Marker Of Neuronal Activation Research Articles

Prevention of age-associated neuronal hyperexcitability with improved learning and attention upon knockout or antagonism of LPAR2

Recent studies suggest that synaptic lysophosphatidic acids (LPAs) augment glutamate-dependent cortical excitability and sensory information processing in mice and humans via presynaptic LPAR2 activation. Here, we studied the consequences of LPAR2 deletion or antagonism on various aspects of cognition using a set of behavioral and electrophysiological analyses. Hippocampal neuronal network activity was decreased in middle-aged LPAR2−/− mice, whereas hippocampal long-term potentiation (LTP) was increased suggesting cognitive advantages of LPAR2−/− mice. In line with the lower excitability, RNAseq studies revealed reduced transcription of neuronal activity markers in the dentate gyrus of the hippocampus in naïve LPAR2−/− mice, including ARC, FOS, FOSB, NR4A, NPAS4 and EGR2. LPAR2−/− mice behaved similarly to wild-type controls in maze tests of spatial or social learning and memory but showed faster and accurate responses in a 5-choice serial reaction touchscreen task requiring high attention and fast spatial discrimination. In IntelliCage learning experiments, LPAR2−/− were less active during daytime but normally active at night, and showed higher accuracy and attention to LED cues during active times. Overall, they maintained equal or superior licking success with fewer trials. Pharmacological block of the LPAR2 receptor recapitulated the LPAR2−/− phenotype, which was characterized by economic corner usage, stronger daytime resting behavior and higher proportions of correct trials. We conclude that LPAR2 stabilizes neuronal network excitability upon aging and allows for more efficient use of resting periods, better memory consolidation and better performance in tasks requiring high selective attention. Therapeutic LPAR2 antagonism may alleviate aging-associated cognitive dysfunctions.

0415 Metabolic Dysfunction and Sleep Disruption in Models of Alzheimer’s Disease

Abstract Introduction Metabolic perturbations and sleep disruptions are a cause and consequence of Alzheimer’s disease pathophysiology. A bidirectional relationship exists where impairments in sleep and metabolism contribute to the development of Alzheimer’s disease while the presence of Alzheimer’s disease pathology leads to decreased cerebral metabolism, peripheral glucose intolerance, and disrupted sleep. While the effects of type 2 diabetes (T2D) and Alzheimer’s disease on sleep have been explored separately, no previous studies have examined the effect of acute glycemic variability, a defining feature of T2D, on sleep in the context of Alzheimer’s disease. The goal of this study is to determine how glycemic variability drives sleep disruptions by modifying the relationship between cerebral glucose metabolism and neuronal activity. Methods Biosensors are implanted bilaterally into the hippocampus of APP/PS1, a model of amyloid-beta (Aβ) overexpression, and P301S, a model of tau deposition and neurodegeneration, mice to measure ISF glucose and lactate, markers of cerebral metabolism and neuronal activity, respectively. Simultaneous cortical EEG/EMG recordings are used for sleep/wake scoring and analysis. Results Glycemic fluctuations cause a decoupling of the typical relationships between cerebral metabolism and neuronal activity, while also increasing arousal in 3-month-old, wildtype mice. The presence of AD-like pathology results in a similar, albeit muted cerebral metabolic response to peripheral glycemic variability, but a diminished effect on wakefulness, likely due to age- and pathology-dependent increases in overall time spent awake. Conversely, in aged, P301S mice, cerebral metabolic responsiveness is lost and a ceiling effect on wakefulness emerges, suggesting differential effects on sleep with tau accumulation. Moreover, aged mice show progressive disruptions to overall sleep quality and quantity, highlighting the synergism existing between AD-like pathology and glucose intolerance in sleep dysfunction. Lactate seems to be a common driver of disruption in this synergistic cycle. Conclusion This study represents a novel approach to defining the dynamic interplay between risk factors for AD and T2D and suggests a feedforward loop of disease progression where disrupted sleep can alter the relationship between neuronal activity, metabolism, and pathology. Support NIH/NIA 1K01AG050719. Harold and Mary Eagle Fund for Alzheimer’s Research New Vision Award through Donors Cure Foundation

PPG neurons in the nucleus of the solitary tract modulate heart rate but do not mediate GLP-1 receptor agonist-induced tachycardia in mice

ObjectiveGlucagon-like peptide-1 receptor agonists (GLP-1RAs) are used as anti-diabetic drugs and are approved for obesity treatment. However, GLP-1RAs also affect heart rate (HR) and arterial blood pressure (ABP) in rodents and humans. Although the activation of GLP-1 receptors (GLP-1R) is known to increase HR, the circuits recruited are unclear, and in particular, it is unknown whether GLP-1RAs activate preproglucagon (PPG) neurons, the brain source of GLP-1, to elicit these effects. MethodsWe investigated the effect of GLP-1RAs on heart rate in anaesthetized adult mice. In a separate study, we manipulated the activity of nucleus tractus solitarius (NTS) PPG neurons (PPGNTS) in awake, freely behaving transgenic Glu-Cre mice implanted with biotelemetry probes and injected with AAV-DIO-hM3Dq:mCherry or AAV-mCherry-FLEX-DTA. ResultsSystemic administration of the GLP-1RA Ex-4 increased resting HR in anaesthetized or conscious mice, but had no effect on ABP in conscious mice. This effect was abolished by β-adrenoceptor blockade with atenolol, but unaffected by the muscarinic antagonist atropine. Furthermore, Ex-4-induced tachycardia persisted when PPGNTS neurons were ablated, and Ex-4 did not induce expression of the neuronal activity marker cFos in PPGNTS neurons. PPGNTS ablation or acute chemogenetic inhibition of these neurons via hM4Di receptors had no effect on resting HR. In contrast, chemogenetic activation of PPGNTS neurons increased resting HR. Furthermore, the application of GLP-1 within the subarachnoid space of the middle thoracic spinal cord, a major projection target of PPG neurons, increased HR. ConclusionsThese results demonstrate that both systemic application of Ex-4 or GLP-1 and chemogenetic activation of PPGNTS neurons increases HR. Ex-4 increases the activity of cardiac sympathetic preganglionic neurons of the spinal cord without recruitment of PPGNTS neurons, and thus likely recapitulates the physiological effects of PPG neuron activation. These neurons therefore do not play a significant role in controlling resting HR and ABP but are capable of inducing tachycardia and so are likely involved in cardiovascular responses to acute stress.

M143. REGIONAL CEREBRAL BLOOD FLOW IN INITIALLY ANTIPSYCHOTIC-NAïVE PATIENTS WITH SCHIZOPHRENIA OR PSYCHOSIS: EFFECTS OF PARTIAL D2 RECEPTOR AGONISM AND ASSOCIATION WITH SYMPTOM IMPROVEMENT

BackgroundSchizophrenia is suggested to stem from dysfunction of cortico-striato-thalamo-cortical networks. Supporting this, we have recently found increased glutamate levels in thalamus in antipsychotic-naïve patients with schizophrenia or psychosis. Moreover, higher baseline glutamate levels were related to less improvement of psychotic symptoms after treatment. Other groups have reported that striatal dopaminergic function in patients with psychosis is increased and associated with psychotic symptoms. Regional cerebral blood flow (rCBF) is considered a marker of neuronal activity and can be used to study the cortico-striato-thalamo-cortical networks.Here, we investigated rCBF in a large group of initially antipsychotic-naïve patients with schizophrenia or psychosis before and after treatment and related the findings to changes in psychotic symptoms.MethodsrCBF was acquired in 49 initially antipsychotic-naïve patients and 50 matched healthy controls at baseline, and in 32 patients and 53 healthy controls after 6 weeks with the pseudo-Continuous Arterial Spin Labelling (pCASL) sequence. Patients were treated with a partial dopamine D2 receptor agonist (aripiprazole) as monotherapy (mean dose: 10.6±5.4mg).Primary regions of interest of rCBF were thalamus, limbic- and associative striatum. In explorative analyses, we estimated rCBF in sensorimotor striatum, hippocampus-amygdala, and frontal lobe.Psychopathology was assessed with the positive and negative syndrome scale (PANSS).A linear mixed model was used to test if the change in rCBF was different in patients compared with healthy controls (significant interaction) with adjustment for age, sex, and whole brain rCBF. Post hoc tests evaluated possible group differences at baseline and after 6 weeks.In a general linear model, we investigated associations between baseline rCBF in striatum and thalamus and improvement in psychotic symptoms.ResultsrCBF changed over 6 weeks in all striatal regions (associative striatum: p=0.023; limbic striatum: p=0.004; sensorimotor striatum: p=0.004). Post hoc tests of associative striatum indicated no baseline group differences in rCBF (SCZ/HC: 63.4/65.1 mL/100g/min, p=0.59), however rCBF was higher in patients after 6 weeks (SCZ/HC: 68.8/63.2 mL/100g/min, p=0.049). Post hoc tests of limbic striatum indicated no baseline group differences in rCBF (SCZ/HC: 62.7/65.3 mL/100g/min, p=0.25) and no group differences rCBF after 6 weeks (SCZ/HC: 69.3/63.3 mL/100g/min, p=0.12).Post hoc tests of sensorimotor striatum indicated no baseline group differences in rCBF (SCZ/HC: 72.6/71.2 mL/100g/min, p=0.18), however, rCBF was significantly higher in patients after 6 weeks (SCZ/HC: 80.0/67.2 mL/100g/min, p<0.001).There were no group differences in the rCBF changes in thalamus (p=0.09), hippocampus-amygdala (p=0.24), frontal lobe (p=0.90), and the whole brain (p=0.14).In patients, higher baseline rCBF in limbic striatum was associated with less improvement in PANSS positive symptoms (p=0.025). We found no other associations between rCBF and psychopathy.DiscussionThe findings suggest that treatment with a partial dopamine D2 agonist increases rCBF in associative and sensorimotor striatum in initially antipsychotic-naïve patients and that higher rCBF in limbic striatum at baseline is related to poorer treatment outcome. Future studies should investigate the associations between different neurotransmitters and rCBF in vivo. This could further characterize disturbances in cortico-striato-thalamo-cortical networks in schizophrenia or psychosis as well as the effect of treatment.

AT1 -R is involved in the development of long-lasting, region-dependent and oxidative stress-independent astrocyte morphological alterations induced by Ketamine.

Astrocytes play an essential role in the genesis, maturation and regulation of the neurovascular unit. Multiple evidence support that astrocyte reactivity has a close relationship to neurovascular unit dysfunction, oxidative stress and inflammation, providing a suitable scenario for the development of mental disorders. Ketamine has been proposed as a single-use antidepressant treatment in major depression, and its antidepressant effects have been associated with anti-inflammatory properties. However, Ketamine long-lasting effects over the neurovascular unit components remain unclear. Angiotensin II AT1 receptor (AT1 -R) blockers have anti-inflammatory, antioxidant and neuroprotective effects. The present work aims to distinguish the acute and long-term Ketamine effects over astrocytes response extended to other neurovascular unit components, and the involvement of AT1 -R, in prefrontal cortex and ventral tegmental area. Male Wistar rats were administered with AT1 -R antagonist Candesartan/Vehicle (days 1-10) and Ketamine/Saline (days 6-10). After 14days drug-free, at basal conditions or after Ketamine Challenge, the brains were processed for oxidative stress analysis, cresyl violet staining and immunohistochemistry for glial, neuronal activation and vascular markers. Repeated Ketamine administration induced long-lasting region-dependent astrocyte reactivity and morphological alterations, and neuroadaptative changes observed as exacerbated oxidative stress and neuronal activation, prevented by the AT1 -R blockade. Ketamine Challenge decreased microglial and astrocyte reactivity and augmented cellular apoptosis, independently of previous treatment. Overall, AT1 -R is involved in the development of neuroadaptative changes induced by repeated Ketamine administration but does not interfere with the acute effects supporting the potential use of AT1 -R blockers as a Ketamine complementary therapy in mental disorders.

Modulation of fear behavior and neuroimmune alterations in house dust mite exposed A/J mice, a model of severe asthma

Fear-associated conditions such as posttraumatic stress disorder (PTSD) and panic disorder (PD) are highly prevalent. There is considerable interest in understanding contributory risk and vulnerability factors. Accumulating evidence suggests that chronically elevated inflammatory load may be a potential risk factor for these disorders. In this regard, an association of asthma, a chronic inflammatory condition, with PTSD and PD has been reported. Symptoms of PD and PTSD are more prevalent in severe asthmatics, compared to those with mild or moderate asthma suggesting that factors that influence the severity of asthma, may also influence susceptibility to the development of fear-related disorders. There has been relatively little progress in identifying contributory factors and underlying mechanisms, particularly, the translation of severe asthma-associated lung inflammation to central neuroimmune alterations and behavioral manifestations remains unclear. The current study investigated the expression of behaviors relevant to PD and PTSD (CO2 inhalation and fear conditioning/extinction) in A/J mice using a model of severe allergic asthma associated with a mixed T helper 2 (Th2) and Th17 immune response. We also investigated the accumulation of Th2- and Th17-cytokine expressing cells in lung and brain tissue, microglial alterations, as well as neuronal activation marker, delta FosB (ΔFosB)) in fear and panic regulatory brain areas. HDM-exposed mice elicited higher freezing during fear extinction. CO2-associated spontaneous and conditioned freezing, as well as anxiety or depression-relevant exploratory and coping behaviors were not altered by HDM treatment. A significant increase in brain Th17-associated inflammatory mediators was observed prior to behavioral testing, accompanied by microglial alterations in specialized blood brain barrier-compromised circumventricular area, subfornical organ. Post extinction measurements revealed increased ΔFosB staining within the medial prefrontal cortex and basolateral amygdala in HDM-treated mice. Collectively, our data show modulation of brain immune mechanisms and fear circuits by peripheral airway inflammation, and is relevant to understanding the risk and comorbidity of asthma with fear-associated disorders such as PTSD.

Mechanical allodynia in mice with tenascin-X deficiency associated with Ehlers-Danlos syndrome

Tenascin-X (TNX) is a member of the extracellular matrix glycoprotein tenascin family, and TNX deficiency leads to Ehlers-Danlos syndrome, a heritable human disorder characterized mostly by skin hyperextensibility, joint hypermobility, and easy bruising. TNX-deficient patients complain of chronic joint pain, myalgia, paresthesia, and axonal polyneuropathy. However, the molecular mechanisms by which TNX deficiency complicates pain are unknown. Here, we examined the nociceptive behavioral responses of TNX-deficient mice. Compared with wild-type mice, TNX-deficient mice exhibited mechanical allodynia but not thermal hyperalgesia. TNX deficiency also increased pain sensitivity to chemical stimuli and aggravated early inflammatory pain elicited by formalin. TNX-deficient mice were significantly hypersensitive to transcutaneous sine wave stimuli at frequencies of 250 Hz (Aδ fiber responses) and 2000 Hz (Aβ fiber responses), but not to stimuli at frequency of 5 Hz (C fiber responses). In addition, the phosphorylation levels of extracellular signal-related kinase, an active neuronal marker, and the activity of NADPH-diaphorase, a neuronal nitric oxide activation marker, were enhanced in the spinal dorsal horns of TNX-deficient mice. These results suggest that TNX deficiency contributes to the development of mechanical allodynia and hypersensitivity to chemical stimuli, and it induces hypersensitization of myelinated A fibers and activation of the spinal dorsal horn.

Focal transcranial magnetic stimulation in awake rats: Enhanced glucose uptake in deep cortical layers

BackgroundTranscranial magnetic stimulation (TMS) is an emerging neuromodulation tool. However, preclinical models of TMS are limited. ObjectiveTo develop a method for performing TMS in awake rats and to characterize neuronal response to TMS by mapping glucose uptake following TMS administration. MethodsA headpost was implanted into rat skull serving as a refence to guide TMS target. Motor threshold measurement was used as the metric to assess the consistency in TMS delivery across animals and across sessions. Using a fluorescent glucose analogue (2-NBDG) as a marker of neuronal activity, we mapped glucose uptake in response to TMS of the rat motor cortex. ResultsThe average motor threshold (n = 41) was 34.6 ± 6.3 % of maximum stimulator output (MSO). The variability of motor threshold across animals was similar to what has been reported in human studies. Furthermore, there was no significant difference in motor threshold measured across 3 separate days. Enhancement in fluorescent signals were TMS dose (power)-dependent, which centered around the motor cortex, covering an area medial-laterally 2 mm, rostral-caudally 4 mm at 55 % MSO, and 3 mm at 35 % MSO. The count of total cells with significant fluorescent signal was: 107 ± 23 (55 % MSO), 73 ± 11 (35 % MSO) and 42 ± 11 (sham, 5% MSO). ConclusionsOur method allows for consistent motor threshold assessment for longitudinal studies. Notably, cells with fluorescent signal enhancement were consistently aggregated in deep cortical layers, with minimal enhancement in superficial layers Comparisons with existing method(s)To our knowledge, this is the first study of focal TMS in awake rodents.

Effect of desacyl-ghrelin on food anticipatory activity in a mouse model of chronic food restriction

Background The physiological mechanisms involved in the adaptation to chronic food restriction are largely unknown. Anorexia nervosa (AN) is characterized by elevated plasma concentrations of ghrelin, a gut orexigenic hormone. AN patients maintain a restricted feeding behavior, usually associated with elevated physical activity in meal anticipation called food anticipatory activity (FAA). Ghrelin exists in two forms: acylghrelin (AG), carrying an octanoyl group, and des-acylghrelin (DAG) which predominates in plasma and having an opposite action on energy homeostasis. The respective role of AG and DAG in the initiation of FAA remains unknown. Objectives We used a mouse model, which combined chronic food restriction and voluntary activity mimicking most of the alterations of AN1. We evaluated: – the change in plasma AG and/or DAG concentrations in relation with FAA; – determine whether injections of AG and/or DAG elicit FAA and interact on hypothalamic (ARC: energy homeostasis) and mesolimbic (VTA: reward/motivation) neurons. Methods Eight-week old female mice were placed in a cage containing a running wheel in free access for 20 days. Mice were exposed to a progressive food restriction (FRW, n = 24) compared to ad libitum fed condition (ALW, n = 24). On D10, blood samples were collected before the onset of FAA (9 a.m.) and during FAA (5 p.m). AG and DAG were measured with selective immunoassays. On D20, AG (10 nmol/10 g BW), DAG (10 nmol/10 g BW), AG + DAG (respectively 10 and 20 nmol/10 g BW), or saline solution were injected subcutaneously both in ALW and FRW mice (n = 6/group); followed by 2 h of the evaluation of physical activity. Immunocytochemical detection of c-Fos protein, a marker of neuronal activation, was obtained in the ARC and VTA. Data were analyzed with a One-Way ANOVA test (Statview software, P Results FRW mice displayed a stable FAA from D6 of restriction. Plasma concentrations of AG and DAG significantly increased between 9 a.m and 5 p.m in FRW mice compared to ALW mice. AG and DAG concentrations were the highest in mice displaying the highest FAA. Interestingly, only the injection of DAG was able to elicit: – an increase in physical activity (P = 0.06) associated with significant neuronal activation in the VTA (P – to decrease glycemia (P = 0.06) and low neuronal activation in the ARC contrary to AG (P Conclusion These results suggest that DAG might have an unexpected function in the induction of physical activity in meal anticipation.

Microglia contribute to cancer cachexia through affecting PVN neurons and POMC neurons

BackgroundCancer cachexia is a state of involuntary weight loss, muscle wasting, and severe malnutrition. Browning of adipocytes and anorexia are major contributors to cancer cachexia through thermogenesis‐induced increasing energy consumption and decreasing food intake, respectively. Increased neural activity of hypothalamic paraventricular nucleus (PVN) is crucially involved in sympathoexcitation causing the browning of adipocytes. Stimulation of pro‐opiomelanocortin (POMC) neurons in arcuate nucleus (ARC) of hypothalamus negatively regulates feeding. Activation of microglia in the hypothalamus can be associated with activity of PVN neurons and POMC neurons. Although microglial activation has been observed in systemic inflammatory animal models, it remains unclear whether microglia contribute to cachexia through affecting PVN neurons and POMC neurons in the cancer model with cancer‐associated systemic inflammation.Methods and ResultsWe implanted AH‐130 hepatoma cells intraperitoneally in Wistar‐Hannover rats to create an established cancer cachexia model exhibiting systemic inflammation. To examine the activity of microglia in hypothalamus, Iba1 immunostaining was performed at day 10 after implantation. In both PVN and ARC, a decreased cell perimeter and an increased cell circularity of microglia were observed in tumor‐bearing rats compared with controls (perimeter, 920.5±77.5 vs 1395±49.7 μm; circularity, 1.20±0.11 vs 0.80±0.04 *10−2; n=2, 5; p&lt;0.05), suggesting microglial activation in cancer cachexia. Then, to test whether the microglial activation contributes to cachexia and prognosis in tumor‐bearing rats, continuous intracerebroventricularinfusion of minocycline, an inhibitor of microglial activation, or vehicle was initiated 3 days before tumor implantation (tumor‐mino, n=8; tumor‐vehicle, n=8). As a control, the vehicle intracerebroventricularinfusion was performed without tumor implantation (control, n=6). We evaluated survival rate up to 12 days after implantation and obtained the tissues from the survived rats at day 12. Minocycline showed a trend increasing survival in tumor‐bearing rats. Food intake had been lower in tumor‐vehicle group than in control group from day 1. Minocycline attenuated the decrease of food intake in tumor‐bearing rats (food intake at day 11, 12.3±1.04 vs 5.45±1.30 g/day; n=7, 6; p&lt;0.05 vs vehicle treatment). Although we obtained the tissues from a limited number of tumor‐bearing rats due to the low survival rate, body weight, heart weight and skeletal muscle weight (gastrocnemius and soleus) were obviously smaller in tumor‐vehicle group than in controls. The immunostaining study demonstrated that, in tumor‐vehicle group compared with control group, microglial activity assessed by morphological analysis was increased in the PVN and ARC, and c‐Fos, a marker of neuronal activity, was also increased in the PVN neurons and ACTH‐positive POMC neurons. Minocycline attenuated these tumor‐induced body and tissue wasting, microglial activation, and neuronal activation.ConclusionThese preliminary data suggest that microglia contribute to cancer cachexia probably through affecting sympathoexcitatory PVN neurons and anorexigenic POMC neurons.

CHARACTERIZING THE LEPTIN‐INDUCED ACTIVATION OF PRE‐SYMPATHETIC CARDIOVASCULAR AND METABOLIC‐RELATED HYPOTHALAMIC NEURONS IN MICE

Obesity has risen 75% in the United States since 1980 and an estimated 80 million American adults are considered obese. Obesity activates the sympathetic nervous system and is associated with neurogenic hypertension. Leptin is an obesity‐related neuropeptide released from fat cells which reduces appetite and increases metabolism. Leptin activates metabolic and cardiovascular responsive pre‐sympathetic neurons within the hypothalamus. Although leptin increases metabolism and curbs appetite, it is also reported to increase blood pressure and heart rate. Considering that the common goal of obesity treatments is to diminish the cardiovascular‐related co‐morbidities increases in blood pressure and heart rate are unacceptable side effects for any potential treatment. Thus, a better understanding of the role hypothalamic sites involved in obesity‐related hypertension is necessary for the development of successful treatments. In this study, we tested the hypothesis that leptin activates pre‐sympathetic hypothalamic neurons that control either metabolic (raphe pallidus‐projecting) or cardiovascular activity (RVLM, rostroventrolateral medulla‐projecting). First, to identify leptin receptor expressing neurons we created a line of transgenic reporter mice using the cre‐lox recombination system to express tdTomato under the control of the leptin (ObRb) receptor gene. Then, to determine if exogenous leptin treatment activates pre‐sympathetic metabolic or cardiovascular‐related hypothalamic neurons we performed neuroanatomical tracer studies in these mice. Metabolically‐related neurons were identified by microinjection (50 nl) of green FluoSpheres™ (505/515) into the raphe nucleus and cardiovascular‐related neurons were identified by microinjection (50 nl) of magenta FluoSpheres™ (660/680) into the rostroventrolateral medulla (RVLM). We then treated these mice with an acute dose of leptin (25 μg/mouse IP) and then performed fluorescence immunohistochemical labelling to identify leptin‐induced neuronal activation of cFos, a marker of neuronal activation. Using this approach we identified both a population of RVLM‐projecting and a population of raphe‐projecting leptin expressing neurons. These data provide additional information to suggest that the leptin‐induced increases in cardiovascular and metabolic activity is mediated in part by activation of pre‐sympathetic hypothalamic neurons.Support or Funding InformationETSU RDC 19‐030 (Zahner MR) NHLBI R15 HL145645‐01 (Zahner MR)

Characterizing the microbiota‐gut‐brain axis in a murine model of pediatric inflammatory bowel disease

BackgroundInflammatory bowel diseases (IBD) are chronic intestinal diseases affecting over 1.4 million Americans. Pediatric patients are especially vulnerable to the psychiatric effects seen in IBD as they are still undergoing brain maturation and neuroplasticity during periods of significant GI inflammation. A murine model of pediatric IBD and the mechanism(s) in which the microbiota‐gut‐brain axis is altered during this developmental time have yet to be identified.AimOur goal was to develop a pediatric model of IBD and assess the effect on the microbiota‐gut‐brain axis at adulthood.HypothesisWe hypothesized that induction of ulcerative colitis by dextran sodium sulfate (DSS) at weanling mice (P21) would lead to lasting effects on the microbiota‐gut‐brain axis into adulthood, including behavioral deficits.MethodsC57BL/6 mice (male and female) were weaned (P21) and treated with 2% DSS (or vehicle) for 5 days in the drinking water. In early adulthood (6–8 weeks of age), behavioral tests were performed (novel object recognition task [NOR], light/dark [L/D] box and open field test [OFT]) and tissue samples collected for PCR, immunofluorescence and microbiota analysis.ResultsDSS‐treated mice showed impaired object recognition and increased anxiety‐like behaviors compared to vehicle controls. Hippocampal gene expression showed increased expression of the pattern recognition receptors (Nod1 and Nod2), inflammatory cytokines (IL17ar, IL23 and IL22) along with a decreased expression of the neuronal activation markers (cFOS). Cell proliferation in the hippocampal dentate gyrus as quantified by doublecortin (DCX) staining showed significantly less DCX than vehicle controls. Finally, microbiota assessment identified persistent dysbiosis in DSS‐treated mice on alpha diversity, taxa abundance and phylum abundance compared to vehicle controls.ConclusionsTaken together, these results indicate that acute DSS‐induced colitis in early adolescence can have long‐lasting effects on the microbiota‐gut‐brain axis in adulthood. Future studies will assess whether administration of probiotics can ameliorate these colitis‐induced microbiota‐gut‐brain axis deficits.Support or Funding InformationFunding provided by Crohn’s &amp; Colitis Foundation (CCFA)

The super-cooling compound icilin stimulates c-Fos and Egr-1 expression and activity involving TRPM8 channel activation, Ca2+ ion influx and activation of the ternary complex factor Elk-1

The TRPM8 cation channel can be activated by the cooling compound icilin. Recently, we showed that stimulation of TRPM8 channels induces a signaling cascade leading to the activation of the transcription factor AP-1. Additionally, expression of the AP-1 constituent c-Fos has been shown to be induced following TRPM8 stimulation. c-Fos is frequently used as a marker for neuronal activity. Here, we have analyzed the mechanism connecting TRPM8 stimulation and c-Fos expression. Furthermore, we analyzed the expression of the neuronal activity-responsive transcription factor Egr-1 following TRPM8 activation. The results show that icilin-induced stimulation of TRPM8 channels increased c-Fos promoter activity and induced c-Fos expression. Moreover, icilin stimulation increased Egr-1 promoter activity and induced the expression of Egr-1. Pharmacological inhibition of TRPM8 blocked the icilin-induced expression of Egr-1 and c-Fos. An influx of Ca2+ ions into the cells via TRPM8 was necessary to stimulate Egr-1 and c-Fos expression following icilin treatment. Genetic experiments revealed that serum response elements within the Egr-1 and c-Fos promoters are crucial to couple TRPM8 stimulation with enhanced transcription of both the Egr-1 and c-Fos genes. These data were corroborated by experiments showing that TRPM8 stimulation increased the transcriptional activation potential of Elk-1, a SRE binding protein. c-Fos is important for neuronal excitability and survival. Egr-1 plays an important role in synaptic plasticity, consolidation and reconsolidation of long-term memory. Elk-1 may preserve neurons against toxic insults but may also induce depressive behaviour. The fact that TRPM8 stimulation activates the transcription factors c-Fos, Egr-1, and Elk-1 connects TRPM8 signaling with maintaining important brain functions.

Cognitive appraisal in fish: stressor predictability modulates the physiological and neurobehavioural stress response in sea bass.

The role of cognitive factors in triggering the stress response is well established in humans and mammals (aka cognitive appraisal theory) but very seldom studied in other vertebrate taxa. Predictability is a key factor of the cognitive evaluation of stimuli. In this study, we tested the effects of stressor predictability on behavioral, physiological and neuromolecular responses in the European sea bass (Dicentrarchus labrax). Groups of four fish were exposed to a predictable (signalled) or unpredictable (unsignalled) stressor. Stressor predictability elicited a lower behavioural response and reduced cortisol levels. Using the expression of immediate early genes (c-fos, egr-1, bdnf and npas4) as markers of neuronal activity, we monitored the activity of three sea bass brain regions known to be implicated in stressor appraisal: the dorsomedian telencephalon, Dm (putative homologue of the pallial amygdala); and the dorsal (Dld) and ventral (Dlv) subareas of the dorsolateral telencephalon (putative homologue of the hippocampus). The activity of both the Dm and Dlv significantly responded to stressor predictability, suggesting an evolutionarily conserved role of these two brain regions in information processing related to stressor appraisal. These results indicate that stressor predictability plays a key role in the activation of the stress response in a teleost fish, hence highlighting the role of cognitive processes in fish stress.

Rislenemdaz treatment in the lateral habenula improves despair-like behavior in mice.

The specific GluN2B antagonist rislenemdaz (Ris; a.k.a. MK-0657 and CERC-301) is in phase II clinical trial as an antidepressive drug, but the working mechanism for its antidepressant effects is not clearly understood. Given the important role of the lateral habenula (LHb) in the pathogenesis of depression and the fact that GluN2B-containing N-methyl-D-aspartate receptors and brain-derived neurotrophic factor (BDNF) are expressed in the LHb, we conducted a study to examine whether the LHb mediates Ris' antidepressant effects in a chronic restraint stress (CRS)-induced depressive-like mouse model. In this study, Ris was administered systemically or locally into the LHb. Short hairpin RNAs were used to knockdown BDNF in the LHb. Depressive-like behaviors were assessed with the open field test, forced swimming test, tail suspension test, and sucrose preference test. Expression of GluN2B, BDNF, and c-Fos in the LHb were analyzed with western blotting and immunohistochemistry under condition with Ris administered systemically or with BDNF knockdown in the LHb. We found that both systemic and intra-LHb administration of Ris alleviated CRS-induced despair-like behavior and that systemic Ris reduced LHb expression of GluN2B, BDNF, and c-Fos (a neuronal activity marker). Specific knockdown of BDNF in the LHb prevented CRS-induced despair-like behavior, while preventing CRS-induced increases in BDNF and c-Fos expression in the LHb. Together these results suggest that Ris may exert its antidepressant effects through affecting the LHb such as downregulating BDNF expression in the LHb.

Dipeptide tyrosyl-leucine exhibits antidepressant-like activity in mice

Depression is a worldwide health problem. In the present study, we found that a dipeptide, tyrosyl leucine (Tyr-Leu, YL), administered orally, intracerebroventricularly, or intraperitoneally exhibited a potent antidepressant-like activity in the forced swim and tail suspension tests in naïve mice. YL increased the amount of cells expressing c-Fos, a marker for neuronal activity, in the dentate gyrus of the hippocampus. YL increased bromo-2′-deoxyuridine-positive cells and doublecortin expression in the dentate gyrus of the hippocampus, suggesting that YL enhanced the proliferation of hippocampal progenitor cells in vivo and in vitro. YL did not affect hippocampal mRNA and protein expression of BDNF, which is a regulatory factor of both neurogenesis and depression-like behavior. Intriguingly, YL suppressed activation of the hypothalamo-pituitary-adrenal axis by forced swim stress. Moreover, other aromatic amino acid-leucines, Phe-Leu and Trp-Leu, also exhibited antidepressant-like activities, suggesting that the structure of aromatic amino acid-leucine may be important for antidepressant activity. In addition, bovine milk casein-derived peptide, Tyr-Leu-Gly (YLG), an anxiolytic peptide, exhibited an antidepressant-like activity. Our findings demonstrate that YL exhibits an antidepressant-like effect, moderates the stress response, and induces hippocampal neuronal proliferation through a signal pathway independent of BDNF.

Changes in brain peptides associated with reproduction and energy homeostasis: Putative roles of gonadotrophin-releasing hormone-II and tyrosine hydroxylase in determining reproductive performance in response to daily food availability times in diurnal zebra finches.

Previous studies have demonstrated 'quality-quantity' trade-offs with daily food availability times in zebra finches. Compared with food access ad lib., zebra finch pairs with restricted food access for 4hours in the morning produced poor quality offspring, whereas those with the same food access in the evening produced fewer but better quality offspring. The present study investigated whether food-time-dependent differential effects on reproductive performance involved brain peptides associated with reproduction and energy homeostasis in zebra finches. We measured peptide/protein expression of gonadotrophin-releasing hormone (GnRH)-I, GnRH-II, gonadotrophin-inhibitory hormone (GnIH), tyrosine hydroxylase (TH), neuropeptide Y (NPY), cocaine- and amphetamine regulated transcript (CART) and ZENK (a neuronal activation marker) by immunohistochemistry and mRNA expression of genes coding for the type 2 (DIO2) and type 3 (DIO3) deiodinase by a quantitative polymerase chain reaction in male and female zebra finches that were paired and kept under a 12:12 hour light/dark photocycle at 24±2°C temperature for >12months with access to food ad lib., or for only 4hours in the morning or evening. In both sexes, GnRH-I, DIO2 and DIO3 expression did not differ significantly between the three feeding conditions, although levels showed an overall food effect. However, in males, GnIH expression was significantly higher in evening-fed birds compared to ad lib. fed birds. Interestingly, GnRH-II and TH levels were significantly lower in restricted feeding compared to the ad lib. group and, importantly, GnRH-II and TH-immunoreactivity levels were negatively and positively correlated with egg laying latency and reproductive success (offspring/brood/pair), respectively. At the same time, we found no effect on the hypothalamic expression of orexigenic (NPY) and anorexigenic (CART) peptides, or ZENK protein (ie, the neuronal activity marker). These results suggest the involvement of reproductive neuropeptides, with putative roles for GnRH-II and TH, in the food-time-dependent effect on reproductive performance, albeit with subtle sex differences, in diurnal zebra finches, which possess the ability to reproduce year-round, in a manner similar to other continuously breeding vertebrates.

Significance of the orexinergic system in modulating stress-related responses in an animal model of post-traumatic stress disorder

Converging evidence indicates that orexins (ORXs), the regulatory neuropeptides, are implicated in anxiety- and depression-related behaviors via the modulation of neuroendocrine, serotonergic, and noradrenergic systems. This study evaluated the role of the orexinergic system in stress-associated physiological responses in a controlled prospective animal model. The pattern and time course of activation of hypothalamic ORX neurons in response to predator-scent stress (PSS) were examined using c-Fos as a marker for neuronal activity. The relationship between the behavioral response pattern 7 days post-exposure and expressions of ORXs was evaluated. We also investigated the effects of intracerebroventricular microinfusion of ORX-A or almorexant (ORX-A/B receptor antagonist) on behavioral responses 7 days following PSS exposure. Hypothalamic levels of ORX-A, neuropeptide Y (NPY), and brain-derived neurotrophic factor (BDNF) were assessed. Compared with rats whose behaviors were extremely disrupted (post-traumatic stress disorder [PTSD]-phenotype), those whose behaviors were minimally selectively disrupted displayed significantly upregulated ORX-A and ORX-B levels in the hypothalamic nuclei. Intracerebroventricular microinfusion of ORX-A before PSS reduced the prevalence of the PTSD phenotype compared with that of artificial cerebrospinal fluid or almorexant, and rats treated with almorexant displayed a higher prevalence of the PTSD phenotype than did untreated rats. Activated ORX neurons led to upregulated expressions of BDNF and NPY, which might provide an additional regulatory mechanism for the modulation of adaptive stress responses. The study indicates that the activated ORX system might promote adaptive responses to PSS probably via stimulation of BDNF and NPY secretion, and early intervention with ORX-A reduces the prevalence of the PTSD phenotype and increases the prevalence of adaptive phenotypes. The findings provide some insights into the mechanisms underlying the involvement of the ORX system in stress-related disorders.

Behavioral deficits in left hemispatial neglect are related to a reduction of spontaneous neuronal activity in the right superior parietal lobule

Focal brain lesions may induce dysfunctions in distant brain regions leading to behavioral impairments. Based on this concept of ‘diaschisis’, spatial neglect following stroke has been related to structural damage of the right-lateralized ventral attention network (VAN) and disrupted inter-hemispheric functional connectivity (FC) in the bilateral dorsal attention network (DAN). We questioned whether neglect-related behavioral deficits may be determined by local dysfunction of a specific region within these brain networks.We investigated acute right-hemisphere stroke patients with left hemispatial neglect using resting-state functional MRI, neuropsychological tests of spatial attention and clinical assessment of neglect-related functional disability. In addition to conventional FC analyses between different cortical regions of interest (ROIs) in the DAN/VAN, we extracted the fractional amplitude of low frequency fluctuations (fALFF) from each ROI as a marker of regional spontaneous neuronal activity. Although DAN regions (as opposed to the VAN regions) were largely spared from structural brain damage, they exhibited a significant reduction of inter-hemispheric FC. However, significant fMRI-behavior correlations were revealed specifically for the fALFF of one DAN-ROI in the right superior parietal lobule (SPL): the smaller the fALFF in the right posterior intraparietal sulcus, the more severe the patient's pathological attention bias and neglect-related functional impairment.In line with ‘diaschisis’, our findings confirm a crucial role of the non-lesioned but dysfunctional right SPL for the emergence of spatial neglect and its behavioral consequences. They further support targeting the SPL dysfunction by non-invasive brain stimulation in neglect rehabilitation.

Medial Prefrontal Cortex Neural Plasticity, Orexin Receptor 1 Signaling, and Connectivity with the Lateral Hypothalamus Are Necessary in Cue-Potentiated Feeding.

Cognitive processes contribute to the control of feeding behavior and help organism's survival when they support physiological needs. They can become maladaptive, such as when learned food cues drive feeding in the absence of hunger. Associative learning is the basis for cue-driven food seeking and consumption, and behavioral paradigms with Pavlovian cue-food conditioning are well established. Yet, the neural mechanisms underlying circuit plasticity across cue-food learning, cue memory recall, and subsequent food motivation are unknown. Here, we demonstrated the medial prefrontal cortex (mPFC) is a site of learning-induced plasticity and signaling of the neuropeptide orexin within the mPFC mediates cue potentiated feeding (CPF). First, using a marker of neuronal activation, c-fos, we confirmed that the mPFC is activated during CPF. Next, to assess whether the same mPFC neuronal ensemble is activated during cue-food learning and later CPF, we used the Daun02 chemogenetic inactivation method in c-fos-lacZ transgenic male and female rats. Selective inactivation of the mPFC neurons that were active during the last cue-food training session abolished CPF during test, demonstrating that the mPFC is a site of plasticity. We postulated that integration of food cue memory and feeding motivation requires mPFC communications with lateral hypothalamus and showed that disconnection of that system abolished CPF. Then we showed that lateral hypothalamus orexin-producing neurons project to the mPFC. Finally, we blocked orexin receptor 1 signaling in the mPFC and showed that it is a neuromodulator necessary for the cue-driven consumption. Together, our findings identify a causal function for the mPFC in the cognitive motivation to eat.SIGNIFICANCE STATEMENT Obesity has reached epidemic proportions, and the associated health consequences are serious and costly. The causes of obesity are complex because, in addition to physiological energy and nutrient needs, environmental cues can drive feeding through hedonic and cognitive processes. Learned food cues from the environment can powerfully stimulate appetite and food consumption in the absence of hunger. Using an animal model for cue-potentiated feeding, the current study determined the mPFC neuronal plasticity and neuropeptide orexin signaling are critical circuit and neurotransmitter mechanisms involved in this form of cognitive motivation to eat. These findings identify key targets for potential treatment of excessive appetite and overeating.