Neuropeptide mRNA Research Articles

Evaluation of the SH-SY5Y cell line as an in vitro model for potency testing of a neuropeptide-expressing AAV vector

Viral vectors have become important tools for basic research and clinical gene therapy over the past years. However, in vitro testing of vector-derived transgene function can be challenging when specific post-translational modifications are needed for biological activity. Similarly, neuropeptide precursors need to be processed to yield mature neuropeptides. SH-SY5Y is a human neuroblastoma cell line commonly used due to its ability to differentiate into specific neuronal subtypes. In this study, we evaluate the suitability of SH-SY5Y cells in a potency assay for neuropeptide-expressing adeno-associated virus (AAV) vectors. We looked at the impact of neuronal differentiation and compared single-stranded (ss) AAV and self-complementary (sc) AAV transduction at increasing MOIs, RNA transcription kinetics, as well as protein expression and mature neuropeptide production. SH-SY5Y cells proved highly transducible with AAV1 already at low MOIs in the undifferentiated state and even better after neuronal differentiation. Readouts were GFP or neuropeptide mRNA expression. Production of mature neuropeptides was poor in undifferentiated cells. By contrast, differentiated cells produced and sequestered mature neuropeptides into the medium in a MOI-dependent manner.

Unraveling the periprandial changes in brain serotonergic activity and its correlation with food intake-related neuropeptides in rainbow trout Oncorhynchus mykiss.

This study explored changes in brain serotonin content and activity together with hypothalamic neuropeptide mRNA abundance around feeding time in rainbow trout, as well as the effect of one-day fasting. Groups of trout fed at two (ZT2) and six (ZT6) hours after lights on were sampled from 90 minutes before to 240 minutes after feeding, while additional groups of non-fed trout were also included in the study. Changes in brain amine and metabolite contents were measured in hindbrain, diencephalon and telencephalon, while in the diencephalon the mRNA abundance of tryptophan hydroxylase (tph1, tph2), serotonin receptors (5htr1a, 5htr1b and 5htr2c) and several neuropeptides (npy, agrp1, cartpt, pomca1, crfb) involved in the control of food intake were also assessed. The results showed changes in the hypothalamic neuropeptides that were consistent with the expected role for each in the regulation of food intake in rainbow trout. Serotonergic activity increased rapidly at the time of food intake in the diencephalon and hindbrain and remained high for much of the postprandial period. This increase in serotonin abundance was concomitant with elevated levels of pomca1 mRNA in the diencephalon, suggesting that serotonin might act on brain neuropeptides to promote a satiety profile. Furthermore, serotonin synthesis and neuronal activity appear to increase already before the time of feeding, suggesting additional functions for this amine before and during food intake. Exploration of serotonin receptors in the diencephalon revealed only small changes for gene expression of 5htr1b and 5htr2c receptors during the postprandial phase. Therefore, the results suggest that serotonin may play a relevant role in the regulation of feeding behavior in rainbow trout during periprandial time, but a better understanding of its interaction with brain centers involved in receiving and processing food-related signals is still needed.

Malocclusion impairs cognitive behavior via AgRP signaling in adolescent mice.

Occlusal disharmony induced by deteriorating oral health conditions, such as tooth loss and decreased masticatory muscle due to sarcopenia, is one of the causes of cognitive impairment. Chewing is an essential oral function for maintaining cognitive function not only in the elderly but also in young people. Malocclusion is an occlusal disharmony that commonly occurs in children. The connection between a decline in cognitive function and malocclusion in children has been shown with chronic mouth breathing, obstructive sleep apnea syndrome, and thumb/digit sucking habits. However, the mechanism of malocclusion-induced cognitive decline is not fully understood. We recently reported an association between feeding-related neuropeptides and cognitive decline in adolescent mice with activity-based anorexia. The aim of the present study was to assess the effects of malocclusion on cognitive behavior and clarify the connection between cognitive decline and hypothalamic feeding-related neuropeptides in adolescent mice with malocclusion. Four-week-old mice were randomly assigned to the sham-operated solid diet-fed (Sham/solid), sham-operated powder diet-fed (Sham/powder), or malocclusion-operated powder diet-fed (Malocclusion/powder) group. We applied composite resin to the mandibular anterior teeth to simulate malocclusion. We evaluated cognitive behavior using a novel object recognition (NOR) test, measured hypothalamic feeding-related neuropeptide mRNA expression levels, and enumerated c-Fos-positive cells in the hypothalamus 1 month after surgery. We also evaluated the effects of central antibody administration on cognitive behavior impairment in the NOR test. The NOR indices were lower and the agouti-related peptide (AgRP) mRNA levels and number of c-Fos-positive cells were higher in the malocclusion/powder group than in the other groups. The c-Fos-positive cells were also AgRP-positive. We observed that the central administration of anti-AgRP antibody significantly increased the NOR indices. The present study suggests that elevated cerebral AgRP signaling contributes to malocclusion-induced cognitive decline in adolescents, and the suppression of AgRP signaling can be a new therapeutic target against cognitive decline in occlusal disharmony.

Differential vulnerability of neuronal subpopulations of the subiculum in a mouse model for mesial temporal lobe epilepsy.

Selective loss of inhibitory interneurons (INs) that promotes a shift toward an excitatory predominance may have a critical impact on the generation of epileptic activity. While research on mesial temporal lobe epilepsy (MTLE) has mostly focused on hippocampal changes, including IN loss, the subiculum as the major output region of the hippocampal formation has received less attention. The subiculum has been shown to occupy a key position in the epileptic network, but data on cellular alterations are controversial. Using the intrahippocampal kainate (KA) mouse model for MTLE, which recapitulates main features of human MTLE such as unilateral hippocampal sclerosis and granule cell dispersion, we identified cell loss in the subiculum and quantified changes in specific IN subpopulations along its dorso-ventral axis. We performed intrahippocampal recordings, FluoroJade C-staining for degenerating neurons shortly after status epilepticus (SE), fluorescence in situ hybridization for glutamic acid decarboxylase (Gad) 67 mRNA and immunohistochemistry for neuronal nuclei (NeuN), parvalbumin (PV), calretinin (CR) and neuropeptide Y (NPY) at 21 days after KA. We observed remarkable cell loss in the ipsilateral subiculum shortly after SE, reflected in lowered density of NeuN+ cells in the chronic stage when epileptic activity occurred in the subiculum concomitantly with the hippocampus. In addition, we show a position-dependent reduction of Gad67-expressing INs by ∼50% (along the dorso-ventral as well as transverse axis of the subiculum). This particularly affected the PV- and to a lesser extent CR-expressing INs. The density of NPY-positive neurons was increased, but the double-labeling for Gad67 mRNA expression revealed that an upregulation or de novo expression of NPY in non-GABAergic cells with a concomitant reduction of NPY-positive INs underlies this observation. Our data suggest a position- and cell type-specific vulnerability of subicular INs in MTLE, which might contribute to hyperexcitability of the subiculum, reflected in epileptic activity.

Modulatory effect of long-term treatment with escitalopram and clonazepam on the expression of anxiety-related neuropeptides: neuromedin U, neuropeptide S and their receptors in the rat brain

Newly identified multifunctional peptidergic modulators of stress responses: neuromedin U (NMU) and neuropeptide S (NPS) are involved in the wide spectrum of brain functions. However, there are no reports dealing with potential molecular relationships between the action of diverse anxiolytic or antidepressant drugs and NMU and NPS signaling in the brain. The present work was therefore focused on local expression of the aforementioned stress-related neuropeptides in the rat brain after long-term treatment with escitalopram and clonazepam. Studies were carried out on adult, male Sprague-Dawley rats that were divided into 3 groups: animals injected with saline (control) and experimental individuals treated with escitalopram (at single dose 5mg/kg daily), and clonazepam (at single dose 0.5mg/kg). All individuals were sacrificed under anaesthesia and the whole brain excised. Total mRNA was isolated from homogenized samples of amygdala, hippocampus, hypothalamus, thalamus, cerebellum and brainstem. Real time-PCR method was used for estimation of related NPS, NPS receptor (NPSR), NMU, NMU and receptor 2 (NMUR2) mRNA expression. The whole brains were also sliced for general immunohistochemical assessment of the neuropeptides expression. Chronic administration of clonazepam resulted in an increase of NMU mRNA expression and formation of NMU-expressing fibers in the amygdala, while escitalopram produced a significant decrease in NPSR mRNA level in hypothalamus. Long-term escitalopram administration affects the local expression of examined neuropeptides mRNA in a varied manner depending on the brain structure. Pharmacological effects of escitalopram may be connected with local at least partially NPSR-related alterations in the NPS/NMU/NMUR2 gene expression at the level selected rat brain regions. A novel alternative mode of SSRI action can be therefore cautiously proposed.

Escitalopram alters local expression of noncanonical stress-related neuropeptides in the rat brain via NPS receptor signaling.

Neuropeptide S (NPS) is a multifunctional regulatory factor that exhibits a potent anxiolytic activity in animal models. However, there are no reports dealing with the potential molecular relationships between the anxiolytic activity of selective serotonin reuptake inhibitors (SSRIs) and NPS signaling, especially in the context of novel stress-related neuropeptides action. The present work therefore focused on gene expression of novel stress neuropeptides in the rat brain after acute treatment with escitalopram and in combination with neuropeptide S receptor (NPSR) blockade. Studies were carried out on adult, male Sprague-Dawley rats that were divided into five groups: animals injected with saline (control) and experimental rats treated with escitalopram (at single dose 10mg/kg daily), escitalopram and SHA-68, a selective NPSR antagonist (ata single doseof 40mg/kg), SHA-68 alone and corresponding vehicle (solvent SHA-68) control. To measure anxiety-like behavior and locomotor activity the open field test was performed. All individuals were killed under anaesthesia and the whole brainwas excised. Total mRNA was isolated from homogenized samples ofthe amygdala, hippocampus, hypothalamus, thalamus, cerebellum, and brainstem. Real-time PCR was used for estimation of related NPS, NPSR, neuromedin U (NMU), NMU receptor 2 (NMUR2) and nesfatin-1 precursor nucleobindin-2 (NUCB2) gene expression. Acute escitalopram administration affects the local expression of the examined neuropeptides mRNA in a varied manner depending on brain location. An increase in NPSR and NUCB2 mRNA expression in the hypothalamus and brainstem was abolished by SHA-68 coadministration, while NMU mRNA expression was upregulated after NPSR blockade in the hippocampus and cerebellum. The pharmacological effects of escitalopram may be connected with local NPSR-related alterations in NPS/NMU/NMUR2 and nesfatin-1 gene expression at the level of selected rat brain regions. A novel alternative mode of SSRI action can be therefore cautiously proposed.

Chronic Undernutrition Inhibits KNDy Neurons in Ovariectomized Ewe Lambs

Undernutrition negatively impacts reproductive success, at least in part, through a central suppression of GnRH secretion. Given that GnRH neurons are devoid of receptors for peripheral metabolic hormones such as leptin and insulin, nutritional regulation of GnRH secretion must be through afferent input. Neurons which co-express kisspeptin, neurokinin B (NKB), and dynorphin, termed KNDy neurons, are a unique population of cells in the arcuate nucleus (ARC) of the hypothalamus and are believed to play a critical role in GnRH/LH pulse generation. With our recent evidence that chronic feed restriction reduced kisspeptin and NKB in young, castrated male sheep, we hypothesized that nutrient restriction would inhibit expression of the KNDy neuron peptides kisspeptin and NKB, while increasing dynorphin expression in young, ovariectomized female sheep. Fifteen ewe lambs were ovariectomized and were fed to maintain body weight (n=7; Fed) or feed-restricted to lose 20% of pre-study body weight (FR; n=8). Blood samples were taken weekly every 12 minutes for 4.5 hours via jugular venipuncture and plasma was stored at -20°C until assessment of LH using radioimmunoassay. Body weights were recorded weekly and feed amounts were adjusted to achieve desired body weights. Following blood collection at Week 13, animals were euthanized, brain tissue was perfused with 4% paraformaldehyde, and tissue containing the hypothalamus was collected for assessment of KNDy neuropeptide mRNA abundance by in situ hybridization (RNAscope). At Week 13, the average percent change in body weight was clearly evident (Fed, 7.35 +/- 2.2% vs FR, -21.36 +/- 0.9%), and mean LH concentrations were lower in FR ewes (10.70 +/- 3.1 ng/ml) compared to Fed controls (20.98 +/- 3.8 ng/ml). Data analyzed to date for kisspeptin, NKB, and dynorphin in the ARC show that feed restriction reduced the number of kisspeptin mRNA-expressing cells (Fed, 165 +/- 25 vs FR, 9.25 +/- 6), the number of NKB mRNA-expressing cells (Fed, 141 +/- 28 vs FR, 24 +/- 5), and the number of dynorphin mRNA-expressing cells (Fed, 109 +/- 41 vs FR, 29.5 +/- 24). Together, these findings demonstrate that chronic feed restriction suppresses KNDy neurons and supports a role for these key reproductive neurons in the central mechanism governing GnRH/LH secretion during undernutrition in female sheep.

Gut microbiota depletion affects nutritional and behavioral responses to activity-based anorexia model in a sex-dependent manner

In the last decade, the role of the microbiota-gut-brain axis in eating behavior and anxiety-depressive disorders has gained increasing attention. Although a gut microbiota dysbiosis has been reported in anorectic patients, its pathophysiological role remains poorly understood. Thus, we aimed to characterize the potential role of gut microbiota by evaluating the effects of its depletion in the Activity-Based Anorexia (ABA) mouse model both in male and female mice. Male and female C57Bl/6 mice were submitted (ABA group) or not (CT group) to the ABA protocol, which combines access to a running wheel with a progressive limited food access. Gut microbiota was previously depleted or not by a cocktail of antibiotics (ATB) delivered by oral gavages. We monitored body composition, anxiety-like behavior, leptin and adiponectin plasma levels, hypothalamic and hippocampal neuropeptides mRNA levels, as well as dopamine (DRD) and serotonin (5HT1 and 4) receptors mRNA expression. In response to the ABA model, the body weight loss was less pronounced in ATB-treated ABA compared to untreated ABA, while food intake remained unaffected by ATB treatment. ATB-treated ABA exhibited increased fat mass and decreased lean mass compared to untreated ABA both in male and female mice, whereas but plasma adipokine concentrations were affected in a sex-dependent manner. Only male ABA mice showed a reduced anticipatory physical activity in response to ATB treatment. Similarly, anxiety-like behavior was mainly affected in ATB-treated ABA male mice compared to ATB-treated ABA female mice, which was associated with male-specific alterations of hypothalamic CRH mRNA and hippocampal DRD and 5-HT1A mRNA levels. Our study provides evidence that ATB-induced gut microbiota depletion triggers alterations of nutritional and behavioral responses to the activity-based anorexia model in a sex-dependent manner.

Neonatal Serotonin Depletion Induces Hyperactivity and Anxiolytic-like Sex-Dependent Effects in Adult Rats.

The serotoninergic system plays an important role in the ontogeny of the mammalian central nervous system, and changes in serotonin production during development may lead to permanent changes in brain cytoarchitecture and function. The present study investigated the programming effects of neonatal serotonin depletion on behavior and molecular components of the serotoninergic system in adult male and female rats. Subcutaneous para-chlorophenylalanine (pCPA) administration (100mgkg-1) was performed daily on postnatal days 8-16 to deplete brain serotonin content. During adulthood, elevated plus-maze, open field, social interaction, forced swimming, and food, saline, and sucrose intake tests were performed. Relative expression of serotonin neurotransmission components in several brain areas was determined by qPCR. Additionally, serotonin immunofluorescence and neuropeptide mRNA expression were assessed in dorsal raphe (DRN) and paraventricular (PVN) nuclei, respectively. Rat performance in behavioral tests demonstrated a general increase in locomotor activity and active escape behavior as well as decreased anxiety-like behavior after neonatal brain serotonin depletion. The behavioral programming effects due to neonatal serotonin depletion were more pronounced in females than males. At the gene expression level, the mRNA of Tph1 and Tph2 were lower in DRN while Htr2c was higher in the amygdala of pCPA-treatedmales, while Htr1a, Htr2c, Oxt, Avp, Crh, and Trh were not different in any treatments or sex in PVN. The results indicate that neonatal serotonin depletion has long-term consequences on locomotion and anxiety-like behavior associated with long-lasting molecular changes in the brain serotoninergic system in adult rats.

Sex Differences in Escalated Methamphetamine Self-Administration and Altered Gene Expression Associated With Incubation of Methamphetamine Seeking.

Methamphetamine (METH) use disorder is prevalent worldwide. There are reports of sex differences in quantities of drug used and relapses to drug use among individuals with METH use disorder. However, the molecular neurobiology of these potential sex differences remains unknown. We trained rats to self-administer METH (0. 1 mg/kg/infusion, i.v.) on an fixed-ratio-1 schedule for 20 days using two 3-hour daily METH sessions separated by 30-minute breaks. At the end of self-administration training, rats underwent tests of cue-induced METH seeking on withdrawal days 3 and 30. Twenty-four hours later, nucleus accumbens was dissected and then used to measure neuropeptide mRNA levels. Behavioral results show that male rats increased the number of METH infusions earlier during self-administration training and took more METH than females. Both male and female rats could be further divided into 2 phenotypes labeled high and low takers based on the degree of escalation that they exhibited during the course of the METH self-administration experiment. Both males and females exhibited incubation of METH seeking after 30 days of forced withdrawal. Females had higher basal mRNA levels of dynorphin and hypocretin/orexin receptors than males, whereas males expressed higher vasopressin mRNA levels than females under saline and METH conditions. Unexpectedly, only males showed increased expression of nucleus accumbens dynorphin after METH self-administration. Moreover, there were significant correlations between nucleus accumbens Hcrtr1, Hcrtr2, Crhr2, and Avpr1b mRNA levels and cue-induced METH seeking only in female rats. Our results identify some behavioral and molecular differences between male and female rats that had self-administered METH. Sexual dimorphism in responses to METH exposure should be considered when developing potential therapeutic agents against METH use disorder.

Na+/K+-ATPase is involved in the regulation of food intake in rainbow trout but apparently not through brain glucosensing mechanisms

To assess the hypothesis that Na+/K+-ATPase (NKA) is involved in the central regulation of food intake in fish, we observed in a first experiment with rainbow trout (Oncorhynchus mykiss) that intracerebroventricular (ICV) treatment with ouabain decreased food intake. We hypothesized that this effect relates to modulation of glucosensing mechanisms in brain areas (hypothalamus, hindbrain, and telencephalon) involved in food intake control. Therefore, we evaluated in a second experiment, the effect of ICV administration of ouabain, in the absence or in the presence of glucose, on NKA activity, mRNA abundance of different NKA subunits, parameters related to glucosensing, transcription factors, and appetite-related neuropeptides in brain areas involved in the control of food intake. NKA activity and mRNA abundance of nkaα1a and nkaα1c in brain were inhibited by ouabain treatment and partially by glucose. The anorectic effect of ouabain is opposed to the orexigenic effect reported in mammals. The difference might relate to the activity of glucosensing as well as downstream mechanisms involved in food intake regulation. Ouabain inhibited glucosensing mechanisms, which were activated by glucose in hypothalamus and telencephalon. Transcription factors and neuropeptides displayed responses comparable to those elicited by glucose when ouabain was administered alone, but not when glucose and ouabain were administered simultaneously. Ouabain might therefore affect other processes, besides glucosensing mechanisms, generating changes in membrane potential and/or intracellular pathways finally modulating transcription factors and neuropeptide mRNA abundance leading to modified food intake.

Effects of γ-aminobutyric acid on aggressive behaviour, jejunum villus morphology, serum biochemical indicators and hippocampal neuropeptide mRNA levels in piglets at weaning with mixing

The effects of γ-aminobutyric acid (GABA) on weanling piglets after mixing stress were investigated and the underlying molecular mechanism was analyzed. Sixty weaning piglets were randomly assigned to either the control group (weaning and mixing with a 3 : 3 sex ratio) or the GABA supplement group (30 mg GABA/kg body weight/day + weaning and mixing with a 3 : 3 sex ratio). Aggressive behaviours have been recorded for 2 days and the number of lesions for 3 days. The diarrhea rate on day 6 post-weaning and mixing was analyzed. Serum biochemical indicators, antioxidant variables, jejunum villus morphology and mRNA levels of stress-related neuropeptide genes of the hippocampus were investigated. The GABA addition decreased serum adrenocorticotropic hormone concentrations (P < 0.05), aggressive behaviours of weaned piglets 5 h after mixing (P < 0.05), lesion scores over the entire 3-day period (P < 0.01) and diarrhea rate (P < 0.01) and improved jejunum villus integrity. Serum neuropeptide Y (NPY) concentration (P < 0.05) and total superoxide dismutase activity (P < 0.01) were increased in the GABA supplement group, whereas serum malondialdehyde concentration had a decreasing tendency (0.05 < P < 0.1), and glutathione peroxidase activity had an increasing tendency (0.05 < P < 0.1). The GABA treatment group had increased mRNA levels of NPY (P < 0.05) and peptide YY (PYY) (P < 0.05) in the hippocampus, which may contribute to insights into the regulatory mechanism of GABA in weaning and mixing stress. The addition of GABA is beneficial to reduce weaning and mixing stress in piglets, and NPY and PYY may mediate the process.

The Protein Status of Rats Affects the Rewarding Value of Meals Due to their Protein Content

Protein status is controlled by the brain, which modulates feeding behavior to prevent protein deficiency. This study tested in rats whether protein status modulates feeding behavior through brain reward pathways. Experiments were conducted in male Wistar rats (mean ± SD weight; 230±16 g). In experiment 1, rats adapted for 2 wk to a low-protein (LP; 6% of energy) or a normal-protein (NP; 14% of energy) diet were offered a choice between 3 cups containing high-protein (HP; 50% of energy), NP, or LP feed; their intake was measured for 24 h. In 2 other experiments, the rats were adapted for 2 wk to NP and either HP or LP diets and received, after overnight feed deprivation, a calibrated HP, NP, or LP meal daily. After the meal, on the last day, rats were killed and body composition and blood protein, triglycerides, gut neuropeptides, and hormones were determined. In the brain, neuropeptide mRNAs in the hypothalamus and c-Fos protein and opioid and dopaminergic receptor mRNAs in the nucleus accumbens (NAcc) were measured. Rats fed an LP compared with an NP diet had 7% lower body weight, significantly higher protein intake in a choice experiment (mean ± SD: 30.5%±0.05% compared with 20.5%±0.05% of energy), higher feed-deprived blood ghrelin, lower postmeal blood leptin, and higher neuropeptide Y (Npy) and corticotropin-releasing hormone (Crh) mRNA expression in the hypothalamus. In contrast to NP, rats fed an LP diet showed postmeal c-Fos protein expression in the NAcc, which was significantly different between meals, with LP < NP < HP. In contrast, in rats adapted to an HP diet compared with an NP diet, energy intake was lower; and in the NAcc, meal-induced c-Fos protein expression was 20% lower, and mRNA expression was 17% higher for dopamine receptor 2 (Drd2) receptors and 38% lower for κ opioid receptor (Oprk1) receptors. A protein-restricted diet induced a reward system-driven appetite for protein, whereas a protein-rich diet reduced the meal-induced activation of reward pathways and lowered energy intake in male rats.

The role of the transient receptor potential ankyrin type-1 (TRPA1) channel in migraine pain: evaluation in an animal model

BackgroundClinical and experimental studies have pointed to the possible involvement of the transient receptor potential ankyrin type-1 (TRPA1) channels in migraine pain. In this study, we aimed to further investigate the role of these channels in an animal model of migraine using a novel TRPA1 antagonist, ADM_12, as a probe.MethodsThe effects of ADM_12 on nitroglycerin-induced hyperalgesia at the trigeminal level were investigated in male rats using the quantification of nocifensive behavior in the orofacial formalin test. The expression levels of the genes coding for c-Fos, TRPA1, calcitonin gene-related peptide (CGRP) and substance P (SP) in peripheral and central areas relevant for migraine pain were analyzed. CGRP and SP protein immunoreactivity was also evaluated in trigeminal nucleus caudalis (TNC).ResultsIn rats bearing nitroglycerin-induced hyperalgesia, ADM_12 showed an anti-hyperalgesic effect in the second phase of the orofacial formalin test. This effect was associated to a significant inhibition of nitroglycerin-induced increase in c-Fos, TRPA1 and neuropeptides mRNA levels in medulla-pons area, in the cervical spinal cord and in the trigeminal ganglion. No differences between groups were seen as regards CGRP and SP protein expression in the TNC.ConclusionsThese findings support a critical involvement of TRPA1 channels in the pathophysiology of migraine, and show their active role in counteracting hyperalgesia at the trigeminal level.

LRRK2: An Emerging New Molecule in the Enteric Neuronal System That Quantitatively Regulates Neuronal Peptides and IgA in the Gut

Leucine-rich repeat kinase 2 (LRRK2) is a recently discovered molecule associated with familial and sporadic Parkinson's disease. It regulates many central neuronal functions such as cell proliferation, apoptosis, autophagy, and axonal extension. However, in contrast to the well-documented function of LRRK2 in central neurons, it is unclear whether LRRK2 is expressed in enteric neurons and affects the physiology of the gut. By examining LRRK2-KO mice, this study investigated whether enteric neurons express LRRK2 and whether intestinal neuronal peptides and IgA are quantitatively changed. Intestinal protein lysates and sections prepared from male C57BL/6J mice were analyzed by Western blotting and immunostaining using anti-LRRK2 antibody, respectively. Intestinal neuronal peptide-mRNAs were quantified by real-time PCR in wild-type mice and LRRK2-KO mice. Intestinal IgA was quantified by ELISA. Lamina propria mononuclear cells (LPMCs) were analyzed by flow cytometry to evaluate the ratio of B1 to B2 B cells. Western analysis and immunostaining revealed that LRRK2 is expressed in enteric neurons. The amounts of mRNA for vasoactive intestinal peptide, neuropeptide Y, and substance P were increased in LRRK2-KO mice accompanied by an increment of IgA. However, the intestinal B cell subpopulations were not altered in LRRK2-KO mice. For the first time, we have revealed that LRRK2 is expressed in enteric neurons and related to quantitative alterations of neuronal peptide and IgA. Our study highlights the importance of LRRK2 in enteric neurons as well as central neurons.

Plasticity in gastrointestinal morphology and enzyme activity in lactating striped hamsters (Cricetulus barabensis).

In small mammals, marked phenotypic plasticity of digestive physiology has been shown to make it easier for them to cope with energetically stressful periods, such as lactation. It has been proposed that the capacity of the gut to digest and absorb food is not the limiting factor to sustained energy intake (SusEI) during peak lactation. In this study, plasticity in energy intake and gastrointestinal morphology was examined in striped hamsters at different stages of reproduction and when raising litters of different sizes. Mechanisms associated with digestive enzymes and neuroendocrine hormones underpinning the plasticity were also examined. Females significantly increased energy intake, digestibility, digestive tract mass and the activity of stomach pepsin and small intestine maltase, sucrase and aminopeptidase in peak lactation compared with the non-productive and post-lactating periods. Further, females raising large litters significantly increased energy intake, digestibility, gastrointestinal mass and activity of digestive enzymes, and weaned heavier offspring compared with those nursing small and medium litters, indicating that the significant plasticity of digestive physiology increased reproductive performance. Agouti-related protein (AgRP) mRNA expression in the hypothalamus was up-regulated significantly in females raising large litters relative to those raising small litters. Serum leptin levels, and mRNA expression of hypothalamus neuropeptide Y (NPY) and the anorexigenic neuropeptides pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) did not differ among females raising small, medium and large litters. Leptin levels in lactation may only reflect a state of energy balance rather than being the prime driver of hyperphagia. Some hypothalamic neuropeptides, such as NPY, POMC and CART, may be involved in the limits to the SusEI during lactation.

Adolescent chronic stress causes hypothalamo–pituitary–adrenocortical hypo-responsiveness and depression-like behavior in adult female rats

Adolescence is a period of substantial neuroplasticity in stress regulatory neurocircuits. Chronic stress exposure during this period leads to long-lasting changes in neuroendocrine function and emotional behaviors, suggesting adolescence may be a critical period for development of stress vulnerability. This study investigated the effects of exposure to 14 days of chronic variable stress (CVS) in late-adolescent (pnd 45–58) female rats on neuroendocrine function, neuropeptide mRNA expression and depressive-like behavior in adolescence (pnd 59) and in adulthood (pnd 101). Adult females exposed to CVS in adolescence have a blunted hypothalamo-pituitary-adrenocortical (HPA) axis in response to a novel stressor and increased immobility in the forced swim test. Blunted HPA axis responses were accompanied by reduced vasopressin mRNA expression in the paraventricular nucleus of the hypothalamus (PVN), suggesting decreased central drive. Adolescent females tested immediately after CVS did not exhibit differences in stress reactivity or immobility in the forced swim test, despite evidence for enhanced central HPA axis drive (increased CRH mRNA expression in PVN). Overall, our study demonstrates that exposure to chronic stress in adolescence is sufficient to induce lasting changes in neuroendocrine drive and behavior, potentially altering the developmental trajectory of stress circuits as female rats age into adulthood.

Uninephrectomy in Rats on a Fixed Food Intake Potentiates Both Anorexia and Circulating Cytokine Subsets in Response to LPS

Recent human studies have suggested that mild reduction in kidney function can alter immune response and increase susceptibility to infection. The role of mild reduction in kidney function in altering susceptibility to bacterial lipopolysaccharide (LPS) responses was investigated in uninephrectomized rats compared to Sham-operated controls rats 4 weeks after surgery. Throughout the 4 weeks, all rats were maintained under mild food restriction at 90% of ad libitum intake to ensure the same caloric intake in both groups. In comparison to Sham, uninephrectomy (UniNX) potentiated LPS-induced anorexia by 2.1-fold. The circulating anorexigenic cytokines granulocyte-macrophage colony stimulating factor, interferon-γ, tumor necrosis factor-α, and complement-derived acylation-stimulating protein were elevated after LPS in UniNX animals compared to Sham animals. Interleukin(IL)1β and IL6 pro-inflammatory cytokines were transiently increased. Anti-inflammatory cytokines IL4 and IL10 did not differ or had a tendency to be lower in UniNX group compared to Sham animals. LPS-induced anorexia was associated with increased anorexigenic neuropeptides mRNA for pro-opiomelanocortin, corticotrophin-releasing factor, and cocaine–amphetamine-regulated transcript in the hypothalamus of both Sham and UniNX groups, but at higher levels in the UniNX group. Melanocortin-4-receptor mRNA was markedly increased in the UniNX group, which may have contributed to the enhanced anorexic response to LPS of the UniNX group. In summary, UniNX potentiates pro-inflammatory cytokine production, anorexia, and selected hypothalamic anorexigenic neuropeptides in response to LPS.

A novel role for xenopsin: Stimulation of food intake

Xenopsin (XPN), an extract from frog skin, is comprised of 80 amino acids and exerts effects on the mammalian digestive tract. The purpose of the study presented here was to determine if XPN would affect food intake using chicks as models. Chicks which had been fasted for 180min did not change food or water intake after central injection of XPN. However, ab libitum fed chicks which received 1 and 3nmol central XPN increased food intake while water intake was not affected. When the dose was increased to 9nmol chicks did not increase food intake but their water intake was reduced suggesting malaise. Chicks injected with XPN had increased c-Fos immunoreactivity in the lateral hypothalamus, but other hypothalamic appetite-associated nuclei were not affected. When XPN was directly injected into the lateral hypothalamus food intake was increased, suggesting a primary site of action. When the expression of appetite-associated neuropeptide mRNA was quantified chicks injected with XPN had increased proopiomelanocortin mRNA. Lastly, a comprehensive behavior analysis was performed and while XPN injected chicks had an increase in the number of feeding pecks, jumping, preening, deep rest and sitting were all decreased. Thus, we conclude that exogenous XPN functions as an orexigenic factor in chicks and its effects are mediated by the lateral hypothalamus.

The expression of leptin, hypothalamic neuropeptides and UCP1 before, during and after fattening in the Daurian ground squirrel (Spermophilus dauricus)

The Daurian ground squirrel (Spermophilus dauricus) accumulates large amounts of body fat during pre-hibernation fattening. Leptin, an adipose-derived hormone, plays important roles in energy balance and thermogenesis. We predicted that body fat accumulation would lead to the elevation of leptin concentration while its effect on satiety would be suppressed in hypothalamus during fattening. In addition, the uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) would increase and correlated positively with leptin concentration before hibernation. Here, we measured serum leptin concentration and leptin mRNA in white adipose tissue (WAT), hypothalamic neuropeptides involved in energy regulation and UCP1 in BAT before, during and after fattening in squirrels. The fat mass gradually increased during fattening but serum leptin increased mainly in the late phase of fattening, which was consistent with leptin mRNA expression in WAT. During fattening, the mRNA of hypothalamic leptin receptor was up-regulated and correlated positively with serum leptin. Orexigenic neuropeptide Y mRNA increased by 67%; however agouti-related peptide remained unchanged before hibernation. There was no significant change in anorexigenic neuropeptide mRNA. No change in suppressor of cytokine signaling-3 and protein tyrosine phosphatase-1B was detected. UCP1 mRNA expression and protein content in BAT increased significantly after fattening. These changes were independent of environmental conditions and serum leptin concentration. Our results suggest that the dissociation of leptin production and adiposity during fattening may facilitate fat accumulation. No evidence of suppressed leptin signal was found in fattening squirrels. The UCP1 recruitment in post-fattening squirrels could occur without winter-like acclimation and increased leptin.