c-Fos Immunoreactive Neurons Research Articles

Evidence for a Role of 5-HT-glutamate Co-releasing Neurons in Acute Stress Mechanisms.

A major subpopulation of midbrain 5-hydroxytryptamine (5-HT) neurons expresses the vesicular glutamate transporter 3 (VGLUT3) and co-releases 5-HT and glutamate, but the function of this co-release is unclear. Given the strong links between 5-HT and uncontrollable stress, we used a combination of c-Fos immunohistochemistry and conditional gene knockout mice to test the hypothesis that glutamate co-releasing 5-HT neurons are activated by stress and involved in stress coping. Acute, uncontrollable swim stress increased c-Fos immunoreactivity in neurons co-expressing VGLUT3 and the 5-HT marker tryptophan hydroxylase 2 (TPH2) in the dorsal raphe nucleus (DRN). This effect was localized in the ventral DRN subregion and prevented by the antidepressant fluoxetine. In contrast, a more controllable stressor, acute social defeat, had no effect on c-Fos immunoreactivity in VGLUT3-TPH2 co-expressing neurons in the DRN. To test whether activation of glutamate co-releasing 5-HT neurons was causally linked to stress coping, mice with a specific deletion of VGLUT3 in 5-HT neurons were exposed to acute swim stress. Compared to wildtype controls, the mutant mice showed increased climbing behavior, a measure of active coping. Wildtype mice also showed increased climbing when administered fluoxetine, revealing an interesting parallel between the behavioral effects of genetic loss of VGLUT3 in 5-HT neurons and 5-HT reuptake inhibition. We conclude that 5-HT-glutamate co-releasing neurons are recruited by exposure to uncontrollable stress. Furthermore, natural variation in the balance of 5-HT and glutamate co-released at the 5-HT synapse may impact stress susceptibility.

Corticotropin-releasing factor potentiates glutamatergic input and excitability of presympathetic neurons in the hypothalamus in spontaneously hypertensive rats

Hyperactivity of presympathetic neurons in the hypothalamic paraventricular nucleus (PVN) plays a key role in generating excess sympathetic output in hypertension. However, the mechanisms driving hyperactivity of PVN presympathetic neurons in hypertension are unclear. In this study, we determined the role of corticotropin-releasing factor (CRF) in the PVN in augmented glutamatergic input, neuronal excitability and sympathetic outflow in hypertension. The number of CRF or c-Fos immunoreactive neurons and CRF/c-Fos double-labeled neurons in the PVN was significantly greater in spontaneously hypertensive rats (SHRs) than in normotensive Wistar-Kyoto (WKY) rats. Blocking glutamatergic input reduced the CRF-potentiated excitability of spinally projecting PVN neurons. Furthermore, CRF knockdown via Crispr/Cas9 in the PVN decreased the frequencies of spontaneous firing and miniature excitatory postsynaptic currents (mEPSCs) in spinally projecting PVN neurons in SHRs. In addition, the mRNA and protein levels of CRFR1, but not CRFR2, in the PVN were significantly higher in SHRs than in WKY rats. Blocking CRFR1 with NBI-35965, but not blocking CRFR2 with Antisauvagine-30, reduced the frequencies of spontaneous firing and mEPSCs of spinally projecting PVN neurons in SHRs. Also, microinjection of NBI-35965 into the PVN significantly reduced arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) in anesthetized SHRs, but not in WKY rats. However, microinjection of Antisauvagine-30 into the PVN had no effect on ABP or RSNA in WKY rats and SHRs. Our findings suggest that endogenous CRF in the PVN potentiates glutamatergic input and firing activity of PVN presympathetic neurons via CRFR1, resulting in augmented sympathetic outflow in hypertension.

Zerumbone Neuro-modulates Spinal Motor and Sensory Neurons of Rats: a c-Fos study

Background: A natural sesquiterpene Zerumbone (ZER) is abundant in rhizomes of Zingiber zerumbet (L) Smith, well-known for its beneficial effects. Very few reported studies of Zerumbone (ZER) as well as its bioactive compounds focusing on its neuromodulatory activities based on it functional neuro-anatomy. Objective: The aim of this study was to investigate the neuroanatomical functional site of the zerumbone in rat spinal motor and sensory neurons by means of c-Fos immunohistochemistry. Results: Our present study demonstrated that i.p., administration of zerumbone (100 mg/kg) significantly (p<0.01) increase in the number of c-Fos immunoreactive neurons in the dorsal and ventral nuclei of the rats. Conclusion: The result of the present study strongly indicates that zerumbone has neuromodulatory effects on sensory and motor neurons. To the best of our knowledge, it is the first report which indicates that zerumbone might play crucial roles in motor and sensory neuromodulation. Our findings warrant further study to reveal the underlying molecular mechanisms of zerumbone induced sensory and motor neuromodulation. We suggest the c-Fos immunohistochemical functional mapping of zerumbone in central nervous system of model animals to reveal its therapeutics potential and mechanisms.

Acute gut inflammation reduces neural activity and spine maturity in hippocampus but not basolateral amygdala

Gastrointestinal tract (gut) inflammation increases stress and threat-coping behaviors, which are associated with altered activity in fear-related neural circuits, such as the basolateral amygdala and hippocampus. It remains to be determined whether inflammation from the gut affects neural activity by altering dendritic spines. We hypothesized that acute inflammation alters dendritic spines in a brain region-specific manner. Here we show that acute gut inflammation (colitis) evoked by dextran sodium sulfate (DSS) did not affect the overall spine density in the CA1 region of hippocampus, but increased the relative proportion of immature spines to mature spines on basal dendrites of pyramidal neurons. In contrast, in animals with colitis, no changes in spine density or composition on dendrites of pyramidal cells was observed in the basolateral amygdala. Rather, we observed decreased spine density on dendrites of stellate neurons, but not the relative proportions of mature vs immature spines. We used cFos expression evoked by the forced swim task as a measure of neural activity during stress and found no effect of DSS on the density of cFos immunoreactive neurons in basolateral amygdala. In contrast, fewer CA1 neurons expressed cFos in mice with colitis, relative to controls. Furthermore, CA1 cFos expression negatively correlated with active stress-coping in the swim task and was negatively correlated with gut inflammation. These data reveal that the effects of acute gut inflammation on synaptic remodeling depend on brain region, neuronal phenotype, and dendrite location. In the hippocampus, a shift to immature spines and hypoactivity are more strongly related to colitis-evoked behavioral changes than is remodeling in basolateral amygdala.

Early maternal separation alters the activation of stress-responsive brain areas in adulthood

The expression of c-Fos protein has been extensively used as a marker of neuronal activation in response to stressful stimuli. Early maternal separation (MS) is a model of early life adversity that affects the responsiveness of the brain areas to stressors. Thus, this study examined the impact of early MS on activating stress-responsive areas in the brain of adult rats in response to physical (ether) or psychological (restraint) stressors. Male pups were divided for the MS or non-handled (NH) groups. The MS was carried out daily between the 2nd and 14th day of postnatal life and consisted in removing the dams from the cage for 180 min. The rats were then subjected to experimental protocols of restraint or ether exposure at 10–12 weeks old. The rats were anesthetized 90 min after exposure to the stressors, and their brains were prepared for immunohistochemical analysis of c-Fos immunoreactive (c-Fos-ir) neurons in the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON), medial preoptic area (MPA), medial amygdaloid nucleus (MeA), locus coeruleus (LC), and nucleus of the solitary tract (NST). The MS-group presented 86%, 125%, 73%, 56%, and 137% higher c-Fos-ir neurons in the LC, PVN, SON, MPA, and MeA, respectively, compared to NH-group in response to the restraint stressor. In addition, the MS-group presented 180%, 137%, 170%, and 138% higher c-Fos-ir neurons for the ether exposure in the LC, PVN, MPA, and MeA, respectively. Our results show a greater increase in neuronal activation in the MS group, indicating that early life adversity can induce reprogramming in the brain response to stress in adulthood.

Emodin Interferes With Nitroglycerin-Induced Migraine in Rats Through CGMP-PKG Pathway.

The purpose of this research was to explore the effect and mechanism of emodin in interfering with nitroglycerin-induced migraine rats. We carried out behavioral research within 2 h post-nitroglycerin (NTG) injection, and blood samples were collected through the abdominal aorta for measurements of nitric oxide (NO), calcitonin gene-related peptide (CGRP), substance P (SP), tumor necrosis factor (TNF-α) and cyclic guanosine monophosphate (cGMP) levels. Immunohistochemistry was adopted to detect the activation of c-Fos immunoreactive neurons in brain tissues. The number and integrated optical density (IOD) of c-Fos positive cells were measured using Image-Pro Plus. Western blotting was applied to detect the levels of PKG protein in rat brain tissues. The results showed that emodin can alleviate the pain response of migraine rats and significantly reduce the levels of NO, CGRP, SP, TNF-α and cGMP in migraine rats. In addition, emodin can significantly reduce the number of c-Fos positive cells and the IOD value. Moreover, the expression of PKG protein was significantly inhibited by emodin. Therefore, it is inferred that emodin can relieve migraine induced by NTG through the cGMP-PKG pathway, and can be used as a potential botanical medicine for the treatment of migraine.

Patterns of Nerve Fibre Impairments and Neuronal Activation in Male Diabetic Rats With and Without Mechanical Allodynia: A Comparative Study

BackgroundIt is still clinically difficult to definitively distinguish between painful and nonpainful diabetic peripheral neuropathy (DPN). In this study, pain patterns were further categorized; diabetic rats were grouped according to the presence or absence of mechanical allodynia (MA), which is a hallmark of neuropathic pain, and comparisons were made between diabetic rats with and without MA. MethodsDiabetes was established in rats by a single intraperitoneal streptozotocin injection, and vehicle injection was administered in the control group. Pain behaviour tests for MA and thermal hyperalgesia were performed every week. At week 4, the diabetic group with thermal hyperalgesia was further grouped by the presence of MA. Morphometric analysis of sciatic nerves and myelin basic protein quantification were conducted. The numbers of c-Fos–immunoreactive neurons in both the dorsal root ganglion (DRG) and the spinal dorsal horn (SDH) were determined. ResultsDiabetic rats with MA had more severe impairment of myelinated nerve fibres than those without MA (p<0.001). Higher percentages of NF-200–positive neurons in the DRG were c-Fos immunoreactive in the DPN-with-MA group than in the DNP-without-MA group (p=0.014). The numbers of c-Fos–immunoreactive neurons in both the superficial (I–II) and deeper (III–V) laminae of the SDH in the DPN-with-MA group were larger than those in the subgroups without MA (p<0.001). ConclusionsThe impairment of primary myelinated fibres and the pattern of neuronal activation in both the DRG and the SDH are specific features that differentiate diabetic rats with MA from those without MA.

Social interaction masking contributes to changes in the activity of the suprachiasmatic nucleus and impacts on circadian rhythms

Light is the most powerful temporal cue that entrains physiology and behavior through modulation of the suprachiasmatic nucleus (SCN) of the hypothalamus. However, on a daily basis, individuals face a combination of light and several non-photic cues, such as social interaction. In order to investigate whether SCN activity and SCN-driven rhythms are altered by social interaction, adult male C57BLJ/6 mice were maintained in groups of 3–4 animals per cage or 1 animal per cage (socially isolated) under 12:12 h / light:dark (LD) cycles or constant darkness (DD). Analysis of the two anatomical subdivisions (ventral, v and dorsal, d) of the medial SCN revealed an effect of housing conditions on the d-SCN but not on the v-SCN on the number of c-Fos immunoreactive (ir) neurons. As such, 2 h after the light-phase onset d-SCN c-Fos-ir number was lower in single-housed mice under LD. Importantly, under DD there were no effect of housing conditions in the number of c-Fos-ir SCN neurons. Social isolation increased the amplitude and strength of SCN-driven rhythm of body temperature (Tc) entrained to LD and it advanced its onset, uncoupling with spontaneous locomotor activity (SLA) rhythm, without altering endogenous Tc and SLA rhythms expressed under DD. Associated with reduced Tc in the light phase, single-housed mice showed reduced body weight. However, these phenotypes were not accompanied by changes in the number of c-Fos-ir neurons in the preoptic area (POA), which are known to regulate energy metabolism and Tc. Altogether, these results imply that the social interaction masking effect on the d-SCN is added to that of light stimulus, in order to achieve full c-Fos expression in the SCN, which, in turn seems to be required to maintain daily-phase coherence between the photo-entrained rhythms of Tc and SLA. There might be an inter-relationship between masking (social interaction) and entrainment stimulus (light) that impacts the circadian parameters of the photo-entrained Tc rhythm. As such, in the absence of social interactions a more robust Tc rhythm is shown. This inter-relationship seems to occur in the dorsal subdivision of the SCN but not in the POA.

Social Experience Interacts with Serotonin to Affect Functional Connectivity in the Social Behavior Network following Playback of Social Vocalizations in Mice.

Past social experience affects the circuitry responsible for producing and interpreting current behaviors. The social behavior network (SBN) is a candidate neural ensemble to investigate the consequences of early-life social isolation. The SBN interprets and produces social behaviors, such as vocalizations, through coordinated patterns of activity (functional connectivity) between its multiple nuclei. However, the SBN is relatively unexplored with respect to murine vocal processing. The serotonergic system is sensitive to past experience and innervates many nodes of the SBN; therefore, we tested whether serotonin signaling interacts with social experience to affect patterns of immediate early gene (IEG; cFos) induction in the male SBN following playback of social vocalizations. Male mice were separated into either social housing of three mice per cage or into isolated housing at 18–24 d postnatal. After 28–30 d in housing treatment, mice were parsed into one of three drug treatment groups: control, fenfluramine (FEN; increases available serotonin), or pCPA (depletes available serotonin) and exposed to a 60-min playback of female broadband vocalizations (BBVs). FEN generally increased the number of cFos-immunoreactive (-ir) neurons within the SBN, but effects were more pronounced in socially isolated mice. Despite a generalized increase in cFos immunoreactivity, isolated mice had reduced functional connectivity, clustering, and modularity compared with socially reared mice. These results are analogous to observations of functional dysconnectivity in persons with psychopathologies and suggests that early-life social isolation modulates serotonergic regulation of social networks.

Oral application of clozapine-N-oxide using the micropipette-guided drug administration (MDA) method in mouse DREADD systems.

The designer receptor exclusively activated by designer drugs (DREADD) system is one of the most widely used chemogenetic techniques to modulate the activity of cell populations in the brains of behaving animals. DREADDs are activated by acute or chronic administration of their ligand, clozapine-N-oxide (CNO). There is, however, a current lack of a non-invasive CNO administration technique that can control for drug timing and dosing without inducing substantial distress for the animals. Here, we evaluated whether the recently developed micropipette-guided drug administration (MDA) method, which has been used as a non-invasive and minimally stressful alternative to oral gavages, may be applied to administer CNO orally to activate DREADDs in a dosing- and timing-controlled manner. Unlike standard intraperitoneal injections, administration of vehicle substances via MDA did not elevate plasma levels of the major stress hormone, corticosterone, and did not attenuate exploratory activity in the open field test. At the same time, however, administration of CNO via MDA or intraperitoneally was equally efficient in activating hM3DGq-expressing neurons in the medial prefrontal cortex, as evident by time-dependent increases in mRNA levels of neuronal immediate early genes (cFos, Arc and Zif268) and cFos-immunoreactive neurons. Compared to vehicle given via MDA, oral administration of CNO via MDA was also found to potently increase locomotor activity in mice that express hM3DGq in prefrontal neurons. Taken together, our study confirms the effectiveness of CNO given orally via MDA and provides a novel method for non-stressful, yet well controllable CNO treatments in mouse DREADD systems.

Neuropeptide S Displays as a Key Neuromodulator in Olfactory Spatial Memory.

Neuropeptide S (NPS) is an endogenous peptide recently recognized to be presented in the brainstem and believed to play an important role in maintaining memory. The deletion of NPS or NPS receptor (NPSR) in mice shows a deficit in memory formation. Our recent studies have demonstrated that central administration of NPS facilitates olfactory function and ameliorates olfactory spatial memory impairment induced by muscarinic cholinergic receptor antagonist and N-methyl-D-aspartate receptor antagonist. However, it remains to be determined if endogenous NPS is an indispensable neuromodulator in the control of the olfactory spatial memory. In this study, we examined the effects of NPSR peptidergic antagonist [D-Val5]NPS (10 and 20 nmol, intracerebroventricular) and nonpeptidergic antagonist SHA 68 (10 and 50 mg/kg, intraperitoneal) on the olfactory spatial memory using computer-assisted 4-hole-board olfactory spatial memory test in mice. Furthermore, immunofluorescence was employed to identify the distributions of c-Fos and NPSR immunoreactive (-ir) neurons in olfactory system and hippocampal formation known to closely relate to the olfactory spatial memory. [D-Val5]NPS dosing at 20 nmol and SHA 68 dosing at 50 mg/kg significantly decreased the number of visits to the 2 odorants interchanged spatially, switched odorants, in recall trial, and simultaneously reduced the percentage of Fos-ir in NPSR-ir neurons, which were densely distributed in the anterior olfactory nucleus, piriform cortex, subiculum, presubiculum, and parasubiculum. These findings suggest that endogenous NPS is a key neuromodulator in olfactory spatial memory.

Systemic Glucose Administration Alters Water Diffusion and Microvascular Blood Flow in Mouse Hypothalamic Nuclei - An fMRI Study.

The hypothalamus is the principal regulator of global energy balance, enclosing additionally essential neuronal centers for glucose-sensing and osmoregulation. Disturbances in these tightly regulated neuronal networks are thought to underlie the development of severe pandemic syndromes, including obesity and diabetes. In this work, we investigate in vivo the response of individual hypothalamic nuclei to the i.p. administration of glucose or vehicle solutions, using two groups of adult male C57BL6/J fasted mice and a combination of non-invasive T2∗-weighted and diffusion-weighted functional magnetic resonance imaging (fMRI) approaches. MRI parameters were assessed in both groups of animals before, during and in a post-stimulus phase, following the administration of glucose or vehicle solutions. Hypothalamic nuclei depicted different patterns of activation characterized by: (i) generalized glucose-induced increases of neuronal activation and perfusion-markers in the lateral hypothalamus, arcuate and dorsomedial nuclei, (ii) cellular shrinking events and decreases in microvascular blood flow in the dorsomedial, ventromedial and lateral hypothalamus, following the administration of vehicle solutions and (iii) increased neuronal activity markers and decreased microperfusion parameters in the ARC nuclei of vehicle-administered animals. Immunohistochemical studies performed after the post-stimulus phase confirmed the presence of c-Fos immunoreactive neurons in the arcuate nucleus (ARC) from both animal groups, with significantly higher numbers in the glucose-treated animals. Together, our results reveal that fMRI methods are able to detect in vivo diverse patterns of glucose or vehicle-induced effects in the different hypothalamic nuclei.

Reversible Functional Changes Evoked by Anodal Epidural Direct Current Electrical Stimulation of the Rat Auditory Cortex.

Rat auditory cortex was subjected to 0.1 mA anodal direct current in seven 10-min sessions on alternate days. Based on the well-known auditory cortex control of olivocochlear regulation through corticofugal projections, auditory brainstem responses (ABRs) were recorded as an indirect test of the effectiveness and reversibility of the multisession protocol of epidural stimulation. Increases of 20–30 dB ABR auditory thresholds shown after epidural stimulation reverted back to control levels 10 min after a single session. However, increases in thresholds revert 4 days after multisession stimulation. Less changes in wave amplitudes and threshold shifts were shown in ABR recorded contralaterally to the electrically stimulated side of the brain. To assess tissue effects of epidural electric stimulation on the brain cortex, well characterized functional anatomical markers of glial cells (GFAP/astrocytes and Iba1/microglial cells) and neurons (c-Fos) were analyzed in alternate serial sections by quantitative immunocytochemistry. Restricted astroglial and microglial reactivity was observed within the cytoarchitectural limits of the auditory cortex. However, interstitial GFAP overstaining was also observed in the ventricular surface and around blood vessels, thus supporting a potential global electrolytic stimulation of the brain. These results correlate with extensive changes in the distribution of c-Fos immunoreactive neurons among layers along sensory cortices after multisession stimulation. Quantitative immunocytochemical analysis supported this idea by showing a significant increase in the number of positive neurons in supragranular layers and a decrease in layer 6 with no quantitative changes detected in layer 5. Our data indicate that epidural stimulation of the auditory cortex induces a reversible decrease in hearing sensitivity due to local, restricted epidural stimulation. A global plastic response of the sensory cortices, also reported here, may be related to electrolytic effects of electric currents.

Effect of adenosine A1 receptor agonist induced‐hibernation on the arousal pathway in the Arctic Ground Squirrel

Hibernation is a seasonal phenomenon and in the arctic ground squirrel (AGS), the effect of seasonal changes is very evident. In late fall AGS decrease their metabolic demand followed by the entry into hibernation in winter. Previously in the lab it has been shown that N6‐cyclohexyladenosine (CHA), an A1 adenosine receptor agonist, induces hibernation in AGS in a seasonally dependent manner. In winter AGS treated with CHA experience a decrease in metabolic demand and temperature as seen during natural hibernation. However, in summer CHA triggers a brief and transient metabolic suppression that does not lead to hibernation onset.As adenosine is known to promote sleep, we decided to investigate if the sleep‐arousal pathways were affecting the seasonal difference in response to CHA. The tuberomammillary nucleus (TMN) is a nucleus of the ascending arousal pathway characterized by fast firing rates to promote wakefulness. Previously we showed a decrease in neuronal activation in the TMN in AGS treated with CHA in both summer and winter seasons.Here we test the hypothesis that seasonal response to CHA is associated with difference in activation in another nucleus of the ascending arousal pathway, the locus coeruleus (LC).AGS implanted with body temperature (Tb) transmitter, were treated with CHA or vehicle (0.5mg/kg, IP). AGS were perfused with 4% paraformaldehyde and brains were dissected and cryoprotected in 5%, 10%, 15%, 20%, 30% sucrose. We performed free‐floating immunohistochemistry on 40μm brain slices in four experimental groups: winter CHA, winter control, summer CHA and summer control. We sectioned brains from eight AGS per group for a total of 32 animals and identified active nuclei with cFos immunoreactivity (mouse anti‐cFos 1:20,000, Santa Cruz) and active tyrosine hydroxylase neurons (rabbit anti‐TH). Blinded analysis of cFos immunoreactive neurons was done using bright field microscopy and Metamorph software. Statistical analysis was performed on R.CHA produced a hibernation‐like response measured by a decrease in Tb in winter AGS but not in the summer. We did not observe any significant difference in neuronal activation in the LC, thus CHA is associated with a decreased neuronal activity in the TMN, resulting in the TMN being the major wakefulness center affected by CHA but not by seasons.Support or Funding InformationResearch was supported by NIH R03 NS081637, NSF IOS‐1258179, RL5GM118990 and Alaska INBRE P20GM103395. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

C-Fos expression in visual cortex of infant rhesus monkeys with myopia induced by hyperopic defocus

Objective To observe c-Fos expression in visual cortex of infant rhesus monkeys with myopia induced by hyperopic defocus and preliminarily investigate the possibility of visual cortex participating in myopia. Methods Eight SPF grade healthy infant rhesus monkeys aged 20 to 30 days were randomly divided into hyperopic defocused group and control group, 4 monkeys for each group.The monkeys in hyperopic defocused group wore -3 D spectacle lenses.The monkeys in control group wore 0 D lenses.The monkeys' refractive error, corneal topography, vitreous chamber depth were measured at the start of lens wear and at 2, 4, 6, 8, 12 weeks post-treatment.At 12 weeks post-treatment, the visual cortex tissues were removed for c-Fos protein measurement by immunohistochemistry and Western blot assays.The results were analyzed semiquantitatively to compare the differences of c-Fos expression between hyperopic defocused group and control group.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.This study protocol was approved by Ethic Committee of Zhongshan Ophthalmic Center (No.2013-014). Results After 12 weeks' lens wear, the vitreous chamber elongation amplitude of hyperopic defocused group monkeys was more obvious than that of the control group ([0.93±0.24]mm vs.[0.72±0.09]mm; t=2.292, P=0.047). The decrease of hyperopic degrees of hyperopic defocused group monkeys was more obvious than that of the control group ([-3.23±1.36]D vs.[-1.55±0.52]D; t=-3.273, P=0.006). The eyes of hyperopic defocused group monkeys appeared a remarkable myopic shift after treatment.The number of c-Fos immunoreactive neurons was less in the hyperopic defocused group than that in the control group, with a statistically significant difference between them ([1 843±191]/mm2 vs.[2 296±503]/mm2; t=2.381, P=0.041). Western blot assay showed that the optical density of c-Fos protein in the hyperopic defocused group was significantly less than that in the control group (0.50±0.17 vs.0.99±0.22; t=-4.982, P<0.01). Conclusions Hyperopic defocus, as an abnormal visual stimulus, can induce the onset of myopia in infant rhesus monkeys and inhibit c-Fos expression in visual cortex.Visual cortex may participate in myopia induced by hyperopic defocus. Key words: c-Fos; Myopia; Visual cortex; Rhesus monkey; Optical defocus

Positive reinforcement and c‐Fos expression following abuse‐like thinner inhalation in mice: Behavioural and immunohistochemical assessment

Thinners are organic solvents widely used in industrial applications, but they have also been subject to abuse by inhalation for their psychoactive and rewarding properties. In spite of the prevalence of inhalant abuse, the addictive potential and pathways mediating their reinforcing effects are not yet fully understood and thus still subject of further investigations. Here, we assessed in mice the locomotor activity and the ability of paint thinner to reinforce the conditioning in the place preference paradigm following acute (1day), subchronic (6weeks) and chronic (12weeks) exposures to 300 and 600ppm of thinner vapor. While locomotor activity was unaffected by the different thinner treatments, a positive conditioned place preference to inhaled thinner was found upon subchronic and chronic exposures. To investigate the activated brain structures underlying such behavioural changes, we analyzed the distribution of c-Fos immunoreactivity, a marker for neuronal activation, following acute and repeated exposures to 600ppm of thinner. Notably, thinner exposure increased the number of c-Fos immunoreactive neurons with increasing duration of exposure in the majority of structures examined; including those typically involved in the processing of rewarding or emotionally stimuli (e.g., ventral tegmental area, core and shell of nucleus accumbens, amygdala, bed nucleus of the stria terminalis, and cingulate cortex), and olfactory stimuli (e.g., piriform cortex and olfactory tubercle). Moreover, prolonged, but not acute thinner inhalation significantly increased c-Fos immunoreactivity in all hippocampal subregions. Taken together, the expanded distribution of thinner-induced c-Fos expression may underlie the observed positive reinforcement upon long-term thinner inhalation.

Inhibition of Medial Preoptic Area Improves Physical Performance in Trained Rats

The medial preoptic area (MPO) plays a key role in the regulation of autonomic thermoeffectors and this area seems to be more activated after physical training, as evidenced by greater number of exercise‐induced c‐Fos immunoreactive neurons in trained rats. This observation raised the question as to whether inhibition of MPO can affect physical performance and thermoregulatory responses in trained rats. Following 2 weeks of high‐intensity interval training (HIIT), Wistar rats were submitted to an incremental‐speed exercise on a motor‐driven treadmill until fatigue after bilateral microinjection of muscimol (80 pmol/100 nl) to evaluate their aerobic capacity and thermoregulatory responses. The core body temperature (Tc) and the maximal oxygen uptake (VO2 max) were recorded continuously during exercise. Physical training increased the time to fatigue (43 ± 2 min vs. 64 ± 2 min; p=0.0009; n=6); the maximum speed attained during the incremental exercise (24 ± 1 m/min vs. 31 ± 1 m/min; p=0.0013), the distance traveled (675 ± 36 m vs. 1261 ± 68 m; p=0.0008); and resulted in a higher oxygen uptake (38 ± 1 ml/min/kg vs. 42 ± 1 ml/min/kg; p=0.01). Interestingly, at fatigue, no difference in the absolute Tc was found between the groups (38.28 ± 0.2 °C vs. 38.78 ± 0.1 °C; p=0.1). Bilateral blockade of MPO using muscimol improved physical performance, as observed by the increased time to fatigue (71 ± 6 min muscimol vs. 58 ± 2 min ‐ control, p=0.004; n=3); distance traveled (1528 ± 188 m muscimol vs. 1094 ± 84 m control; p=0.05); and maximum oxygen uptake (49 ± 1 ml/min/kg muscimol vs. 44 ± 2 ml/min/kg control; p=0.04). Regarding the thermoregulatory adjustments, MPO inhibition tended to increase Tc at fatigue (40.37 ± 0.5 °C muscimol vs. 39.35 ± 0.2 °C ‐ control; p=0.08). There was an increase in the Tc of the control animals during exercise but this temperature remained lower than that of the muscimol group (p&lt;0.001). Our findings show that neurons in the MPO are involved in the control of the aerobic capacity of trained rats, however, the improved physical performance of these animals caused by MPO inhibition did not result from changes in thermoregulatory adjustments.Support or Funding InformationConselho Nacional de Desenvolvimento Científico e Tecnlógico ‐ CNPq; grant 434646/2016‐4This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Baicalin Alleviates Nitroglycerin-induced Migraine in Rats via the Trigeminovascular System.

Migraine is a common neurological disorder with a serious impact on quality of life. The aim of this study was to explore the effect of baicalin on nitroglycerin-induced migraine rats. We carried out a behavioral research within 2h post-nitroglycerin injection, and blood samples were drawn for measurements of nitric oxide (NO), calcitonin gene-related peptide, and endothelin (ET) levels. Immunohistochemistry was adopted to detect the activation of C-fos immunoreactive neurons in periaqueductal gray. The number, area size, and integrated optical density of C-fos positive cells were measured using Image-Pro Plus. As a result, baicalin administration (0.22mm/kg) alleviated pain responses of migraine rats. It profoundly decreased NO and calcitonin gene-related peptide levels, increased ET levels, and rebuilt the NO/ET balance in migraine rats. Besides, baicalin pretreatment significantly reduced the number, the stained area size, and integrated optical density value of C-fos positive cells. In brief, this paper supports the possibility of baicalin as a potential migraine pharmacotherapy. Copyright © 2017 John Wiley & Sons, Ltd.

BDNF and AMPA receptors in the cNTS modulate the hyperglycemic reflex after local carotid body NaCN stimulation.

The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation.

C-Fos expression in the parabrachial nucleus following intraoral bitter stimulation in the rat with dietary-induced zinc deficiency

Zinc deficiency causes various symptoms including taste disorders. In the present study, changes in expression of c-Fos immunoreactivity in neurons of the parabrachial nucleus (PBN), one of the relay nuclei for transmission of gustatory information, after bitter stimulation to the dorsal surface of the tongue were examined in zinc-deficient rats. Experimental zinc-deficient animals were created by feeding a low-zinc diet for 4weeks, and showed the following symptoms of zinc deficiency: low body weight, low serum zinc content and behavioral changes to avoid bitter stimulation. In normal control animals, intraoral application of 1mM quinine caused increased numbers of c-Fos-immunoreactive (c-Fos-IR) neurons in the external lateral subnucleus and external medial subnucleus of the PBN (elPBN and emPBN, respectively) compared with application of distilled water. However, in the zinc-deficient animals, the numbers of c-Fos-IR neurons in the elPBN and emPBN did not differ significantly between application of quinine and distilled water. After feeding the zinc-deficient animals a normal diet for 4weeks, the symptoms of zinc deficiency recovered, and the expression of c-Fos-IR neurons following intraoral bitter stimulation became identical to that in the normal control animals. The present results indicate that dietary zinc deficiency causes alterations to neuronal activities in the gustatory neural circuit, and that these neuronal alterations can be reversed by changing to a normal diet.