Lack of neuropeptide FF signalling in mice leads to reduced repetitive behavior, altered drinking behavior, and fuel type selection.

Although best known for their involvement in modulating nociception, Neuropeptide FF (NPFF) group peptides have been suggested to fulfil a variety of biological functions such as feeding, anxiety behaviors and thermogenesis. However, evidence supporting these functions of NPFF is mostly pharmacological, leaving the physiological relevance unaddressed. Here we examined the physiological impact of lack of NPFF signalling in both genders using a Npff-/- mouse model. NPFF expression in the mouse is restricted to the spinal cord and brainstem while its cognate receptor NPFFR2 has wider distribution throughout the brain. Both male and female Npff-/- mice showed reduced repetitive behaviors evidenced in the marble burying test and self-grooming test. A decrease in anxiety-related behaviors in the Npff-/- mice was also observe in the open field test and to a lesser degree in an elevated plus maze test. Moreover, both male and female Npff-/- mice exhibited increased water intake resulting from increases in drinking size, rather than number of drinking events. During a fasting-refeeding challenge, Npff-/- mice of both genders displayed alterations in reparatory exchange ratio that reflect a greater fuel type flexibility. Npff-/- mice were otherwise wild-type-like regarding body weight, body composition, feeding behaviors, locomotion or energy expenditure. Together, these findings reveal the important physiological roles of NPFF signalling in the regulation of anxiety-related and repetitive behaviors, fluid homeostasis and oxidative fuel selection, highlighting the therapeutical potential of the NPFF system in a number of behavioral and metabolic disorders.

Comparison of ion channel inhibitor combinations for limiting secondary degeneration following partial optic nerve transection.

Following neurotrauma, secondary degeneration of neurons and glia adjacent to the injury leads to further functional loss. A combination of ion channel inhibitors (lomerizine + oxATP + YM872) has been shown to be effective at limiting structural and functional loss due to secondary degeneration. Here we assess efficacy of the combination where oxATP is replaced with Brilliant Blue G (BBG), a more clinically applicable P2X7 receptor inhibitor. Partial optic nerve transection was used to model secondary degeneration in adult female rats. Animals were treated with combinations of lomerizine + YM872 + oxATP or lomerizine + YM872 + BBG, delivered via osmotic mini-pump directly to the injury site. Outcomes assessed were Iba1 + and ED1 + microglia and macrophages, oligodendroglial cell numbers, node/paranode structure and visual function using the optokinetic nystagmus test. The lomerizine + BBG + YM872 combination was at least as effective at the tested concentrations as the lomerizine + oxATP + YM872 combination at preserving node/paranode structure and visual function when delivered locally. However, neither ion channel inhibitor combination significantly improved microglial/macrophage nor oligodendroglial numbers compared to vehicle-treated controls. In conclusion, a locally delivered combination of ion channel inhibitors incorporating lomerizine + BBG + YM872 is at least as effective at limiting secondary degeneration following partial injury to the optic nerve as the combination incorporating oxATP.

NPFF signalling is critical for thermosensory and dietary regulation of thermogenesis.

Thermogenesis is a centrally regulated physiological process integral for thermoregulation and energy homeostasis. However, the mechanisms and pathways involved remain poorly understood. Importantly, in this study we uncovered that in an environment of 28 °C that is within the mouse thermoneutral zone, lack of NPFF signalling leads to significant increases in energy expenditure, resting metabolic rate and brown adipose tissue (BAT) thermogenesis, which is associated with decreased body weight gain and lean tissue mass. Interestingly, when exposed to a high-fat diet (HFD) at 28 °C, Npff-/- mice lost the high energy expenditure phenotype observed under chow condition and exhibited an impaired diet-induced thermogenesis. On the other hand, under conditions of increasing levels of thermal demands, Npff-/- mice exhibited an elevated BAT thermogenesis at mild cold condition (22 °C), but initiated comparable BAT thermogenic responses as WT mice when thermal demand increased, such as an exposure to 4 °C. Together, these results reveal NPFF signalling as a novel and critical player in the control of thermogenesis, where it regulates thermosensory thermogenesis at warm condition and adjusts thermoregulation under positive energy balance to regulate diet-induced thermogenesis.

Central NPFF signalling is critical in the regulation of glucose homeostasis.

OBJECTIVE: Neuropeptide FF (NPFF) group peptides belong to the evolutionary conserved RF-amide peptide family. While they have been assigned a role as pain modulators, their roles in other aspects of physiology have received much less attention. NPFF peptides and their receptor NPFFR2 have strong and localized expression within the dorsal vagal complex that has emerged as the key centre for regulating glucose homeostasis. Therefore, we investigated the role of the NPFF system in the control of glucose metabolism and the histochemical and molecular identities of NPFF and NPFFR2 neurons. METHODS: We examined glucose metabolism in Npff-/- and wild type (WT) mice using intraperitoneal (i.p.) glucose tolerance and insulin tolerance tests. Body composition and glucose tolerance was further examined in mice after 1-week and 3-week of high-fat diet (HFD). Using RNAScope double ISH, we investigated the neurochemical identity of NPFF and NPFFR2 neurons in the caudal brainstem, and the expression of receptors for peripheral factors in NPFF neurons. RESULTS: Lack of NPFF signalling in mice leads to improved glucose tolerance without significant impact on insulin excursion after the i.p. glucose challenge. In response to an i.p. bolus of insulin, Npff-/- mice have lower glucose excursions than WT mice, indicating an enhanced insulin action. Moreover, while HFD has rapid and potent detrimental effects on glucose tolerance, this diet-induced glucose intolerance is ameliorated in mice lacking NPFF signalling. This occurs in the absence of any significant impact of NPFF deletion on lean or fat masses, suggesting a direct effect of NPFF signalling on glucose metabolism. We further reveal that NPFF neurons in the subpostrema area (SubP) co-express receptors for peripheral factors involved in glucose homeostasis regulation such as insulin and GLP1. Furthermore, Npffr2 is expressed in the glutamatergic NPFF neurons in the SubP, and in cholinergic neurons of the dorsal motor nucleus of the vagus (DMV), indicating that central NPFF signalling is likely modulating vagal output to innervated peripheral tissues including those important for glucose metabolic control. CONCLUSIONS: NPFF signalling plays an important role in the regulation of glucose metabolism. NPFF neurons in the SubP are likely to receive peripheral signals and mediate the control of whole-body glucose homeostasis via centrally vagal pathways. Targeting NPFF and NPFFR2 signalling may provide a new avenue for treating type 2 diabetes and obesity.

Ninjin'yoeito, a herbal medicine, enhances glucose tolerance in mice.

The prevalence of Type 2 diabetes increases under conditions of obesity but also due to aging. While a variety of treatment options are being explored there are still many unanswered questions about the underlying mechanisms for the aetiology and progression of this illness. Here we show that pre-treatment with Ninjin'yoeito (NYT), a herbal medicine composed of 12 different ingrediencies, before a glucose challenge results in significantly improved glucose tolerance. This occurs in the absence of significant alterations in insulin excursion compared to vehicle treatment, indicating NYT improves insulin responsiveness and/or insulin-independent glucose disposal. Furthermore, we identify Ginseng - one of the 12 ingredients of NYT - as one key component contributing to NYT's effect on glucose clearance. Importantly, lack of Y4 receptor signalling abolishes the positive effects of NYT on glucose tolerance suggesting Y4 receptor-controlled pathways are crucial in mediating this action of NYT. Using c-fos as neuronal activation marker, we show NYT activates the area postrema - a circumventricular organ in the brainstem that expresses high level of Y4 receptors, supporting an involvement of brainstem Y4 signalling in NYT-activated central networks. Together, these data suggest that NYT is a positive influencer of glucose metabolism in insulin-sensitive tissues and the mechanistic actions of NYT include brainstem Y4 circuitries.

Ninjin'yoeito modulates feeding and activity under negative energy balance conditions via the NPY system.

The central and peripheral neuropeptide Y (NPY) system is critically involved in feeding and energy homeostasis control. Disease conditions as well as aging can lead to reduced functionality of the NPY system and boosting it represents a promising option to improve health outcomes in these situations. Here we show that Ninjin-yoeito (NYT), a Japanese kampo medicine comprising twelve herbs, and known to be effective to treat anorexia and frailty, mediates part of its action via NPY/peptide YY (PYY) related pathways. Especially under negative energy homeostasis conditions NYT is able to promote feeding and reduces activity to conserve energy. These effects are in part mediated via signalling through the NPY system since lack of Y4 receptors or PYY leading to modification in these responses highlighting the possibility for combination treatment to improve aging related conditions on energy homeostasis control.

Dynamic regional alterations in mouse brain neuronal activity following short-term changes in energy balance.

OBJECTIVE: Knowledge of the functional contribution to energy homeostatic control by different brain areas is limited. This study employed a systematic approach to identify brain regions specifically influenced by a positive energy balance. METHODS: The c-fos expression was mapped throughout the mouse brain after varying durations (24 hours to up to 14 days) of high-fat diet (HFD) exposure or after reversal from a 7-day HFD to a chow diet. In parallel, the metabolic and behavioral impacts of these treatments were examined. RESULTS: A HFD elicited rapid and pronounced compensatory responses which were, however, insufficient to overcome the impact of the positive energy balance. Rapid and dynamic responses of c-fos expression throughout the brain were seen over the course of HFD exposure, with some regions showing linear-like responses and some regions exhibiting biphasic responses. The switch from HFD to chow resulted in metabolic compensations mitigating the effects of the negative energy balance and a heightened preference for sweet taste. Interestingly, this diet switch led to a significant c-fos activation in the lateral hypothalamus, an area unresponsive to HFD intervention. CONCLUSIONS: Plasticity exists in the extended brain networks facilitating rapid adaptations dependent on energy availability. Knowledge of these critical control points may provide novel antiobesity treatment targets.

The effects of a combination of ion channel inhibitors on pathology in a model of demyelinating disease.

BACKGROUND: Multiple sclerosis (MS) has been shown to feature oxidative damage, which can be modelled using the cuprizone model of demyelinating disease. Oxidative damage can occur as a result of excessive influx of calcium ions (Ca2+) and oligodendroglia are particularly vulnerable. However, the effects of limiting excess Ca2+ influx on oxidative damage, oligodendroglia and myelin structure are unknown. OBJECTIVE: This study investigated the effects of limiting excess Ca2+ flux on oxidative damage and associated changes in oligodendroglial densities and Node of Ranvier structure in the cuprizone model. METHODS: The effects of three weeks of cuprizone administration and of treatment with a combination of three ion channel inhibitors (Lomerizine, Brilliant Blue G (BBG) and YM872), were semi-quantified immunohistochemically. Outcomes assessed were protein nitration (3-nitrotyrosine (3NT)) oxidative damage to DNA (8-hydroxy deoxyguanosine (8OHDG)), advanced glycation end-products (carboxymethyl lysine (CML)), immunoreactivity of microglia (Iba1) and astrocytes (glial acidic fibrillary protein (GFAP)), densities of oligodendrocyte precursor cells (OPCs) (platelet derived growth factor alpha receptor (PDGFαR) with olig2) and oligodendrocytes (olig2 and CC1), and structural elements of the Node of Ranvier (contactin associated protein (Caspr)). RESULTS: The administration of cuprizone resulted in increased protein nitration, DNA damage, and astrocyte and microglial immunoreactivity, a decrease in the density of oligodendrocytes and OPCs, together with altered structure of the Node of Ranvier and reduced myelin basic protein immunoreactivity. Treatment with the ion channel inhibitor combination significantly lowered protein nitration, increased the density of OPCs and reduced the number of atypical Node of Ranvier complexes; other outcomes were unaffected. CONCLUSION: Our findings suggest that excess Ca2+ influx contributes to protein nitration, and associated changes to OPC densities and Node of Ranvier structure in demyelinating disease.

Comparing modes of delivery of a combination of ion channel inhibitors for limiting secondary degeneration following partial optic nerve transection.

Injury to the central nervous system is exacerbated by secondary degeneration. Previous research has shown that a combination of orally and locally administered ion channel inhibitors following partial optic nerve injury protects the myelin sheath and preserves function in the ventral optic nerve, vulnerable to secondary degeneration. However, local administration is often not clinically appropriate. This study aimed to compare the efficacy of systemic and local delivery of the ion channel inhibitor combination of lomerizine, brilliant blue G (BBG) and YM872, which inhibits voltage-gated calcium channels, P2X7 receptors and Ca2+ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors respectively. Following a partial optic nerve transection, adult female PVG rats were treated with BBG and YM872 delivered via osmotic mini pump directly to the injury site, or via intraperitoneal injection, both alongside oral administration of lomerizine. Myelin structure was preserved with both delivery modes of the ion channel inhibitor combination. However, there was no effect of treatment on inflammation, either peripherally or at the injury site, or on the density of oligodendroglial cells. Taken together, the data indicate that even at lower concentrations, the combinatorial treatment may be preserving myelin structure, and that systemic and local delivery are comparable at improving outcomes following neurotrauma.

Amygdala NPY Circuits Promote the Development of Accelerated Obesity under Chronic Stress Conditions.

Neuropeptide Y (NPY) exerts a powerful orexigenic effect in the hypothalamus. However, extra-hypothalamic nuclei also produce NPY, but its influence on energy homeostasis is unclear. Here we uncover a previously unknown feeding stimulatory pathway that is activated under conditions of stress in combination with calorie-dense food; NPY neurons in the central amygdala are responsible for an exacerbated response to a combined stress and high-fat-diet intervention. Central amygdala NPY neuron-specific Npy overexpression mimics the obese phenotype seen in a combined stress and high-fat-diet model, which is prevented by the selective ablation of Npy. Using food intake and energy expenditure as readouts, we demonstrate that selective activation of central amygdala NPY neurons results in increased food intake and decreased energy expenditure. Mechanistically, it is the diminished insulin signaling capacity on central amygdala NPY neurons under combined stress and high-fat-diet conditions that leads to the exaggerated development of obesity.

Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-ß.

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease, where chronic inflammation plays an essential role in its pathology. A feature of MS is the production of autoantibodies stimulated by an altered-peptide-ligand response and epitope spreading, resulting in loss of tolerance for self-proteins. The involvement of autoantibodies in MS pathogenesis has been suggested to initiate and drive progression of inflammation; however, the etiology of MS remains unknown. The effect of etomoxir and interferon-ß (IFN-ß) was examined in an experimental-autoimmune-encephalomyelitis (EAE) model of MS. Moreover, the impact of etomoxir and IFN-ß on recognition of brain proteins in serum from EAE rats was examined with the purpose of identifying the autoantibody reactivities involved in MS. Animals treated with etomoxir on day 1 exhibited a statistically significantly lower disease score than animals treated with IFN-ß (on day 1 or 5) or placebo. Etomoxir treatment on day 5 resulted in a significantly lower disease score than IFN-ß treatment on day 1. After disease induction antibodies was induced to a broad pallet of antigens in the brain. Surprisingly, by blocking CPT1 and therewith lipid metabolism several alterations in the antibody response was observed suggesting that autoantibodies play a role in the EAE animal model.