Contribution of Hippocampal 5-HT<Subscript>3</Subscript> Receptors in Hippocampal Autophagy and Extinction of Conditioned Fear Responses after a Single Prolonged Stress Exposure in Rats

One of the hypotheses about the pathogenesis of posttraumatic stress disorder (PTSD) is the dysfunction of serotonin (5-HT) neurotransmission. While certain 5-HT receptor subtypes are likely critical for the symptoms of PTSD, few studies have examined the role of 5-HT₃ receptor in the development of PTSD, even though 5-HT₃ receptor is critical for contextual fear extinction and anxiety-like behavior. Therefore, we hypothesized that stimulation of 5-HT₃ receptor in the dorsal hippocampus (DH) could prevent hippocampal autophagy and the development of PTSD-like behavior in animals. To this end, we infused SR57227, selective 5-HT₃ agonist, into the DH after a single prolonged stress (SPS) treatment in rats. Three weeks later, we evaluated the effects of this pharmacological treatment on anxiety-related behaviors and extinction of contextual fear memory. We also accessed hippocampal autophagy and the expression of 5-HT_3A subunit, Beclin-1, LC3-I, and LC3-II in the DH. We found that SPS treatment did not alter anxiety-related behaviors but prolonged the extinction of contextual fear memory, and such a behavioral phenomenon was correlated with increased hippocampal autophagy, decreased 5-HT_3A expression, and increased expression of Beclin-1 and LC3-II/LC3-I ratio in the DH. Furthermore, intraDH infusions of SR57227 dose-dependently promoted the extinction of contextual fear memory, prevented hippocampal autophagy, and decreased expression of Beclin-1 and LC3-II/LC3-I ratio in the DH. These results indicated that 5-HT₃ receptor in the hippocampus may play a critical role in the pathogenesis of hippocampal autophagy, and is likely involved in the pathophysiology of PTSD.     

NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.     

Differential regulation of CaMKII inhibitor β protein expression after exposure to a novel context and during contextual fear memory formation

Understanding of the molecular basis of long‐term fear memory (fear LTM) formation provides targets in the treatment of emotional disorders. Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is one of the key synaptic molecules involved in fear LTM formation. There are two endogenous inhibitor proteins of CaMKII, CaMKII Nα and Nβ, which can regulate CaMKII activity in vitro. However, the physiological role of these endogenous inhibitors is not known. Here, we have investigated whether CaMKII Nβ protein expression is regulated after contextual fear conditioning or exposure to a novel context. Using a novel CaMKII Nβ‐specific antibody, CaMKII Nβ expression was analysed in the naïve mouse brain as well as in the amygdala and hippocampus after conditioning and context exposure. We show that in naïve mouse forebrain CaMKII Nβ protein is expressed at its highest levels in olfactory bulb, prefrontal and piriform cortices, amygdala and thalamus. The protein is expressed both in dendrites and cell bodies. CaMKII Nβ expression is rapidly and transiently up‐regulated in the hippocampus after context exposure. In the amygdala, its expression is regulated only by contextual fear conditioning and not by exposure to a novel context. In conclusion, we show that CaMKII Nβ expression is differentially regulated by novelty and contextual fear conditioning, providing further insight into molecular basis of fear LTM.     

Standardised Extract of Bacopa monniera (CDRI-08) Improves Contextual Fear Memory by Differentially Regulating the Activity of Histone Acetylation and Protein Phosphatases (PP1α, PP2A) in Hippocampus

Contextual fear conditioning is a paradigm for investigating cellular mechanisms involved in hippocampus-dependent memory. Earlier, we showed that standardised extract of Bacopa monniera (CDRI-08) improves hippocampus-dependent learning in postnatal rats by elevating the level of serotonin (5-hydroxytryptamine, 5-HT), activate 5-HT_3A receptors, and cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein. In this study, we have further examined the molecular mechanism of CDRI-08 in hippocampus-dependent memory and compared to the histone deacetylase (HDACs) inhibitor sodium butyrate (NaB). To assess the hippocampus-dependent memory, wistar rat pups were subjected to contextual fear conditioning (CFC) following daily (postnatal days 15–29) administration of vehicle solution (0.5 % gum acacia + 0.9 % saline)/CDRI-08 (80 mg/kg, p.o.)/NaB (1.2 g/kg in PBS, i.p.). CDRI-08/NaB treated group showed enhanced freezing behavior compared to control group when re-exposed to the same context. Administration of CDRI-08/NaB resulted in activation of extracellular signal-regulated kinase ERK/CREB signaling cascade and up-regulation of p300, Ac-H3 and Ac-H4 levels, and down-regulation of HDACs (1, 2) and protein phosphatases (PP1α, PP2A) in hippocampus following CFC. This would subsequently result in an increased brain-derived neurotrophic factor (Bdnf) (exon IV) mRNA in hippocampus. Altogether, our results indicate that CDRI-08 enhances hippocampus-dependent contextual memory by differentially regulating histone acetylation and protein phosphatases in hippocampus.     

Grin1 Receptor Deletion within CRF Neurons Enhances Fear Memory

Corticotropin releasing factor (CRF) dysregulation is implicated in mood and anxiety disorders such as posttraumatic stress disorder (PTSD). CRF is expressed in areas engaged in fear and anxiety processing including the central amygdala (CeA). Complicating our ability to study the contribution of CRF-containing neurons to fear and anxiety behavior is the wide variety of cell types in which CRF is expressed. To manipulate specific subpopulations of CRF containing neurons, our lab has developed a mouse with a Cre recombinase gene driven by a CRF promoter (CRFp3.0Cre) (Martin et al., 2010). In these studies, mice that have the gene that encodes NR1 (Grin1) flanked by loxP sites (floxed) were crossed with our previously developed CRFp3.0Cre mouse to selectively disrupt Grin1 within CRF containing neurons (Cre+/^fGrin1+). We find that disruption of Grin1 in CRF neurons did not affect baseline levels of anxiety, locomotion, pain sensitivity or exploration of a novel object. However, baseline expression of Grin1 was decreased in Cre+/^fGrin1+ mice as measured by RTPCR. Cre+/^fGrin1+ mice showed enhanced auditory fear acquisition and retention without showing any significant effect on fear extinction. We measured Gria1, the gene that encodes AMPAR1 and the CREB activator Creb1 in the amygdala of Cre+/^fGrin1+ mice after fear conditioning. Both Gria1 and Creb1 were enhanced in the amygdala after training. To determine if the Grin1-expressing CRF neurons within the CeA are responsible for the enhancement of fear memory in adults, we infused a lentivirus with Cre driven by a CRF promoter (LV pCRF-Cre/^fGrin1+) into the CeA of floxed Grin1 mice. Cre driven deletion of Grin1 specifically within CRF expressing cells in the CeA also resulted in enhanced fear memory acquisition and retention. Altogether, these findings suggest that selective disruption of Grin1 within CeA CRF neurons strongly enhances fear memory.     

Modulation of Inositol 1,4,5-Trisphosphate Receptor Type 2 Channel Activity by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Phosphorylation

InsP₃-mediated calcium release through the type 2 inositol 1,4,5-trisphosphate receptor (InsP₃R2) in cardiac myocytes results in the activation of associated CaMKII, thus enabling the kinase to act on downstream targets, such as histone deacetylases 4 and 5 (HDAC4 and HDAC5). The CaMKII activity also feedback modulates InsP₃R2 function by direct phosphorylation and results in a dramatic decrease in the receptor-channel open probability (P_o). We have identified S150 in the InsP₃R2 core suppressor domain (amino acids 1–225) as the specific residue that is phosphorylated by CaMKII. Site-directed mutagenesis reveals that S150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Nonphosphorylatable (S150A) and phosphomimetic (S150E) mutations were studied in planar lipid bilayers. The InsP₃R2 S150A channel showed no decrease in activity when treated with CaMKII. Conversely, the phosphomimetic (S150E) channel displayed a very low P_o under normal recording conditions in the absence of CaMKII (2 μm InsP₃ and 250 nm [Ca²⁺]_FREE) and mimicked a WT channel that has been phosphorylated by CaMKII. Phopho-specific antibodies demonstrate that InsP₃R2 Ser-150 is phosphorylated in vivo by CaMKIIδ. The results of this study show that serine 150 of the InsP₃R2 is phosphorylated by CaMKII and results in a decrease in the channel open probability.     

Updating versus Exposure to Prevent Consolidation of Conditioned Fear

Targeting the consolidation of fear memories following trauma may offer a promising method for preventing the development of flashbacks and other unwanted re-experiencing symptoms that characterise Posttraumatic Stress Disorder (PTSD). Research has demonstrated that performing visuo-spatial tasks after analogue trauma can block the consolidation of fear memory and reduce the frequency of flashbacks. However, no research has yet used verbal techniques to alter memories during the consolidation window. This is surprising given that the most effective treatments for PTSD are verbally-based with exposure therapy and trauma-focused cognitive behavioural therapy gaining the most evidence of efficacy. Psychological therapies aim to reduce the conditioned fear response, which is in keeping with the preliminary finding that an increased propensity for fear conditioning may be a vulnerability factor for PTSD. Our research had two aims. We investigated the degree to which individual differences in fear conditioning predict the development of PTSD symptoms. We also compared the preventative effects of two clinically informed psychological techniques administered during the consolidation window: exposure to the trauma memory and updating the meaning of the trauma. 115 healthy participants underwent a fear conditioning paradigm in which traumatic film stimuli (unconditioned stimuli) were paired with neutral stimuli (conditioned stimuli). Participants were randomly allocated to an updating, exposure or control group to compare the effects on the conditioned fear response and on PTSD symptomatology. The results showed that stronger conditioned responses at acquisition significantly predicted the development of PTSD symptoms. The updating group, who verbally devalued the unconditioned stimulus within the consolidation window, experienced significantly lower levels of PTSD symptoms during follow-up than the exposure and control groups. These findings are consistent with clinical interventions for chronic PTSD and have important implications for identifying those at risk as well as for designing novel early interventions to prevent the development of PTSD.     

N-Alkylated arylsulfonamides of (aryloxy)ethyl piperidines: 5-HT7 receptor selectivity versus multireceptor profile

The N-alkylation of the sulfonamide moiety, in a group of arylsulfonamide derivatives of (aryloxy)ethyl piperidines, may be considered as a strategy for the design of selective 5-HT₇ receptor ligands or multifunctional agents to extend a polypharmacological approach to the treatment of complex diseases. The study allowed for the identification of 31 (1-methyl-N-{1-[2-(2-(t-butyl)phenoxy)ethyl]piperidin-4-yl}-N-cyclopropylmethyl-1H-pyrazole-4-sulfonamide), a potent and selective 5-HT₇ receptor antagonist and 33 (1-methyl-N-{1-[2-(biphenyl-2-yloxy)ethyl]piperidin-4-yl}-N-cyclopropylmethyl-1H-pyrazole-4-sulfonamide), as multimodal 5-HT/dopamine receptor ligand, as 5-HT_2A/5-HT₇/D₂ receptor antagonists. Both selected compounds were evaluated in vivo in a forced swim test (FST) in mice and in a novel object recognition (NOR) task in rats, demonstrating distinct antidepressant-like and pro-cognitive properties (MED = 1.25 mg/kg and 1 mg/kg, ip, respectively). These findings warrant further studies to explore the therapeutic potential of N-alkylated arylsulfonamides for the treatment of CNS disorders.     

Synthesis and structure–activity relationships of new carbonyl guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor antagonists. Part 2

We previously reported that the novel dual 5-HT_2B and 5-HT₇ receptor antagonist N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (4) exerted a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs. To develop a synthetic strategy, we performed docking studies of lead compound 4 bound to 5-HT_2B and 5-HT₇ receptors, and observed that the carbonyl guanidine group forms a tight interaction network with an active center Asp (D135:5-HT_2B, D162:5-HT₇), Tyr (Y370:5-HT_2B, Y374:5-HT₇) and aromatic residue (W131:5-HT_2B, F158:5-HT₇). Based on molecular modeling results, we optimized the substituents at the 5- to 8-position and 9-position of the fluorene ring and identified N-(diaminomethylene)-9-hydroxy-9-methyl-9H-fluorene-2-carboxamide (24a) exhibits potent affinity for 5-HT_2B (K_i = 4.3 nM) and 5-HT₇ receptor (K_i = 4.3 nM) with high selectivity over 5-HT_2A, 5-HT_2C, α₁, D₂ and M₁ receptors. Compound 24a reversed the hypothermic effect of 5-carboxamidotryptamine (5-CT) in mice and also showed a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs when orally administered at 30 mg/kg. Compound 24a is therefore a promising candidate for a novel class of anti-migraine agent without any adverse effects.     

Environmental enrichment exerts anxiolytic effects in the Indian field mouse (Mus booduga)

Environmental enrichment (EE) is known to have behavioral and physiological anxiolytic effects in several animal models. However, it is as yet unclear how EE modulates behavior of wild animals and the underlying molecular mechanisms. The adult male field mouse Mus booduga (n = 42) captured at agricultural field, were housed in non-enriched standard condition (SC) for 7 days and considered as directly from wild (DW). Another two groups of mice were housed in either EE or SC for 30 days. Behavioral testing was carried out to assess their anxiety-like behavior in the elevated plus-maze (EPM). We found that on EPM, mice housed in EE display less anxiety like behavior when compared to mice housed in SC. Exposure to plus-maze did not increase the levels of corticosterone (CORT) in prefrontal cortex (PFC) and circulating CORT, and adrenocorticotropic hormone (ACTH) in the mice housed in EE but not in the mice housed in SC. We observed a trend in the EE induced inhibition of expression of corticotropin releasing hormone (CRH), glucocorticoid receptor (GR), E2 ubiquitin-conjugating enzyme (Ubc9) and steroid receptor coactivator-1 (SRC-1) mRNA levels, which are all known to be involved in the stress response signaling pathway. Our study suggests that EE exerts therapeutic and anxiolytic effects against stressors.     

5-HT2C Receptors in the Basolateral Amygdala and Dorsal Striatum Are a Novel Target for the Anxiolytic and Antidepressant Effects of Exercise

Physical activity reduces the incidence and severity of psychiatric disorders such as anxiety and depression. Similarly, voluntary wheel running produces anxiolytic- and antidepressant-like effects in rodent models. The specific neurobiological mechanisms underlying the beneficial properties of exercise, however, remain unclear. One relevant pharmacological target in the treatment of psychiatric disorders is the 5-HT_2C receptor (5-HT_2CR). Consistent with data demonstrating the anxiogenic consequences of 5-HT_2CR activation in humans and rodents, we have previously reported that site-specific administration of the selective 5-HT_2CR agonist CP-809101 in the lateral/basolateral amygdala (BLA) increases shock-elicited fear while administration of CP-809101 in the dorsal striatum (DS) interferes with shuttle box escape learning. These findings suggest that activation of 5-HT_2CR in discrete brain regions contributes to specific anxiety- and depression-like behaviors and may indicate potential brain sites involved in the anxiolytic and antidepressant effects of exercise. The current studies tested the hypothesis that voluntary wheel running reduces the behavioral consequences of 5-HT_2CR activation in the BLA and DS, specifically enhanced shock-elicited fear and interference with shuttle box escape learning. After 6 weeks of voluntary wheel running or sedentary conditions, the selective 5-HT_2CR agonist CP-809101 was microinjected into either the BLA or the DS of adult Fischer 344 rats, and shock-elicited fear and shuttle box escape learning was assessed. Additionally, in-situ hybridization was used to determine if 6 weeks of voluntary exercise changed levels of 5-HT_2CR mRNA. We found that voluntary wheel running reduced the behavioral effects of CP-809101 and reduced levels of 5-HT_2CR mRNA in both the BLA and the DS. The current data indicate that expression of 5-HT_2CR mRNA in discrete brain sites is sensitive to physical activity status of the organism, and implicates the 5-HT_2CR as a target for the beneficial effects of physical activity on mental health.     

Synthesis and activity of di- or trisubstituted N-(phenoxyalkyl)- or N-{2-[2-(phenoxy)ethoxy]ethyl}piperazine derivatives on the central nervous system

Aim of the study was evaluation of anxiolytic, antidepressant, anticonvulsant and analgesic activity in a series of a consistent group of compounds. A series of eleven new N-(phenoxyalkyl)- or N-{2-[2-(phenoxy)ethoxy]ethyl}piperazine derivatives has been obtained. Their affinity towards 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, D₂ and α₁ receptors has been assessed, and then functional assays were performed. The compounds were evaluated in mice, i.p. for their antidepressant-like (forced swim test), locomotor, anxiolytic-like (four-plate test) activities as well as – at higher doses – for anticonvulsant potential (MES) and neurotoxicity (rotarod). Two compounds (3, 6) were also evaluated for their analgesic activity in neuropathic pain models (streptozocin test, oxaliplatin test) and they were found active against allodynia in diabetic neuropathic pain at 30?mg/kg. Among the compounds, anxiolytic-like, anticonvulsant or analgesic activity was observed but antidepressant-like activity was not. One of the two most interesting compounds is 1-{2-[2-(2,4,6-trimethylphenoxy)ethoxy]ethyl}-4-(2-methoxyphenyl)piperazine dihydrochloride (9), exhibiting anxiolytic and anticonvulsant activity in mice, i.p. 30 min after administration (at 2.5?mg/kg and ED₅₀?=?26.33?mg/kg, respectively), which can be justified by the receptor profile: 5-HT_1A K_i?=?5?nM (antagonist), 5-HT₇ K_i?=?70?nM, α₁ K_i?=?15?nM, D₂ K_i?=?189?nM (antagonist). Another interesting compound is 1-[3-(2,4,6-trimethylphenoxy)propyl]-4-(4-methoxyphenyl)piperazine dihydrochloride (3), exhibiting anxiolytic, anticonvulsant and antiallodynic activity in mice, i.p., 30?min after administration (at 10?mg/kg, ED₅₀?=?23.50?mg/kg, at 30?mg/kg, respectively), which can be related with 5-HT_1A weak antagonism (K_i?=?146?nM), or other possible mechanism of action, not evaluated within presented study. Additionally, for the most active compound in the four-plate test (7), molecular modeling was performed (docking to receptors 5-HT_1A, 5-HT_2A, 5-HT₇, D₂ and α_1A).     

Enhanced Histone Acetylation in the Infralimbic Prefrontal Cortex is Associated with Fear Extinction

The molecular processes that establish fear memory are complex and involve a combination of genetic and epigenetic influences. Dysregulation of these processes can manifest in humans as a range of fear-related anxiety disorders like post-traumatic stress disorders (PTSD). In the present study, immunohistochemistry for acetyl H3, H4, c-fos, CBP (CREB-binding protein) in the infralimbic prefrontal cortex (IL-PFC) and prelimbic prefrontal cortex (PL-PFC) of mPFC (medial prefrontal cortex) and basal amygdala (BA), lateral amygdala (LA), centrolateral amygdala (CeL), centromedial amygdala (CeM) of the amygdala was performed to link region-specific histone acetylation to fear and extinction learning. It was found that the PL-PFC and IL-PFC along with the sub-regions of the amygdala responded differentially to the fear learning and extinction. Following fear learning, c-fos and CBP expression and acetylation of H3 and H4 increased in the BA, LA, CeM, and CeL and the PL-PFC but not in the IL-PFC as compared to the naive control. Similarly, following extinction learning, c-fos and CBP expression increased in BA, LA, CeL, and IL-PFC but not in PL-PFC and CeM as compared to the naive control and conditioned group. However, the acetylation of H3 increased in both IL and PL as opposed to H4 which increased only in the IL-PFC following extinction learning. Overall, region-specific activation in amygdala and PFC following fear and extinction learning as evident by the c-fos activation paralleled the H3/H4 acetylation in these regions. These results suggest that the differential histone acetylation in the PFC and amygdala subnuclei following fear learning and extinction may be associated with the region-specific changes in the neuronal activation pattern resulting in more fear/less fear.     

Rotavirus stimulates release of serotonin (5-HT) from human enterochromaffin cells and activates brain structures involved in nausea and vomiting

Rotavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca²⁺ concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4,5-triphosphate (IP₃), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT₃ receptor antagonists.     

Quantitative assay of 5-HT1A receptor gene expression in the brain

5-HT_1A receptors are involved in the regulation of various behaviors and the mechanism of action of anxiolytics and antidepressants. It is rather difficult to study the expression of the 5-HT_1A receptor gene in the brain because of the low concentration of its mRNA. A method developed for quantitating the level of 5-HT_1A receptor gene expression in brain structures involves estimation of the copy number for contaminant genomic DNA, the cDNA of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (a housekeeping gene), and the 5-HT_1A receptor gene cDNA in a cDNA preparation. To estimate the GAPDH and 5-HT_1A receptor cDNA copy numbers, the fluorescent intensity of the corresponding PCR products is calibrated using genomic DNA standards of known concentrations. The expression of the 5-HT_1A receptor gene is corrected for the content of contaminant genomic DNA and presented as a 5-HT_1A receptor cDNA copy number per 100 copies of the GAPDH cDNA. The method was used to demonstrate for the first time that expression of the 5-HT_1A receptor gene is increased in the frontal cortex and the amygdala of mice knocked-out in the monoamine oxidase A gene.