers of CD68(hi) microglia examined by flow cytometry. CD68 is a marker for phagocytic activity. Indeed, these cells ex vivo showed elevated phagocytosis, confirming the increased activation status of chronic SD microglia. Finally, acute SD but not chronic SD increased microglial proliferation, which occurred selectively in telencephalic stress-related brain areas. CONCLUSIONS: In the SD paradigm, changes in CNS-resident microglia numbers and activation states might represent the main immunological component of the psychosocial stress-induced depressive state.

PACAP-deficient mice show attenuated corticosterone secretion and fail to develop depressive behavior during chronic social defeat stress.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) regulates activation of the hypothalamic-pituitary-adrenal (HPA) axis and the adrenal gland in response to various stressors. We previously found that in response to acute psychological stress (restraint), elevated corticotrophin-releasing hormone (CRH) mRNA levels in the hypothalamic paraventricular nucleus (PVN) as well as elevated plasma corticosterone (CORT) were profoundly attenuated in PACAP-deficient mice. To determine whether HPA axis responses and stress-induced depressive-like behaviors in a chronic stress paradigm are affected by PACAP deficiency, we subjected mice to 14 days of social defeat stress. Defeat-exposed PACAP-/- mice showed a marked attenuation of stress-induced increases in serum CORT levels, cellular PVN ΔFosB immunostaining, and depressive-like behaviors (social interaction and forced swim tests) compared to wild-type control mice. The PACAP-/- mice showed reduced PVN FosB-positive cell numbers, but relatively elevated cell counts in several forebrain areas including the medial prefrontal cortex, after social stress. PACAP appears to be specific for mediating HPA activation only in psychological stress because marked elevations in plasma CORT after a systemic stressor (lipopolysaccharide administration) occurred regardless of genotype. We conclude that chronically elevated CORT is a key component of depressive effects of social defeat, and that attenuation of the CORT response at the level of the PVN, as well as extrahypothalamic forebrain regions, in PACAP-deficient mice protects from development of depressive behavior.

Environmental enrichment confers stress resiliency to social defeat through an infralimbic cortex-dependent neuroanatomical pathway.

Enriched environmental (EE) housing dampens stress-induced alterations in neurobiological systems, promotes adaptability, and extinguishes submissive behavioral traits developed during social defeat stress (SD). In the present study, we hypothesized that enrichment before SD can confer stress resiliency and, furthermore, that neuronal activity in the prefrontal cortex (PFC) is requisite for this resiliency. To test these hypotheses, mice were housed in EE, standard (SE), or impoverished (IE) housing and then exposed to SD. EE conferred resilience to SD as measured in several behavioral tasks. EE-housed mice expressed elevated FosB/ΔFosB immunostaining in areas associated with emotional regulation and reward processing, i.e., infralimbic, prelimbic, and anterior cingulate cortices, amygdala, and nucleus accumbens, and this expression was mostly preserved in mice receiving EE followed by SD. In contrast, in SE- or IE-housed animals, SD increased maladaptive behaviors and greatly reduced FosB/ΔFosB staining in the forebrain. We tested the putative involvement of the PFC in mediating resilience by lesioning individual regions of the PFC either before or after EE housing and then exposing the mice to SD. We found that discrete lesions of the infralimbic but not prelimbic or cingulate cortex made before but not after EE abolished the behavioral resiliency to stress afforded by EE and attenuated FosB/ΔFosB expression in the accumbens and amygdala while increasing it in the paraventricular hypothalamic nucleus. These data suggest that pathological ventromedial PFC outputs to downstream limbic targets could predispose an individual to anxiety disorders in stressful situations, whereas enhanced ventromedial PFC outputs could convey stress resilience.

NF-kappaB activity affects learning in aversive tasks: possible actions via modulation of the stress axis.

The role of altered activity of nuclear factor kappaB (NF-kappaB) in specific aspects of motivated behavior and learning and memory was examined in mice lacking the p50 subunit of the NF-kappaB/rel transcription factor family. Nfkb1-deficient mice are unable to produce p50 and show specific susceptibilities to infections and inflammatory challenges, but the behavioral phenotype of such mice has been largely unexamined, owing in large part to the lack of understanding of the role of NF-kappaB in nervous system function. Here we show that Nfkb1 (p50) knockout mice more rapidly learned to find the hidden platform in the Morris water maze than did wildtype mice. The rise in plasma corticosterone levels after the maze test was greater in p50 knockout than in wildtype mice. In the less stressful Barnes maze, which tests similar kinds of spatial learning, the p50 knockout mice performed similarly to control mice. Adrenalectomy with corticosterone replacement eliminated the differences between p50 knockout and wildtype mice in the water maze. Knockout mice showed increased levels of basal anxiety in the open-field and light/dark box tests, suggesting that their enhanced escape latency in the water maze was due to activation of the stress (hypothalamic-pituitary-adrenal) axis leading to elevated corticosterone production by strongly but not mildly anxiogenic stimuli. The results suggest that, as in the immune system, p50 in the nervous system normally serves to dampen NF-kappaB-mediated intracellular activities, which are manifested physiologically through elevated stress responses to aversive stimuli and behaviorally in the facilitated escape performance in learning tasks.

Reduced evoked fos expression in activity-related brain regions in animal models of behavioral depression.

A previous study showed that two mouse models of behavioral depression, immune system activation and depletion of brain monoamines, are accompanied by marked reductions in stimulated neural activity in brain regions involved in motivated behavior. The present study tested whether this effect is common to other depression models by examining the effects of repeated forced swimming, chronic subordination stress or acute intraventricular galanin injection - three additional models - on baseline or stimulated c-fos expression in several brain regions known to be involved in motor or motivational processes (secondary motor, M2, anterior piriform cortex, APIR, posterior cingulate gyrus, CG, nucleus accumbens, NAC). Each of the depression models was found to reduce the fos response stimulated by exposure to a novel cage or a swim stress in all four of these brain areas but not to affect the response of a stress-sensitive region (paraventricular hypothalamus, PVH) that was included for control purposes. Baseline fos expression in these structures was either unaffected or affected in an opposite direction to the stimulated response. Pretreatment with either desmethylimipramine (DMI) or tranylcypromine (tranyl) attenuated these changes. It is concluded that the pattern of a reduced neural function of CNS motor/motivational regions with an increased function of stress areas is common to 5 models of behavioral depression in the mouse and is a potential experimental analog of the neural activity changes occurring in the clinical condition.

Depressive behavior in mice due to immune stimulation is accompanied by reduced neural activity in brain regions involved in positively motivated behavior.

BACKGROUND: Immune stimulation inhibits positively motivated behavior and induces depressive illness. To help clarify the mechanism of these effects, neural activity in response to a positive stimulus was examined in brain regions associated with positively motivated activity defined on the basis of prior behavioral studies of central alpha1-adrenoceptor action. METHODS: Mice pretreated with either lipopolysaccharide or, for comparison, reserpine were exposed to a motivating stimulus (fresh cage) and subsequently assayed for fos expression and mitogen-activated protein kinase (MAPK) phosphorylation, two measures associated with alpha1-adrenoceptor-dependent neural activity, in several positive-activity-related (motor, piriform, cingulate cortex, nucleus accumbens, locus coeruleus) and stress-related brain regions (paraventricular hypothalamus, bed nucleus stria terminalis). RESULTS: Both lipopolysaccharide and reserpine pretreatment abolished fresh cage-induced fos expression and MAPK activation in the positive activity-related brain regions but enhanced these measures in the stress-related areas. CONCLUSIONS: The results support the hypothesis that immune activation reduces alpha1-adrenoceptor-related signaling and neural activity in brain regions associated with positive activity while it increases these functions in stress-associated areas. It is suggested that neural activities of these two types of brain regions are mutually antagonistic and that a reciprocal shift toward the stress regions is a factor in the loss of positively motivated behaviors in sickness behavior and depressive illness.

Role of CNS alpha1-adrenoceptor activity in central fos responses to novelty.

The present study investigated, by use of fos immunohistochemistry, whether the functional activity of alpha(1)-adrenoceptors is elevated during heightened behavioral activity in brain regions shown earlier to contain motoric alpha(1)-receptors. In confirmation, marked c-fos responses that were blocked by an alpha(1)-antagonist (prazosin) were found in four of these brain regions (secondary motor, cingulate, piriform cortices, and nucleus accumbens) of animals exposed to a mildly novel environment (clean cage), which elicits a high degree of sustained exploratory activity. Experimental restriction of exploratory activity in the novel cage by a small enclosure did not reduce the fos responses in these areas, and in fact, enhanced gene expression when carried out in home-caged animals suggesting that the fos response may be more closely associated with the motivation to be active rather than activity itself. Experiments with locally administered alpha(1)-agonists and antagonists in the cortex by reverse dialysis showed that the above mentioned alpha(1)-dependent-fos responses were the result of activation of local alpha(1)-receptors in these brain regions. Unlike the aforementioned brain regions, the fos response of the locus coeruleus was not blocked by prazosin, and this nucleus also showed a marked fos increase to prazosin itself possibly as a compensatory response to the blockade of forebrain alpha(1)-receptors.

Co-regulation of female sexual behavior and pregnancy induction: an exploratory synthesis.

This paper will review both new and old data that address the question of whether brain mechanisms involved in reproductive function act in a coordinated way to control female sexual behavior and the induction of pregnancy/pseudopregnancy (P/PSP) by vaginocervical stimulation. Although it is clear that female sexual behavior, including pacing behavior, is important for induction of P/PSP, there has been no concerted effort to examine whether or how common mechanisms may control both functions. Because initiation of P/PSP requires that the female receive vaginocervical stimulation, central mechanisms controlling P/PSP may be modulated by or interactive with those that control female sexual behavior. This paper presents a synthesis of the literature and recent data from our lab for the purpose of examining whether there are interactions between behavioral and neuroendocrine mechanisms which reciprocally influence both reproductive functions.