Probes for C-FOS

Although anesthesia is commonly used in the fields of medicine and scientific research, the neural mechanisms and circuits through which it produces analgesia is still unclear. Utilizing c-fos labeling of neuronal activity, this project aimed to investigate the brain regions of C57BL/6 mice, which become activated subsequent to isoflurane anesthesia. RNAscope in situ hybridization was used to examine c-fos mRNA activation in the brain. Confocal microscopy was utilized to locate and characterize brain regions displaying c-Fos activation. Finally, manual quantification of c-fos activation in identified brain regions was conducted through Fiji software. The brain regions identified resemble brain areas that have been associated with pain regulation in literature, including the central nucleus of amygdala (CeA), paraventricular nucleus of the hypothalamus (PVN), centrally-projecting Edinger-Westphal nucleus (EWcp), piriform cortex (PC), and para-supraoptic nucleus (ParaSON). Furthermore, the CeA displayed the greatest average number of positive cells and the densest activation, supporting its importance in pain and analgesia. The identified brain regions validate the prominent findings of prior studies, which also found c-Fos activation subsequent to isoflurane anesthesia in the CeA, PVN, and ParaSON (Hua et al., Nat Neurosci, 2020). New regions of c-fos activation, including the EWcp and PC, found in this study are in need of further exploration. PC activation may also be caused by smell from isoflurane. The connections and coordination which the identified brain regions have in producing analgesia is also an area for future investigation. This study is supported by Duke University Anesthesiology Fund and NIH grant R01-DE29342. This study is supported by Duke University Anesthesiology Fund and NIH grant R01-DE29342.

Twenty-five to fifty percent of patients undergoing chemotherapy will develop anticipatory nausea and vomiting (ANV), in which symptoms occur in anticipation of treatment. ANV is triggered by environmental cues and shows little response to traditional antiemetic therapy, suggesting that unique neural pathways mediate this response. Understanding the underlying neural mechanisms of this disorder is critical to the development of novel therapeutic interventions. The purpose of the present study was to identify brain areas activated during ANV and characterize sex differences in both the behavior and the brain areas activated during ANV. We used a rat model of ANV by pairing a novel context with the emetic drug lithium chloride (LiCl) to produce conditioned nausea behaviors in the LiCl-paired environment. We quantitated gaping, an analog of human vomiting, after acute or repeated LiCl in a unique environment. To identify brain regions associated with gaping, we measured c-fos activation by immunochemical staining after these same treatments. We found that acute LiCl activated multiple brain regions including the supraoptic nucleus of the hypothalamus, central nucleus of the amygdala, nucleus of the solitary tract and area postrema, none of which were activated during ANV. ANV activated c-fos expression in the frontal cortex, insula and paraventricular nucleus of the hypothalamus of males but not females. These data suggest that therapies such as ondansetron which target the area postrema are not effective in ANV because it is not activated during the ANV response. Further studies aimed at characterizing the neural circuits and cell types that are activated in the conditioned nausea response will help identify novel therapeutic targets for the treatment of this condition, improving both quality of life and outcomes for patients undergoing chemotherapy.

Within the hindbrain, serotonin (5-HT) functions as a modulator of the central glucagon-like peptide-1 (GLP-1) system. This interaction between 5-HT and GLP-1 is achieved via 5-HT2C and 5-HT3 receptors and is relevant for GLP-1-mediated feeding behavior. The central GLP-1 system is activated by various stressors, activates the hypothalamic pituitary adrenocortical (HPA) axis, and contributes to stress-related behaviors. Whether 5-HT modulates GLP-1's role in the stress response in unknown. We hypothesized that the serotonergic modulation of GLP-1-producing neurons (i.e., PPG neurons) is stimuli-specific and that stressed-induced PPG activity is one of the modalities in which 5-HT plays a role. In this study, we investigated the roles of 5-HT2C and 5-HT3 receptors in mediating the activation of PPG neurons in the nucleus tractus solitarius (NTS) following exposure to three different acute stressors: lithium chloride (LiCl), noncontingent cocaine (Coc), and novel restraint stress (RES). Results showed that increased c-Fos expression in PPG neurons following LiCl and RES-but not Coc-is dependent on hindbrain 5-HT2C and 5-HT3 receptor signaling. Additionally, stressors that depend on 5-HT signaling to activate PPG neurons (i.e., LiCl and RES) increased c-Fos expression in 5-HT-expressing neurons within the caudal raphe (CR), specifically in the raphe magnus (RMg). Finally, we showed that RMg neurons innervate NTS PPG neurons and that some of these PPG neurons lie in close proximity to 5-HT axons, suggesting RMg 5-HT-expressing neurons are the source of 5-HT input responsible for engaging NTS PPG neurons. Together, these findings identify a direct RMg to NTS pathway responsible for the modulatory effect of 5-HT on the central GLP-1 system-specifically via activation of 5-HT2C and 5-HT3 receptors-in the facilitation of acute stress responses.

Acetylcholine is an important modulator of striatal activity and it is vital to controlling striatal-dependent behaviors, including motor and cognitive functions. Despite this significance, the mechanisms determining how acetylcholine impacts striatal signaling are still not fully understood. In particular, little is known about the role of nicotinic acetylcholine receptors (nAChRs) expressed by striatal interneurons. In the present study, we used fluorescent in situ hybridization (FISH) to determine which neuronal types express the most prevalent beta2 nicotinic subunit in the mouse striatum. Our data support a common view that nAChR expression is mostly restricted to striatal interneurons. Surprisingly though, cholinergic interneurons (CINs) were identified as a population with the highest expression of beta2 nicotinic subunit. To investigate the functional significance of beta2-containing nAChRs in striatal interneurons, we deleted them by injecting the AAV-Cre vector into the striatum of beta2-flox/flox male mice. The deletion led to alterations in several behavioral domains, namely to an increased anxiety-like behavior, decrease in sociability ratio, deficit in discrimination learning and increased amphetamine-induced hyperlocomotion and c-Fos expression in mice with beta2 deletion. Further colocalization analysis showed that the increased c-Fos expression was present in both medium spiny neurons and presumed striatal interneurons. The present study concludes, that despite being relatively rare, beta2-containing nAChRs are primarily expressed in striatal neurons by CINs and play a significant role in behavior.SIGNIFICANCE STATEMENTA large variety of nicotinic acetylcholine receptors are expressed in the striatum, a brain region that is crucial in the control of behavior. The complexity of receptors with different functions is hindering our understanding of mechanisms through which striatal acetylcholine modulates behavior. We focused on the role of a small population of beta2-containing nicotinic acetylcholine receptors. We identified neuronal types expressing these receptors and determined their impact in the control of explorative behavior, anxiety-like behavior, learning and sensitivity to stimulants. Additional experiments showed that these alterations were associated with an overall increased activity of striatal neurons. Thus, the small population of nicotinic receptors represents an interesting target for a modulation of response to stimulant drugs and other striatal-based behavior.

Although itch and pain have many similarities, they are completely different in perceptual experience and behavioral response. In recent years, we have a deep understanding of the neural pathways of itch sensation transmission. However, there are few reports on the role of non-neuronal cells in itch. Microglia are known to play a key role in chronic neuropathic pain and acute inflammatory pain. It is still unknown whether microglia are also involved in regulating the transmission of itch sensation. In the present study, we used several kinds of transgenic mice to specifically deplete CX3CR1+ central microglia and peripheral macrophages together (whole depletion), or selectively deplete central microglia alone (central depletion). We observed that the acute itch responses to histamine, compound 48/80 and chloroquine were all significantly reduced in mice with either whole or central depletion. Spinal c-fos mRNA assay and further studies revealed that histamine and compound 48/80, but not chloroquine elicited primary itch signal transmission from DRG to spinal npr1- and somatostatin-positive neurons relied on microglial CX3CL1-CX3CR1 pathway. Our results indicated that central microglia were involved in multiple types of acute chemical itch transmission, while the underlying mechanisms for histamine dependent and non-dependent itch transmission were different that the former required the CX3CL1-CX3CR1 signal pathway.

Introduction The control of energy balance involves communication of peripheral hormones with brain regions controlling food intake and energy expenditure such as the arcuate nucleus of the hypothalamus (ARC). Within the ARC, two primary neuronal subpopulations control energy balance: proopiomelanocortin (POMC) neurons, which reduce food intake and increase energy expenditure; and agouti-related protein (AgRP) neurons, which inhibit POMC neurons and conversely increase food intake and suppress energy expenditure. These circuits are typically disrupted by high fat diet (HFD) leading to a chronic state of energy imbalance and obesity. Accumulating evidence suggests that HFD-induced obesity is associated with deficiency of angiotensin (Ang)-(1-7), a protective renin-angiotensin system hormone. Our recent data show that systemically administered Ang-(1-7) induces adipose thermogenesis to enhance energy expenditure and promote weight loss. We propose that effects of Ang-(1-7) on energy balance involve activation of ARC neurocircuits, but this has not been tested. Additionally, the localization and neuronal subpopulations expressing Ang-(1-7) mas receptors (MasR) in the ARC is unknown. In this study, we hypothesized that: Ang-(1-7) activates ARC neurons; MasR are expressed in the ARC and are primarily colocalized with POMC neurons; and the ability of Ang-(1-7) to activate ARC neurons as well as co-localization of MasR with POMC neurons is disrupted following chronic HFD. Methods Male C57Bl/6J mice were fed a 60% HFD or matched control diet ad libitum for 12 weeks. Mice then received subcutaneous injection of Ang-(1-7) [2 mg/kg] to induce neuronal activation in the ARC, as measured by c-fos gene expression (n=4-6/group). In a second cohort of mice, RNAscope in situ hybridization was performed on coronal ARC sections to determine co-localization of MasR mRNA within POMC versus AgRP neurons (n=5/group). Results We found that Ang-(1-7) increases the number of c-fos positive cells in the ARC (39±6 vs. 19±3 saline; p=0.022) in control diet mice. Ang-(1-7)-mediated activation of ARC neurons was attenuated in HFD mice (34±3 vs. 23±4 saline; p=0.185). The rostral-medial-caudal distribution of ARC MasR was similar between control diet and HFD mice, with no difference in percentage of MasR positive neurons between groups (18±1 and 15±5%, respectively; p=0.733). MasR were more highly co-localized to POMC versus AgRP neurons, with HFD tending to reduce these co-localizations (MasR/POMC: 49±10 control vs. 33±5% HFD, p=0.199; MasR/AgRP: 36±11 control vs.16±7% HFD, p=0.209). Conclusions These findings suggest that chronic HFD reduces the ability of Ang-(1-7) to acutely activate neurons in the ARC. Further, HFD disrupts co-localization of MasR with POMC and AgRP neurons in the ARC indicating disconnect in the endogenous neurocircuitry controlling energy balance. Further studies are needed to explore the importance of MasR in these neuronal subpopulations for energy balance, to determine the potential for targeting of Ang-(1-7) as an innovative pharmacological strategy for obesity treatment.

Pain and itch are distinct sensations arousing evasion and compulsive desire for scratching, respectively. It's unclear whether they could invoke different neural networks in the brain. Here, we use the type 1 herpes simplex virus H129 strain to trace the neural networks derived from two types of dorsal root ganglia (DRG) neurons: one kind of polymodal nociceptors containing galanin (Gal) and one type of pruriceptors expressing neurotensin (Nts). The DRG microinjection and immunosuppression were performed in transgenic mice to achieve a successful tracing from specific types of DRG neurons to the primary sensory cortex. About one-third of nuclei in the brain were labeled. More than half of them were differentially labeled in two networks. For the ascending pathways, the spinothalamic tract was absent in the network derived from Nts-expressing pruriceptors, and the two networks shared the spinobulbar projections but occupied different subnuclei. As to the motor systems, more neurons in the primary motor cortex and red nucleus of the somatic motor system participated in the Gal-containing nociceptor-derived network, while more neurons in the nucleus of the solitary tract (NST) and the dorsal motor nucleus of vagus nerve (DMX) of the emotional motor system was found in the Nts-expressing pruriceptor-derived network. Functional validation of differentially labeled nuclei by c-Fos test and chemogenetic inhibition suggested the red nucleus in facilitating the response to noxious heat and the NST/DMX in regulating the histamine-induced scratching. Thus, we reveal the organization of neural networks in a DRG neuron type-dependent manner for processing pain and itch.

Background Aberrant prefrontal cortex (PFC) activity occurs in patients with neuropsychiatric disorders during sustained attention tasks, suggesting that PFC dysfunction underlies attention deficits in these patients. However, the mechanisms by which the PFC regulates sustained attention remain unclear. Methods Behavioral testing and c-Fos immunohistochemistry during performance of a touchscreen sustained attention task (rCPT) in mice. In vivo calcium and norepinephrine imaging to assess patterns of activity during the rCPT. In vivo electrophysiology to detect how the medial PFC (mPFC) and locus coeruleus (LC) communicate during the rCPT. For assessment of molecular function in subsets of mPFC neurons that receive contact from the LC, we used RNAscope and bulk RNA-sequencing. Results We found that the LC and mPFC synchronized their activity during the rCPT, and imaging of neuronal activity in the mPFC revealed that mPFC neurons have heterogeneous response patterns during rCPT performance, with some neurons increasing their calcium activity during stimulus orientation and some neurons increasing their calcium activity during behavioral responses. To determine the molecular identities of mPFC neurons that connect with the LC, we used RNAscope to find that mPFC neurons receiving LC contact are primarily GABAergic, while mPFC neurons projecting to the LC are primarily excitatory. Using bulk RNA-sequencing, we further found that depolarization of LC inputs to the mPFC caused enrichment of a host of transcripts in mPFC tissue. Conclusions We uncover unique biomarkers of neuronal function in the LC-mPFC circuit, providing insight into potential therapeutic targets for attentional regulation in disorders such as ADHD, major depressive disorder, and schizophrenia.

While toll-like receptors (TLRs), which mediate innate immunity, are known to play an important role in host defense, recent work suggest their involvement in some integrated behaviors, including anxiety, depressive and cognitive functions. Here, we investigated the potential involvement of the flagellin receptor, TLR5, in anxiety, depression and cognitive behaviors using male TLR5 knock-out (KO) mice. We aobserved a specific low level of basal anxiety in TLR5 KO mice with an alteration of the hypothalamo-pituitary axis (HPA) response to acute restraint stress, illustrated by a decrease of both plasma corticosterone level and c-fos expression in the hypothalamic paraventricular nucleus where TLR5 was expressed, compared to WT littermates. However, depression and cognitive-related behaviors were not different between TLR5 KO and WT mice. Nor there were significant changes in the expression of some cytokines (IL-6, IL-10 and TNF-α) and other TLRs (TLR2, TLR3 and TLR4) in the prefrontal cortex, amygdala and hippocampus of TLR5 KO mice compared to WT mice. Moreover, mRNA expression of BDNF and glucocorticoid receptors in the hippocampus and amygdala, respectively, was not different. Finally, acute intracerebroventricular administration of flagellin, a specific TLR5 agonist, or chronic neomycin treatment did not exhibit a significant main effect, only a significant main effect of genotype was observed between TLR5 KO and WT mice. Together, those findings suggest a previously undescribed and specific role of TLR5 in anxiety and open original prospects in our understanding of the brain-gut axis function.

Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells (IHCs), are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons; in this case onto the sparse type II SG neurons. However, unlike glutamate signaling at the inner hair cell (IHC) -type I SG synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-type II SG synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-type II SG pathway to signal to the brain in response to moderate, non-damaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the vesicular glutamate transporters (VGLUTs) associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to type II SG neurons in knockout mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus (CN) in response to sound. From these analyses, we show for the first time that glutamatergic signaling at the OHC-type II SG synapse is capable of activating CN neurons even at moderate sound levels.SIGNIFICANCE STATEMENTEvidence suggests that cochlear outer hair cells (OHC) release glutamate onto type II spiral ganglion neurons only when exposed to loud sound, and that type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGLUT3 for synaptic glutamate release. We then used a genetic mouse line in which OHCs, but not IHCs, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC - type II SG pathway. Together these data indicate that glutamate signaling at the OHC-type II afferent synapse participates in auditory function at moderate sound levels.

Background: The Psychiatric Ratings using Intermediate Stratified Markers (PRISM) project focuses on understanding the biological background behind social deficits, specifically social withdrawal irrespective of diagnosis. Reduced connectional integrity in fiber tracts such as Forceps minor has been indicated in low social individuals as a part of the PRISM 1 project. These fiber tracts are also involved in the Default Mode Network (DMN) and the Social network and they share a common region, the Orbitofrontal Cortex (OFC).This study aims to back-translate the clinical data to preclinical studies and associate social dysfunction in rodents with DMN and particularly OFC. Parvalbumin interneurons are targeted based on their fundamental role in maintaining Excitatory Inhibitory (E/I) balance in brain circuits. Numerous studies indicate behavioral impairment in rodents by increasing excitability of PV+ interneurons. Methods: As an initial step, we characterized the population of projection neurons within OFCs by combining Cholera Toxin subunit B (CTB) as a retrograde tracer and In situ hybridization (ISH) technique (RNAscope). We identified the expression of mRNAs marking glutamatergic (vesicular glutamate transporter [VGLUT]) and GABAergic (vesicular GABA transporter [VGAT]) by using Slc17a7 and Slc32a1 probes. CTB was injected unilaterally in the left OFC (AP=2.68, ML=-0.8, DV=2.2). after 10 days mice were perfused and RNAscope assay was performed using RNAscope™ Multiplex Fluorescent kit (ACDBio™).For inducing hypoactivation of OFC, we introduced an excitatory DREADD (designer receptors exclusively activated by designer drugs) to PV+ interneurons by using a PV-Cre mouse line. Mice were injected either AAV-hSyn-DIO-hM3D(Gq)-mCherry virus (n=12) or AAV-hSyn-DIO-mCherry (n=12) as control virus. As a novel behavioral tool, Radiofrequency identification (RFID)-assisted SocialScan combined with video tracking has been used, which provides a long-term observation of social behaviors. Monitoring the behavior in groups of four was performed for 7 days in total. After two pre-application days, Clozapine-N-oxide (CNO) was injected three times on consecutive days intraperitoneally (5mg/kg) as an activator of hM3D. application days were followed by two post-application days. Mice were perfused and RNAscope was performed to visualize c-fos mRNA expression as neuronal activity marker, and PV expression to validate our virus and mouse line efficacy. Results: ISH results indicated VGLUT1 has the highest expression within projection neurons (81%). 6% are VGAT+ and only 3% are both VGLUT1/VGAT positive neurons. Despite demonstrating the GABAergic projection neurons as a minority, their crucial role as local interneurons to moderate the excitatory neurons is indisputable.In in vivo study, CNO administration induced social dysregulation in DREAAD mice, demonstrated by a reduction in different social parameters (approach, fight, etc.) in terms of duration. During post-application days, DREAAD mice showed significantly higher social interaction in all definedparameters (Social Approach: p=0.0009, unpaired T-test) and locomotion as a non-social parameter (p= 0.0207).Results from ISH support our hypothesis that DREADD activation of PV+ interneurons is followed by high expression of neuronal activity markers in these targeted interneurons. Conclusion: This study indicates that manipulation of PV+ interneurons using artificially engineered activating protein receptors, generates in effect activation of these interneurons, and this manipulation particularly in OFC could cause social dysfunction in mice.