Lishay G. Alaluf

Single intranasal neuropeptide Y infusion attenuates development of PTSD-like symptoms to traumatic stress in rats

Exposure to severe stress leads to development of neuropsychiatric disorders, including depression and Post-Traumatic Stress Disorder (PTSD) in at-risk individuals. Neuropeptide Y (NPY) is associated with resilience or improved recovery. Therefore exogenous administration to the brain has therapeutic potential although peripheral administration can trigger undesirable side effects. Here, we established conditions with intranasal (IN) NPY infusion to rats to obtain CSF concentrations in the proposed anxiolytic range without significant change in plasma NPY. Rats were pretreated with IN NPY or vehicle before exposure to single prolonged stress (SPS) animal model of PTSD and compared to untreated controls. The IN NPY appeared to lessen the perceived severity of stress, as these animals displayed less time immobile in forced swim part of the SPS. Thirty minutes after SPS the elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone was not as pronounced in NPY-infused rats and the induction of tyrosine hydroxylase (TH) in locus coeruleus (LC) was attenuated. Seven days after SPS, they displayed lower depressive-like behavior on Forced Swim Test and reduced anxiety-like behavior on Elevated Plus Maze. The prolonged effect of SPS on Acoustic Startle Response was also lower in NPY-infused rats. Plasma ACTH, corticosterone, and hippocampal glucocorticoid receptor levels were significantly above controls only in the vehicle - but not IN NPY-treated group 1week after SPS. Baseline TH mRNA levels in LC did not differ among groups, but increased with forced swim in the vehicle - but not NPY-pretreated animals. Administration of IN NPY after exposure to SPS led to similar, but not identical, reduction in development of anxiety, depressive-like behavior and hyperarousal. The results show that single IN NPY can alter stress-triggered dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and activation of central noradrenergic activity. These findings provide proof of concept for potential of IN NPY for non-invasive prophylactic treatment or early intervention in response to traumatic stress.

Intranasal neuropeptide Y reverses anxiety and depressive-like behavior impaired by single prolonged stress PTSD model

PTSD is a debilitating neuropsychiatric disorder and many patients do not respond sufficiently to current treatments. Neuropeptide Y (NPY) is suggested to provide resilience to the development of PTSD and co-morbid depression. Injections of NPY to the rodent brain are anxiolytic. Recently we showed that intranasal delivery of NPY to rats before or immediately after exposure to single prolonged stress (SPS) animal model of PTSD prevented development of many biochemical and behavioral symptoms of PTSD, indicating its prophylactic potential. Here, we investigated whether intranasal NPY might provide benefits once symptoms have already developed. One week after exposure to SPS stressors, animals were given intranasal NPY or vehicle and tested on elevated plus maze 2h or 2 days later. The NPY treated rats had lower anxiety-like behavior than vehicle treated rats as indicated by more entries into open arms and fewer into closed arms, lower anxiety index, higher risk assessment and unprotected head dips and reduced grooming time. Their anxiety index was similar to that of unstressed controls. On most of these variables there was no effect of time interval and rats displayed similar overall changes 2h or 2 days after the infusion. Moreover, intranasal NPY led to reduced depressive-like behavior, assessed by forced swim test. Thus, intranasal NPY reversed several behavioral impairments triggered by the traumatic stress of SPS and has potential for non-invasive PTSD therapeutic intervention.

Intranasal infusion of melanocortin receptor four (MC4R) antagonist to rats ameliorates development of depression and anxiety related symptoms induced by single prolonged stress

Brain melanocortinergic systems and specifically melanocortin receptor four (MC4R) are implicated in modulation of anxiety- and depressive-like behavior induced by mild or moderate stress. Here we examine whether blockage of central MC4Rs with HS014 before severe traumatic stress may protect against development of anxiety and depression co-morbid with post-traumatic stress disorder (PTSD). Male rats were treated intranasally (IN) with vehicle or varied doses of HS014, 30min prior to single prolonged stress (SPS) animal model of PTSD. IN administration of 100μg HS014 pre-SPS improved despair behavior in forced swim (FS) immediately after immobilization stress part of SPS protocol. During all 4 intervals of 20min FS these rats spent less time immobile than rats given vehicle or 3.5ng HS014. This dose of HS014 also had a long-term beneficial effect manifested as reduction of immobility time in forced swim test performed after SPS. However, both HS014 doses were effective in ameliorating development of anxiety-like behavior after traumatic stress. Thus, rats given IN HS014 prior to SPS exhibited less open arms (OA) visits in elevated plus maze (EPM), spent longer time in OA and less in closed arms, had lower anxiety index, higher risk assessment and more head dips over borders in OA. They also spent longer time in the center of the open field and defecated less. Reduced grooming behavior in EPM was observed with 100μg HS014. This is the first study revealing pronounced resilience effects of HS014 on development of behavioral symptoms co-morbid with PTSD.

Early Intervention With Intranasal NPY Prevents Single Prolonged Stress-Triggered Impairments in Hypothalamus and Ventral Hippocampus in Male Rats

Intranasal administration of neuropeptide Y (NPY) is a promising treatment strategy to reduce traumatic stress-induced neuropsychiatric symptoms of posttraumatic stress disorder (PTSD). We evaluated the potential of intranasal NPY to prevent dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis, a core neuroendocrine feature of PTSD. Rats were exposed to single prolonged stress (SPS), a PTSD animal model, and infused intranasally with vehicle or NPY immediately after SPS stressors. After 7 days undisturbed, hypothalamus and hippocampus, 2 structures regulating the HPA axis activity, were examined for changes in glucocorticoid receptor (GR) and CRH expression. Plasma ACTH and corticosterone, and hypothalamic CRH mRNA, were significantly higher in the vehicle but not NPY-treated group, compared with unstressed controls. Although total GR levels were not altered in hypothalamus, a significant decrease of GR phosphorylated on Ser232 and increased FK506-binding protein 5 mRNA were observed with the vehicle but not in animals infused with intranasal NPY. In contrast, in the ventral hippocampus, only vehicle-treated animals demonstrated elevated GR protein expression and increased GR phosphorylation on Ser232, specifically in the nuclear fraction. Additionally, SPS-induced increase of CRH mRNA in the ventral hippocampus was accompanied by apparent decrease of CRH peptide particularly in the CA3 subfield, both prevented by NPY. The results show that early intervention with intranasal NPY can prevent traumatic stress-triggered dysregulation of the HPA axis likely by restoring HPA axis proper negative feedback inhibition via GR. Thus, intranasal NPY has a potential as a noninvasive therapy to prevent negative effects of traumatic stress.

Potential of neuropeptide Y for preventing or treating post-traumatic stress disorder

There is extensive evidence that NPY in the brain can modulate the responses to stress and play a critical role in resistance to, or recovery from, harmful effects of stress. Development of PTSD and comorbid depression following exposure to traumatic stress are associated with low NPY. This review discusses putative mechanisms for NPYs anti-stress actions. Recent preclinical data indicating potential for intranasal delivery of NPY to brain as a promising non-invasive strategy to prevent a variety of neuroendocrine, molecular and behavioral impairments in PTSD model are summarized.

Locus coeruleus response to single-prolonged stress and early intervention with intranasal neuropeptide Y.

Dysregulation of the central noradrenergic system is a core feature of post‐traumatic stress disorder (PTSD). Here, we examined molecular changes in locus coeruleus (LC) triggered by single‐prolonged stress (SPS) PTSD model at a time when behavioral symptoms are manifested, and the effect of early intervention with intranasal neuropeptide Y (NPY). Immediately following SPS stressors, male SD rats were administered intranasal NPY (SPS/NPY) or vehicle (SPS/V). Seven days later, TH protein, but not mRNA, was elevated in LC only of the SPS/V group. Although 90% of TH positive cells expressed GR, its levels were unaltered. Compared to unstressed controls, LC of SPS/V, but not SPS/NPY, expressed less Y2 receptor mRNA with more CRHR1 mRNA in subset of animals, and elevated corticotropin‐releasing hormone (CRH) in central nucleus of amygdala. Following testing for anxiety on elevated plus maze (EPM), there were significantly increased TH, DBH and NPY mRNAs in LC of SPS‐treated, but not previously unstressed animals. Their levels highly correlated with each other but not with behavioral features on EPM. Thus, SPS triggers long‐term noradrenergic activation and higher sensitivity to mild stressors, perhaps mediated by the up‐regulation influence of amygdalar CRH input and down‐regulation of Y2R presynaptic inhibition in LC. Results also demonstrate the therapeutic potential of early intervention with intranasal NPY for traumatic stress‐elicited noradrenergic impairments.

Comparative effects of intranasal neuropeptide Y and HS014 in preventing anxiety and depressive-like behavior elicited by single prolonged stress.

Stress triggered neuropsychiatric disorders are a serious societal problem. Prophylactic treatment or early intervention has great potential in increasing resilience to traumatic stress and reducing its harmful impact. Previously, we demonstrated proof of concept that intranasal administration of neuropeptide Y (NPY) or the melanocortin receptor four (MC4R) antagonist, HS014, prior to single prolonged stress (SPS) rodent post-traumatic stress disorder (PTSD) model, can prevent or attenuate many PTSD associated impairments. Here, we compare effects of NPY or HS014 given 30 min before or immediately after SPS stressors on development of anxiety, depressive-like behavior and associated biochemical abnormalities. SPS triggered anxiety on elevated plus maze (EPM) was reduced by intranasal administration of 100 μg NPY and to even greater extent HS014 (3.5 ng or 100 μg). The SPS-elicited depressive-like behavior on forced swim test was prevented with 100 μg NPY or the high dose HS014. Combined administration of low HS014 and NPY, ineffective by themselves, prevented development of depressive-like behavior. Reductions in stress triggered activation of locus coeruleus/noradrenergic system and HPA axis were observed with both HS014 and NPY. In contrast to NPY which has been showed earlier, infusion of HS014 immediately after SPS did not prevent the development of anxiogenic behavior on EPM. However, HS014 given after SPS stressors effectively even at very low dose, prevented development of depressive-like behavior. Thus, both MC4R antagonist and NPY, alone or combined, have potential for prophylactic treatment against traumatic stress triggered anxiety or depressive-like behaviors, while NPY has more widespread potential for early intervention.

Blockage of melanocortin-4 receptors by intranasal HS014 attenuates single prolonged stress-triggered changes in several brain regions.

Melanocortin receptor four (MC4R) is implicated in regulation of stress‐related functions. We previously demonstrated that intranasal infusion of MC4R antagonist HS014, shortly before single prolonged stress (SPS) animal model of post‐traumatic stress disorder, lessened the development of anxiety‐ and depression‐like behavior depending on the dose. Here, we evaluated effects of HS014 on SPS‐elicited changes in hypothalamic‐pituitary‐adrenal axis and expression of several genes of interest in mediobasal hypothalamus, hippocampus, and locus coeruleus. Rats were given intranasal infusion of HS014 (3.5 ng or 100 μg) and 30 min later subjected to SPS stressors. Short‐term responses of HS014 rats in comparison with vehicle‐treated, evident 30 min following SPS stressors, included smaller rise in plasma corticosterone (100 μg HS014), absence of induction of corticotrophin‐releasing hormone mRNA in mediobasal hypothalamus and of mRNA for tyrosine hydroxylase and dopamine‐β hydroxylase in locus coeruleus. Long‐term responses found 7 days after SPS stressors, included lower induction corticotrophin‐releasing hormone mRNA levels in the mediobasal hypothalamus without effect on mRNAs for the glucocorticoid receptor (GR) and FK506‐binding protein 51 (FKBP5), a component of GR co‐chaperone complex; and no induction of GR protein in ventral hippocampus. Thus, antagonism of MC4R prior to SPS attenuates development of several abnormalities in gene expression in regions implicated in post‐traumatic stress disorder.