Preclinical Studies on Intranasal NPY and Selective NPY Receptor Agonists in Rodent PTSD Model: Focus on Locus Coeruleus Noradrenergic System

Post-traumatic stress disorder (PTSD) is a debilitating psychological disorder that develops in a subset of individuals after exposure to a traumatic stress. Neuropeptide Y (NPY) an endogenous 36 amino acid neuropeptide expressed in many areas of the brain and periphery especially those involved in stress may confer resilience to the harmful effect of stress. We demonstrated changes in the Single Prolonged Stress (SPS) PTSD model and potential for their modulation by delivery of intranasal NPY to the brain. There was a progressive worsening of anxiety symptoms at two weeks compared to one week after SPS. Previously we showed that 150µg of NPY was effective to reverse anxiety, depressive-like and hyperarousal symptoms one week after SPS, however this dose was not sufficient to reverse anxiety symptoms two weeks after SPS when symptoms have become more severe. We determined that doubling NPY dose to 300µg was sufficient to reverse symptoms of anxiety, depression and hyperarousal two weeks after more severe symptoms have manifested. Activation of the NPY Y1R subtype was sufficient to prevent the development of SPS elicited anxiety, social impairment and depressive-like behavior. Moreover, intranasal delivery of [D-His26] NPY was superior to NPY for preventing depressive-like behavior and has promise as an early intervention therapy following traumatic stress. Significant molecular impairments in gene expression for corticotrophin releasing hormone (CRH) and neuropeptide Y (NPY) systems in the locus coeruleus (LC) and mediobasal hypothalamus were observed two weeks following SPS. There was a divergence in the expression levels of the norepinephrine transporter (NET) mRNA in the LC after SPS, some animals had significantly higher NET mRNA levels and some had levels were xii similar or lower than unstressed controls. Nevertheless, NET protein levels were reduced in the hippocampus; projection region, likely by increasing noradrenergic activation. The SPS triggered hyperarousal was associated with the changes in NET gene expression. Animals with increased startle response also had increased NET mRNA. Within the subgroup of animals that had a lower acoustic startle response, there was a significant negative correlation with NET mRNA expression. Furthermore, SPS showed a potential effect in the epigenetic regulation of the NET. Increased methylation of the NET gene promoter region was observed in the lower NET responsive group and was associated with a reduction in NET mRNA expression. Overall these results shed new insights into mechanisms for resilience or susceptibility to development of hyperarousal, a diagnostic feature of PTSD and that NPY or a specific Y1R agonist (D-His26)NPY can effectively treat core symptoms of PTSD in an animal model

Single Prolonged Stress as a Prospective Model for Posttraumatic Stress Disorder in Females

Sex plays an important role in susceptibility to stress triggered disorders. Posttraumatic Stress disorder (PTSD), a debilitating psychiatric disorder developed after exposure to a traumatic event, is two times more prevalent in women than men. However, the vast majority of animal models of PTSD, including single prolonged stress (SPS), were performed mostly with males. Here, we evaluated SPS as an appropriate PTSD model for females in terms of anxiety, depressive symptoms and changes in gene expression in the noradrenergic system in the brain. In addition, we examined intranasal neuropeptide Y (NPY) as a possible treatment in females. Female rats were subjected to SPS and given either intranasal NPY or vehicle in two separate experiments. In the first experiment, stressed females were compared to unstressed controls on forced swim test (FST) and for levels of expression of several genes in the locus coeruleus (LC) 12 days after SPS exposure. Using a separate cohort of animals, experiment two examined stressed females and unstressed controls on the elevated plus maze (EPM) and LC gene expression 7 days after SPS stressors. SPS led to increased anxiety-like behavior on EPM and depressive-like behavior on FST. Following FST, the rats displayed elevated tyrosine hydroxylase (TH), CRHR1 and Y1R mRNA levels in the LC, consistent with increased activation of the noradrenergic system. The expression level of these mRNAs was unchanged following EPM, except Y1R. Intranasal NPY at the doses shown to be effective in males, did not prevent development of depressive or anxiety-like behavior or molecular changes in the LC. The results indicate that while SPS could be an appropriate PTSD model for females, sex differences, such as response to NPY, are important to consider

Intranasal Neuropeptide Y as a Potential Therapeutic for Stress-Triggered Disorders in Females

Objective: Examine whether intranasal neuropeptide Y (NPY) is able to alter development of several stress-triggered behavioral impairments in females. Background: Sex is involved in susceptibility to many stress-elicited, neuropsychiatric disorders. However, most of the studies in animal models examining putative therapeutics excludes females. Previous studies in males demonstrated intranasal NPY provided therapeutic relief of stress-elicited behaviors, but not at the same dose in females. Interestingly, the overwhelming majority of studies found that NPY expression is lower in females than in male rodents in many brain areas. Design/Methods: Sprague Dawley female rats were exposed to the Single Prolonged Stress (SPS) animal model and then were immediately infused intranasally with one of several NPY doses starting with 600 μg/rat, which is double the dose effective in males. In a separate cohort of animals, females were infused intranasally with 600μg NPY, a dipeptidyl peptidase IV (DPP4; NPY protease) inhibitor, or both immediately after the SPS stressors. After 14 days they were tested on several behavioral tests. Results: Intranasal NPY at 600μg prevented the SPS-elicited impairment of social interaction. On the forced swim test (FST), there was a dose-response effect of intranasal NPY, with the 1200μg, but not the 600μg, effectively preventing development of the SPS-elicited increased immobility (depressive-like behavior). However, the DPP4 inhibitor and 600μg NPY combined treatment was sufficient at preventing depressive-like behavior on the FST. Conclusions: The results indicate that in females SPS elicits behavioral manifestations of stress-related disorders, such as depressive-like behavior and social impairment. This was prevented with early intervention with high doses of NPY, indicating its therapeutic potential also for females, although a higher dose will likely be required. Furthermore, NPY degradation may play a role in the higher dose requirement for females

Preclinical Findings on the Potential of Intranasal Neuropeptide Y for Treating Hyperarousal Features of PTSD

Acoustic startle response (ASR) assesses hyperarousal, a core symptom of posttraumatic stress disorder (PTSD). Intranasal neuropeptide Y (NPY) administration was shown to prevent hyperarousal in single prolonged stress (SPS) rodent PTSD model. However, it is unclear how ASR itself alters responses to stress. Rats (A-S-A) were exposed to acoustic startle (AS) 1 day before SPS (ASR1) and 2 weeks afterward (ASR2). Other groups were exposed in parallel to either AS (A-A) or SPS or neither. SPS enhanced ASR2. In relevant brain areas, mRNA levels were determined by qRT-PCR. In mediobasal hypothalamus, AS or SPS each increased CRH mRNA levels without an additive effect. Exposure to AS appeared to dampen some responses to SPS. The SPS-triggered reduction of GR and FKBP5 gene expression was not observed in A-S-A group. In locus coeruleus, SPS increased CRHR1 and reduced Y2R mRNAs, but not in A-S-A group. In both regions, AS altered NPY receptor gene expression, which may mediate dampening responses to SPS. In second experiment, intranasal NPY administered 2 weeks after SPS reversed hyperarousal symptoms for at least 7 days. This study reveals important effects of AS on the NPY system and demonstrates that intranasal NPY elicits long-lasting reversal of traumatic stress-triggered hyperarousal

Activation of NPY Receptor Subtype 1 by [D-His 26]NPY is Sufficient to Prevent Development of Anxiety and Depressive like Effects in the Single Prolonged Stress Rodent Model of PTSD

The neuropeptide Y (NPY) system plays an important role in mediating resilience to the harmful effect of stress in post-traumatic stress disorder (PTSD). It can mediate its effects via several G-protein coupled receptors: Y1R, Y2R, Y4R and Y5R. To investigate the role of individual NPY receptors in the resilience effects of NPY to traumatic stress, intranasal infusion of either Y1R agonists [D-His26]NPY, [Leu31Pro34]NPY, Y2R agonist NPY (3-36) or NPY were administered to male Sprague-Dawley rats immediately following the last stressor of the single prolonged stress (SPS) protocol, a widely used PTSD animal model. After 7 or 14 days, effects of the treatments were measured on the elevated plus maze (EPM) for anxiety, in forced swim test (FST) for development of depressive-like or re-experiencing behavior, in social interaction (SI) test for impaired social behavior, and acoustic startle response (ASR) for hyperarousal. [D-His26]NPY, but not [Leu31Pro34]NPY nor NPY (3-36) Y2R, was effective in preventing the SPS-elicited development of anxiety. Y1R, but not Y2R agonists prevented development of depressive- feature on FST, with [D-His26]NPY superior to NPY. The results demonstrate that [D-His26]NPY was sufficient to prevent development of anxiety, social impairment and depressive symptoms, and has promise as an early intervention therapy following traumatic stress

Variable Response of Norepinephrine Transporter to Traumatic Stress and Relationship to Hyperarousal

The noradrenergic systems play a key role in stress triggered disorders such as post-traumatic stress disorder (PTSD). We hypothesized that traumatic stress will alter expression of norepinephrine transporter (NET) in locus coeruleus (LC) and its target brain regions which could be related to hyperarousal. Male Sprague-Dawley rats were subjected to single prolonged stress (SPS) and several weeks later the LC was isolated. NET mRNA levels in LC, determined by RT-PCR, displayed variable response with high and low responsive subgroups. In different cohort, acoustic startle response (ASR) was measured 2 weeks after SPS and levels of NET mRNA and protein in LC determined. The high NET responsive subgroup had greater hyperarousal. Nevertheless, NET protein levels, as determined by western blots, were lower than unstressed controls in LC, ventral hippocampus and medial prefrontal cortex and displayed considerable variability. Hypermethylation of specific CpG region in promoter of SLC6A2 gene, encoding NET, was present in the low, but not high, NET mRNA responsive subgroup. Taken together, the results demonstrate variability in stress elicited changes in NET gene expression and involvement of epigenetic changes. This may underlie mechanisms of susceptibility and resilience to traumatic stress triggered neuropsychiatric symptoms, especially hyperarousal

Intranasal Neuropeptide Y as a Potential Therapeutic for Depressive Behavior in the Rodent Single Prolonged Stress Model in Females

The susceptibility to stress-elicited disorders is markedly influenced by sex. Women are twice as likely as men to develop posttraumatic stress disorder (PTSD), depression, anxiety disorders, and social impairments following exposure to traumatic stress. However, most of the studies in animal models examining putative therapeutics for stress-triggered impairments, including single prolonged stress (SPS), were performed predominantly with males. Previous studies in males demonstrated that intranasal neuropeptide Y (NPY) can provide therapeutic relief of many SPS-triggered behaviors, but is ineffective in females at the same dose. Thus, females may need a higher dose of exogenous NPY to attain a therapeutically significant concentration since the overwhelming majority of studies found that NPY levels in females in many brain regions are lower than in male rodents. Here, we examined SPS as an appropriate model to elicit many PTSD-associated symptoms in females and whether intranasal NPY at higher doses than with males is able to alter the development of SPS-triggered behavioral impairments. Sprague-Dawley female rats were exposed to SPS only, or in a separate cohort after SPS stressors were immediately infused intranasally with one of several doses of NPY, starting with 600 μg/rat-four times the dose effective in males. In the third cohort of animals, females were infused intranasally with either 600 μg NPY, omarigliptin [a dipeptidyl peptidase IV (DPP4) inhibitor], or both right after the SPS stressors. After 19 days they were tested on several behavioral tests. SPS elicited significant depressive/despair like behavior on the forced swim test (FST), anxiety behavior on the elevated plus maze (EPM), as well as impaired social interaction. On the FST, there was a dose-response effect of intranasal NPY, with 1,200 μg, but not 600 μg, preventing the development of the SPS-elicited depressive-like behavior. The omarigliptin and 600 μg NPY combined treatment, but neither alone, was also sufficient at preventing depressive-like behavior on the FST. The results demonstrate that: (1) SPS elicits several behavioral manifestations of PTSD in females; (2) early intervention with a high dose of intranasal NPY has therapeutic potential also for females; and (3) NPY cleavage by DPP4 may play a role in the higher dose requirement for females

Single Prolonged Stress PTSD Model Triggers Progressive Severity of Anxiety, Altered Gene Expression in Locus Coeruleus and Hypothalamus and Effected Sensitivity to NPY

PTSD is heterogeneous disorder that can be long lasting and often has delayed onset following exposure to a traumatic event. Therefore, it is important to take a staging approach to evaluate progression of biological mechanisms of the disease. Here, we begin to evaluate the temporal trajectory of changes following exposure to traumatic stressors in the SPS rat PTSD model. The percent of animals displaying severe anxiety on EPM increased from 17.5% at one week to 57.1% two weeks after SPS stressors, indicating delayed onset or progressive worsening of the symptoms. The LC displayed prolonged activation, and dysbalance of the CRH/NPY systems, with enhanced CRHR1 gene expression, coupled with reduced mRNAs for NPY and Y2R. In the mediobasal hypothalamus, increased CRH mRNA levels were sustained, but there was a flip in alterations of HPA regulatory molecules, GR and FKBP5 and Y5 receptor at two weeks compared to one week. Two weeks after SPS, intranasal NPY at 300 microg/rat, but not 150 microg which was effective after one week, reversed SPS triggered elevated anxiety. It also reversed SPS elicited depressive/despair symptoms and hyperarousal. Overall, the results reveal time-dependent progression in development of anxiety symptoms and molecular impairments in gene expression for CRH and NPY systems in LC and mediobasal hypothalamus by SPS. With longer time afterwards only a higher dose of NPY was effective in reversing behavioral impairments triggered by SPS, indicating that therapeutic approaches should be adjusted according to the degree of biological progression of the disorder

Transgenic expression of ZBP1 in neurons suppresses cocaine-associated conditioning

To directly address whether regulating mRNA localization can influence animal behavior, we created transgenic mice that conditionally express Zipcode Binding Protein 1 (ZBP1) in a subset of neurons in the brain. ZBP1 is an RNA-binding protein that regulates the localization, as well as translation and stability of target mRNAs in the cytoplasm. We took advantage of the absence of ZBP1 expression in the mature brain to examine the effect of expressing ZBP1 on animal behavior. We constructed a transgene conditionally expressing a GFP-ZBP1 fusion protein in a subset of forebrain neurons and compared cocaine-cued place conditioning in these mice versus noninduced littermates. Transgenic ZBP1 expression resulted in impaired place conditioning relative to nonexpressing littermates, and acutely repressing expression of the transgene restored normal cocaine conditioning. To gain insight into the molecular changes that accounted for this change in behavior, we identified mRNAs that specifically immunoprecipitated with transgenic ZBP1 protein from the brains of these mice. These data suggest that RNA-binding proteins can be used as a tool to identify the post-transcriptional regulation of gene expression in the establishment and function of neural circuits involved in addiction behaviors