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Disproportionate, maladapted, and generalized fear are essential hallmarks of posttraumatic stress disorder (PTSD), which develops upon severe trauma in a subset of exposed individuals. Among the brain areas that are processing fear memories, the hippocampal formation exerts a central role linking emotional-affective with cognitive aspects. In the hippocampus, neuronal excitability is constrained by multiple GABAergic interneurons with highly specialized functions and an extensive repertoire of co-released neuromodulators. Neuropeptide Y (NPY) is one of these co-transmitters that significantly affects hippocampal signaling, with ample evidence supporting its fundamental role in emotional, cognitive, and metabolic circuitries. Here we investigated the role of NPY in relation to GABA, both released from the same interneurons of the dorsal dentate gyrus (DG), in different aspects of fear conditioning. We demonstrated that activation of dentate GABA neurons specifically during fear recall reduced cue-related as well as trace-related freezing behavior, whereas inhibition of the same neurons had no significant effects. Interestingly, concomitant overexpression of NPY in these neurons did not further modify fear recall, neither under baseline conditions nor upon chemogenetic stimulation. However, potentially increased co-release of NPY substantially reduced contextual fear, promoted extinction learning, and long-term suppression of fear in a foreground context–conditioning paradigm. Importantly, NPY in the dorsal DG was not only expressed in somatostatin neurons, but also in parvalbumin-positive basket cells and axoaxonic cells, indicating intense feedback and feedforward modulation of hippocampal signaling and precise curtailing of neuronal engrams. Thus, these findings suggest that co-release of NPY from specific interneuron populations of the dorsal DG modifies dedicated aspects of hippocampal processing by sharpening the activation of neural engrams and the consecutive fear response. Since inappropriate and generalized fear is the major impediment in the treatment of PTSD patients, the dentate NPY system may be a suitable access point to ameliorate PTSD symptoms and improve the inherent disease course.

Return of fear by re-exposure to an aversive event is a major obstacle in the treatment of fear-related disorders. Recently, we demonstrated that local pharmacological stimulation of neuropeptide Y type 2 receptors (Y2R) in anteroventral bed nucleus of stria terminalis (BNSTav) facilitates fear extinction and attenuates retrieval of remote fear with or without concomitant extinction training. Whether Y2R activation could also protect against re-exposure to traumatic events is still unknown. Therefore, we investigated reinstatement of remote fear following early Y2R manipulation in BNSTav in relation to concomitant extinction training in mice. We combined local pharmacological manipulation of Y2Rs in BNSTav with or without extinction training and tested for reinstatement of remote fear 15 days later. Furthermore, we employed immediate early gene mapping to monitor related local brain activation. Y2R stimulation by local injection of NPY3–36 into BNSTav facilitated extinction, reduced fear reinstatement at remote stages, and mimicked the influence of extinction in groups without prior extinction training. In contrast, Y2R antagonism (JNJ-5207787) delayed extinction and increased reinstatement. Y2R treatment immediately before remote fear tests had no effect. Concomitantly, Y2R activation at early time points reduced the number of c-Fos positive neurons in BNSTav during testing of reinstated remote fear. Local Y2R stimulation in BNSTav promotes fear extinction and stabilizes suppression of reinstated fear through a long-term influence, even without extinction training. Thus, Y2Rs in BNST are crucial pharmacological targets for extinction-based remote fear suppression.

The mouse olfactory neuroepithelium (ON) is comprised of anatomically distinct populations of cells in separate regions; apical (sustentacular and microvillar), neuronal (olfactory sensory neurons) and basal (horizontal and globose basal cells). The existence of microvillar cells (MVCs) is well documented but their nature and function remains unclear. An important transcription factor for the differentiation of MVCs is Skn-1a, with loss of function of Skn-1a in mice resulting in a complete loss of Trpm-5 expressing MVCs, while olfactory sensory neuron differentiation is normal. Our previous research has shown that neuropeptide Y (NPY) is expressed in MVCs and is important in the neuroproliferation of olfactory precursors. This study showed that following X-ray irradiation of the snout of wildtype mice, which decreases the proliferation of basal precursor cells, the numbers of Trpm-5-positive MVCs is increased at 2 and 5 weeks post-irradiation compared to controls. Skn-1a expression in the ON following X-ray irradiation also increases at 2 weeks post-irradiation in a regionally specific manner matching the expression pattern of Trpm-5-positive MVCs. In parallel, NPYCre knock-in mice were used to examine the expression of Skn-1a following activation of NPY unilaterally in the ON (unilateral nasal irrigation of AAV-NPY-FLEX). These experiments demonstrated that Skn-1a is only expressed when NPY is activated in MVCs. Therefore the expression of NPY is necessary for the transcription factor-mediated differentiation of olfactory MVCs.

Associative learning is thought to involve different forms of activity-dependent synaptic plasticity. Although previous studies have mostly focused on learning-related changes occurring at excitatory glutamatergic synapses, we found that associative learning, such as fear conditioning, also entails long-lasting functional and structural plasticity of GABAergic synapses onto pyramidal neurons of the murine basal amygdala. Fear conditioning-mediated structural remodeling of GABAergic synapses was associated with a change in mIPSC kinetics and an increase in the fraction of synaptic benzodiazepine-sensitive (BZD) GABAA receptors containing the α2 subunit without altering the intrasynaptic distribution and overall amount of BZD-GABAA receptors. These structural and functional synaptic changes were partly reversed by extinction training. These findings provide evidence that associative learning, such as Pavlovian fear conditioning and extinction, sculpts inhibitory synapses to regulate inhibition of active neuronal networks, a process that may tune amygdala circuit responses to threats.

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the hypothalamus and an important regulator of energy homeostasis; however, the specific contributions of different CART neuronal populations to this process are not known. Here, we show that depolarization of mouse arcuate nucleus (Arc) CART neurons via DREADD technology decreases energy expenditure and physical activity, while it exerts the opposite effects in CART neurons in the lateral hypothalamus (LHA). Importantly, when stimulating these neuronal populations in the absence of CART, the effects were attenuated. In contrast, while activation of CART neurons in the LHA stimulated feeding in the presence of CART, endogenous CART inhibited food intake in response to Arc CART neuron activation. Taken together, these results demonstrate anorexigenic but anabolic effects of CART upon Arc neuron activation, and orexigenic but catabolic effects upon LHA-neuron activation, highlighting the complex and nuclei-specific functions of CART in controlling feeding and energy homeostasis.

Posttraumatic stress disorder is characterized by contextually inappropriate, dys-regulated and generalized fear expression and often resistant to therapy. The hippocampus integrates contextual information into spatial and emotional memories, but how diverse modulatory neurotransmitters are shaping this process is not known. Neuropeptide Y is a peptide-neurotransmitter, which modulates hippocampal excitability by activating several G-protein-coupled receptors. Postsynaptic Y1 receptors create strong anxiolytic and fear-suppressing behavior, while pre-synaptic Y2 receptors (Y2R) are mainly anxiogenic. The role of Y2Rs in spatial compared to emotional learning is, however, still controversial. Here we show that deletion of Y2Rs increased recall, but delayed extinction of contextual fear. Interestingly, spatial memory in the Barnes maze was enhanced during early and late testing, suggesting that Y2Rs suppress learning by hippocampal and extra-hippocampal mechanisms. To demonstrate sufficiency of hippocampal Y2Rs we performed viral vector-mediated, locally restricted re-expression of Y2Rs in the hippocampus of Y2KO mice. This treatment reduced spatial memory to the level of wildtype mice only during early, but not late recall. Furthermore, contextual fear was reduced, while induction of fear extinction appeared earlier. Our results suggest that hippocampal Y2R signaling inhibits learning in a time- and content-specific way, resulting in an early reduction of spatial memory and in a specific suppression of fear, by reducing fear recall and promoting fear extinction. We thus propose that reduction of hippocampal excitability through pre-synaptic Y2Rs may control the integration of contextual information into developing memories.

Stress-induced cortical alertness is maintained by a heightened excitability of noradrenergic neurons innervating, notably, the prefrontal cortex. However, neither the signaling axis linking hypothalamic activation to delayed and lasting noradrenergic excitability nor the molecular cascade gating noradrenaline synthesis is defined. Here, we show that hypothalamic corticotropin-releasing hormone-releasing neurons innervate ependymal cells of the 3rd ventricle to induce ciliary neurotrophic factor (CNTF) release for transport through the brain's aqueductal system. CNTF binding to its cognate receptors on norepinephrinergic neurons in the locus coeruleus then initiates sequential phosphorylation of extracellular signal-regulated kinase 1 and tyrosine hydroxylase with the Ca2+-sensor secretagogin ensuring activity dependence in both rodent and human brains. Both CNTF and secretagogin ablation occlude stress-induced cortical norepinephrine synthesis, ensuing neuronal excitation and behavioral stereotypes. Cumulatively, we identify a multimodal pathway that is rate-limited by CNTF volume transmission and poised to directly convert hypothalamic activation into long-lasting cortical excitability following acute stress.

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.

Purpose to explore whether the NK1 and Y2 receptors are involved in the pathogenesis of laser-induced CNV (choroidal neovascularization) in C57Bl/6N mice. Methods CNV was induced by laser damage of Bruch's membrane and the CNV volume was determined by OCT and/or flatmount preparation. First, the development of the CNV volume over time was evaluated. Second, the CNV development in NK1- and Y2 KO mice was analyzed. Third, the effect on the development as well as the regression of CNV by intravitreal injections of the NK1 antagonist SR140333 and the Y2 antagonist BIIEO246 separately and each in combination with Eylea®, was investigated. Furthermore, flatmount CNV volume measurements were correlated to volumes obtained by the in vivo OCT technique. Results CNV volume peak was observed at day 4 after laser treatment. Compared to wild type mice, NK1 and Y2 KO mice showed significantly smaller CNV volumes. Eylea® and the Y2 antagonist significantly reduced the volume of the developing CNV. In contrast to Eylea® there was no effect of either antagonist on the regression of CNV, additionally no additive effect upon combined Eylea®/antagonist treatment was observed. There was a strong positive correlation between CNV volumes obtained by OCT and flatmount. Conclusion NK1 and Y2 receptors mediate the development of laser-induced CNVs in mice. They seem to play an important role at the developmental stage of CNVs, whereas VEGF via VEGF receptor may be an important mediator throughout the CNV existence. In vivo OCT correlates with flatmount CNV volume, representing a useful tool for in vivo evaluations of CNV over time.

The anterior bed nucleus of stria terminalis (BNST) is involved in reinstatement of extinguished fear, and neuropeptide Y2 receptors influence local synaptic signaling. Therefore, we hypothesized that Y2 receptors in anteroventral BNST (BNSTav) interfere with remote fear memory and that previous fear extinction is an important variable. C57BL/6NCrl mice were fear-conditioned, and a Y2 receptor-specific agonist (NPY3-36) or antagonist (JNJ-5207787) was applied in BNSTav before fear retrieval at the following day. Remote fear memory was tested on day 16 in two groups of mice, which had (experiment 1) or had not (experiment 2) undergone extinction training after conditioning. In the group with extinction training, tests of remote fear memory revealed partial retrieval of extinction, which was prevented after blockade of Y2 receptors in BNSTav. No such effect was observed in the group with no extinction training, but stimulation of Y2 receptors in BNSTav mimicked the influence of extinction during tests of remote fear memory. Pharmacological manipulation of Y2 receptors in BNSTav before fear acquisition (experiment 3) had no effect on fear memory retrieval, extinction or remote fear memory. Furthermore, partial retrieval of extinction during tests of remote fear memory was associated with changes in number of c-Fos expressing neurons in BNSTav, which was prevented or mimicked upon Y2 blockade or stimulation in BNSTav. These results indicate that Y2 receptor manipulation in BNSTav interferes with fear memory and extinction retrieval at remote stages, likely through controlling neuronal activity in BNSTav during extinction training.

In this study, we investigated whether the proangiogenic neuropeptides secretoneurin (SN), substance P (SP), and neuropeptide Y (NPY) contribute to the development of abnormal neovascularization in the oxygen-induced retinopathy (OIR) model in mice. By exposing litters of C57Bl/6N mice to 75% oxygen from postnatal day 7 (P7) until postnatal day 11 (P11) and then returning them to normoxic conditions, retinal ischemia and subsequent neovascularization on the retinal surface were induced. Retinae were dissected on P9, P11, P12–P14, P16 and P20, and the concentrations of SN, SP, NPY and VEGF determined by radioimmunoassay or ELISA. The levels of SN and SP increased in controls from P9 until P16 and from P9 until P14, respectively, whereas the levels of NPY were high at P9 and decreased thereafter until P20, suggesting that NPY may participate in the development of the retina. However, dipeptidyl peptidase IV (DPPIV) and the NPY-Y2 receptor were not detectable in the immature retina indicating that NPY is not involved in the physiological vascularization in the retina. Compared to controls, OIR had no effect on the levels of SN, whereas levels of both SP and NPY slightly decreased during hyperoxia. Normalization of the levels of SP, and to a more pronounced extent of NPY, was significantly delayed during relative hypoxia. This clearly indicates that these three neuropeptides are not involved in the pathogenesis of neovascularization in OIR. Moreover, since there were no differences in the expression of two vessel markers in the retina of NPY knockout mice versus controls at P14, NPY is also not involved in the delayed development of the intermediate and deep vascular plexus in the retina in this animal model.

BACKGROUND AND PURPOSE Neuropeptide Y (NPY) and its receptors have been implicated in the control of emotional-affective processing, but the mechanism is unclear. While it is increasingly evident that stimulation of Y1 and inhibition of Y2 receptors produce prominent anxiolytic and antidepressant effects, the contribution of the individual NPY receptor subtypes in the acquisition and extinction of learned fear are unknown. EXPERIMENTAL APPROACH Here we performed Pavlovian fear conditioning and extinction in NPY knockout (KO) and in NPY receptor KO mice. KEY RESULTS NPY KO mice display a dramatically accelerated acquisition of conditioned fear. Deletion of Y1 receptors revealed only a moderately accelerated acquisition of conditioned fear, while lack of Y2 receptors was without any effect on fear learning. However, the strong phenotype seen in NPY KO mice was reproduced in mice lacking both Y1 and Y2 receptors. In addition, NPY KO mice showed excessive recall of conditioned fear and impaired fear extinction. This behaviour was replicated only after deletion of both Y1 and Y2 receptors. In Y1 receptor single KO mice, fear extinction was delayed and was unchanged in Y2 receptor KO mice. Deletion of NPY and particularly Y2 receptors resulted in a generalization of conditioned fear. CONCLUSIONS AND IMPLICATIONS Our data demonstrate that NPY delays the acquisition, reduces the expression of conditioned fear while promoting fear extinction. Although these effects appear to be primarily mediated by Y1 receptors, the pronounced phenotype of Y1Y2 receptor double KO mice suggests a synergistic role of Y2 receptors in fear acquisition and in fear extinction.

Recently, expression of glutamate decarboxylase-67 (GAD67), a key enzyme of GABA synthesis, was detected in the otherwise glutamatergic mossy fibers of the rat hippocampus. Synthesis of the enzyme was markedly enhanced after experimentally induced status epilepticus. Here, we investigated the expression of GAD67 protein and mRNA in 44 hippocampal specimens from patients with mesial temporal lobe epilepsy (TLE) using double immunofluorescence histochemistry, immunoblotting, and in situ hybridization. Both in specimens with (n = 37) and without (n = 7) hippocampal sclerosis, GAD67 was highly coexpressed with dynorphin in terminal areas of mossy fibers, including the dentate hilus and the stratum lucidum of sector CA3. In the cases with Ammon's horn sclerosis, also the inner molecular layer of the dentate gyrus contained strong staining for GAD67 immunoreactivity, indicating labeling of mossy fiber terminals that specifically sprout into this area. Double immunofluorescence revealed the colocalization of GAD67 immunoreactivity with the mossy fiber marker dynorphin. The extent of colabeling correlated with the number of seizures experienced by the patients. Furthermore, GAD67 mRNA was found in granule cells of the dentate gyrus. Levels, both of GAD67 mRNA and of GAD67 immunoreactivity were similar in sclerotic and nonsclerotic specimens and appeared to be increased compared to post mortem controls. Provided that the strong expression of GAD67 results in synthesis of GABA in hippocampal mossy fibers this may represent a self-protecting mechanism in TLE. In addition GAD67 expression also may result in conversion of excessive intracellular glutamate to nontoxic GABA within mossy fiber terminals. © 2011 Wiley Periodicals, Inc.

Anxiety is integrated in the amygdaloid nuclei and involves the interplay of the amygdala and various other areas of the brain. Neuropeptides play a critical role in regulating this process. Neuropeptide Y (NPY), a 36 aa peptide, is highly expressed in the amygdala. It exerts potent anxiolytic effects through cognate postsynaptic Y1 receptors, but augments anxiety through presynaptic Y2 receptors. To identify the precise anatomical site(s) of Y2-mediated anxiogenic action, we investigated the effect of site-specific deletion of the Y2 gene in amygdaloid nuclei on anxiety and depression-related behaviors in mice. Ablating the Y2 gene in the basolateral and central amygdala resulted in an anxiolytic phenotype, whereas deletion in the medial amygdala or in the bed nucleus of the stria terminalis had no obvious effect on emotion-related behavior. Deleting the Y2 receptor gene in the central amygdala, but not in any other amygdaloid nucleus, resulted in an added antidepressant-like effect. It was associated with a reduction of presumably presynaptic Y2 receptors in the stria terminalis/bed nucleus of the stria terminalis, the nucleus accumbens, and the locus ceruleus. Our results are evidence of the highly site-specific nature of the Y2-mediated function of NPY in the modulation of anxiety- and depression-related behavior. The activity of NPY is likely mediated by the presynaptic inhibition of GABA and/or NPY release from interneurons and/or efferent projection neurons of the basolateral and central amygdala.

There is growing evidence that neuropeptide Y (NPY) acting through Y1 and Y2 receptors has a prominent role in modulating anxiety- and depression-like behavior in rodents. However, a role of other Y-receptors like that of Y4 receptors in this process is poorly understood. We now investigated male Y2, Y4 single and Y2/Y4 double knockout mice in behavioral paradigms for changes in motor activity, anxiety and depression-like behavior. Motor activity was increased in Y2, Y4 and Y2/Y4 knockout mice under changing and stressful conditions, but not altered in a familiar environment. Y4 and Y2 knockout mice revealed an anxiolytic phenotype in the light/dark test, marble burying test and in stress-induced hyperthermia, and reduced depression-like behavior in the forced swim and tail suspension tests. In Y2/Y4 double knockout mice, the response in the light/dark test and in the forced swim test was further enhanced compared with Y4 and Y2 knockout mice, respectively. High levels of Y4 binding sites were observed in brain stem nuclei including nucleus of solitary tract and area postrema. Lower levels were found in the medial amygdala and hypothalamus. Peripheral administration of pancreatic polypeptide (PP) induced Y4 receptor-dependent c-Fos expression in brain stem, hypothalamus and amygdala. PP released peripherally from the pancreas in response to food intake, may act not only as a satiety signal but also modulate anxiety-related locomotion.

A prominent role of epigenetic mechanisms in manifestation of epilepsy has been proposed. Thus altered histone H3 and H4 acetylation has been demonstrated in experimental models of temporal lobe epilepsy (TLE). We now investigated changes in the expression of the class I and class IV histone deacetylases (HDAC) in two complementary mouse TLE models. Unilateral intrahippocampal injection of kainic acid (KA) induced a status epilepticus lasting 6 to 24h, development of spontaneous limbic seizures (2 to 3 days after KA injection) and chronic epilepsy, as revealed by telemetric recordings of the EEGs. Mice were killed at different intervals after KA injection and expression of HDAC mRNAs was investigated by in situ hybridization. We observed marked decreases in the expression of HDACs 1, 2 and 11 (by up to 75%) in the granule cell and pyramidal cell layers of the hippocampus during the acute status epilepticus (2 to 6h after KA injection). This was followed by increased expression of all class I HDAC mRNAs in all principal cell layers of the hippocampus after 12 to 48 h. In the chronic phase, 14 and 28 days after KA, only modest increases in the expression of HDAC1 mRNA were observed in granule and pyramidal cells. Immunohistochemistry using an antibody detecting HDAC2 revealed results consistent with the mRNA data and indicates also expression in glial cells on the injection side. Similar changes as seen in the KA model were observed after a pilocarpine-induced status epilepticus except that decreases in HDACs 2, 3 and 8 were also seen at the chronic 28 day interval. The prominent decreases in HDAC expression during status epilepticus are consistent with the previously demonstrated increased expression of numerous proteins and with the augmented acetylation of histone H4. It is suggested that respective putative gene products could facilitate proconvulsive as well as anticonvulsive mechanisms. The increased expression of all class I HDACs during the "silent phase", on the other hand, may be related to decreased histone acetylation, which could cause a decrease in expression of certain proteins, a mechanism that could also promote epileptogenesis. Thus, addressing HDAC expression may have a therapeutic potential in interfering with a status epilepticus and with the manifestation of TLE.

Background Fear and anxiety are integrated in the amygdaloid nuclei and involve the interplay of the amygdala with various other brain areas. Neuropeptides play a critical role in regulating these processes. Neuropeptide Y (NPY) is highly expressed in limbic brain areas, including the amygdala. Depending on the receptor subtypes involved (Y1, Y2 or Y4), NPY has different, in part opposing effects on anxiety, fear and depression-related behaviors.

Background Neuropeptide Y (NPY), a highly conserved 36 amino acid peptide is widely distributed in the central nervous system. Besides its functions in various metabolic processes NPY has attracted considerable attention in modulating emotional-affective behavior. NPY exerts a pronounced anxiolytic effect most likely mediated by Y1 receptors, whereas stimulation of predominantly pre-synaptic Y2 receptors results in increased anxiety. The role of NPY Y2 receptors in the processing of emotional learning, however, remains still elusive.

Background Previous work has suggested that alterations in GABAergic function within the amygdala underlie fear learning. In particular, it has been shown that Pavlovian fear conditioning induces a downregulation of benzodiazepine binding sites as well as transcripts for gephyrin and some GABAA receptor subunits in the basal nucleus of the amygdala (BA), which were restored to control levels after fear extinction.

Background Neuropeptide Y (NPY), a highly conserved 36 amino acid peptide, is widely distributed in the central nervous system. Besides its functions in various metabolic processes NPY has attracted considerable attention in modulating emotional-affective behavior. NPY exerts a pronounced anxiolytic effect most likely mediated by Y1 receptors, whereas stimulation of predominantly presynaptic Y2 receptors results in increased anxiety. The role of NPY in the processing of fear, however, is still not conclusive. The current study aims to elucidate the role of NPY in Pavlovian fear conditioning, a simple form of associative learning.

Neuropeptide Y (NPY) has been extensively studied in relation to anxiety and depression but of the seven NPY receptors known to date, it is not yet clear which one is mainly involved in mediating its effects in emotional behavior. Mice lacking the NPY-Y2 receptors were previously shown to be less anxious due to their improved ability to cope with stressful situations. In the present study, the behavioral phenotype including the response to challenges was analyzed in NPY-Y2 knockout (KO) mice backcrossed in to congenic C57BL/6 background. In the elevated plus-maze (EPM) and the forced swim test (FST), the anxiolytic-like or antidepressant-like phenotype of the NPY-Y2 KO mice could not be confirmed, although this study differs from the previous one only with regard to the genetic background of the mice. In addition, no differences in response to acute stress or to the antidepressant desipramine in the FST were detected between wild type (WT) and NPY-Y2 KO animals. These results suggest that the genetic background of the animals appears to have a strong influence on the behavioral phenotype of NPY-Y2 KO mice. Additionally, to further characterize the animals by their biochemical response to a challenge, the neurochemical changes induced by the anxiogenic compound yohimbine were measured in the medial prefrontal cortex (mPFC) of NPY-Y2 KO and compared to WT mice. Dopamine (DA) levels were significantly increased by yohimbine in the WT but unaffected in the KO mice, suggesting that NPY-Y2 receptor exerts a direct control over both the tonic and phasic release of DA and that, although the anxiety-like behavior of these NPY-Y2 KO mice is unaltered, there are clear modifications of DA dynamics. However, yohimbine led to a significant increase in noradrenaline (NA) concentration and a slight reduction in serotonin concentration that were identical for both phenotypes.

In different behavioral paradigms including the elevated plus maze (EPM), it was observed previously that deletion of the neuropeptide Y Y2 receptor subtype results in potent suppression of anxiety-related and stress-related behaviors. To identify neurobiological correlates underlying this behavioral reactivtiy, expression of c-Fos, an established early marker of neuronal activation, was examined in Y2 receptor knockout (Y2(-/-)) vs. wildtype (WT) mice. Mice were placed on the open arm (OA) or closed arm (CA) of the EPM for 10 min and the effect on regional c-Fos expression in the brain was investigated. The number of c-Fos positive neurons was significantly increased in both WT and Y2(-/-) lines after OA and CA exposure in 51 of 54 regions quantified. These regions included various cortical, limbic, thalamic, hypothalamic, and hindbrain regions. Genotype influenced c-Fos responses to arm exposures in 6 of the 51 activated regions: the cingulate cortex, barrel field of the primary somatosensory cortex, nucleus accumbens, dorsal lateral septum, amygdala and lateral periaqueductal gray. These differences in neuronal activity responses to the novel environments were more pronounced after OA than after CA exposure. Mice lacking Y2 receptors exhibited reduced neuronal activation when compared to WT animals in response to the emotional stressors. Reduced neuronal excitability in the identified brain areas relevant to the processing of motivated, explorative as well as anxiety-related behaviors is suggested to contribute to the reduced anxiety-related behavior observed in Y2(-/-) mice.

There is growing evidence that neuropeptide Y acting through Y1 and Y2 receptors has a prominent role in modulating anxiety- and depression-like behavior in rodents. However, a role of other Y receptors like that of Y4 receptors in this process is poorly understood. We now investigated male Y2, Y4 single and Y2/Y4 double knockout mice in behavioral paradigms for changes in motor activity, anxiety and depression-like behavior. Y4 and Y2 knockout mice revealed an anxiolytic phenotype in the light/dark test, marble-burying test and motor-activity independent in stress-induced hyperthermia, and reduced depression-like behavior in the forced swim and tail suspension tests. In Y2/Y4 double knockout mice, the response in the light/dark test and in the forced swim test was further enhanced compared to Y4 and Y2 knockout mice, respectively. Motor activity was increased in Y2, Y4 and Y2/Y4 knockout mice under changing and stressful conditions, but not altered in a familiar environment. High levels of Y4 binding sites were observed in brain stem nuclei including nucleus of solitary tract and area postrema. Lower levels were found in the medial amygdala and hypothalamus. Peripheral administration of PP induced Y4 receptor-dependent c-Fos expression in brain stem, hypothalamus and amygdala. PP released peripherally from the pancreas in response to food intake, may act not only as a satiety signal but also modulate anxiety-related locomotion. Lack of central Y4 receptors appears to be responsible for the alterations in behavior seen in Y4 and Y2/Y4 knockout mice suggesting a potential new target to treat anxiety-related disorders.

Neuropeptide Y (NPY) acting through Y1 receptors reduces anxiety- and depression-like behavior in rodents, whereas Y2 receptor stimulation has the opposite effect. This study addressed the implication of Y4 receptors in emotional behavior by comparing female germ line Y4 knockout (Y4-/-) mice with control and germ line Y2-/- animals. Anxiety- and depression-like behavior was assessed with the open field (OF), elevated plus maze (EPM), stress-induced hyperthermia (SIH) and tail suspension tests (TST), respectively. Learning and memory were evaluated with the object recognition test (ORT). In the OF and EPM, both Y4-/- and Y2-/- mice exhibited reduced anxiety-related behavior and enhanced locomotor activity relative to control animals. Locomotor activity in a familiar environment was unchanged in Y4-/- but reduced in Y2-/- mice. The basal rectal temperature exhibited diurnal and genotype-related alterations. Control mice had temperature minima at noon and midnight, whereas Y4-/- and Y2-/- mice displayed only one temperature minimum at noon. The magnitude of SIH was related to time of the day and genotype in a complex manner. In the TST, the duration of immobility was significantly shorter in Y4-/- and Y2-/- mice than in controls. Object memory 6 h after initial exposure to the ORT was impaired in Y2-/- but not in Y4-/- mice, relative to control mice. These results show that genetic deletion of Y4 receptors, like that of Y2 receptors, reduces anxiety-like and depression-related behavior. Unlike Y2 receptor knockout, Y4 receptor knockout does not impair object memory. We propose that Y4 receptors play an important role in the regulation of behavioral homeostasis.

Neuropeptide Y (NPY) is abundant in the nervous system. It acts through Y1, Y2, Y4 and Y5 receptors and is involved in a variety of brain functions. When applied locally into the amygdala, NPY exerts an anxiolytic action, presumably mediated by Y1 receptors. Depletion of Y2 receptors induces an anxiolytic phenotype, possibly by abolishing the release-inhibiting action of presynaptic Y2 receptors. In the present study we aimed to find the exact site of the presumed anxiogenic action mediated by Y2 receptors. We conducted site-specific deletions of Y2 receptors in Y2 mice by local injection of an AAV-Cre vector into the hippocampus and the amygdala. As controls, an AAV-GFP vector was injected in Y2 littermates at the same sites. Expression of Cre and GFP was verified by in situ hybridization and immunohistochemistry. Deletion of Y2 receptors was visualized by receptor autoradiography and in situ hybridization. After bilateral injection of an AAV-Cre vector into the basolateral amygdala, mice revealed a tendency towards an anxiolytic phenotype in the light-dark test (LDT). When deletion of Y2 receptors was confined to the central nucleus of the amygdala, an anxiolytic phenotype was observed in the elevated plus maze and the LDT. Moreover, a better stress coping ability was demonstrated in the tail suspension test. In contrast, no anxiolytic effect was detected after intrahippocampal injections. The experiments indicate that the anxiolytic and antidepressant-like effects of Y2 receptor deletion may be generated in certain subnuclei of the amygdala.

Mesial temporal lobe epilepsy (TLE) represents the most frequent type of focal epilepsies. The most prominent neuropathological characteristics of TLE are severe neurodegenerations in the hippocampus (termed Ammon’s horn sclerosis) and in related brain areas such as the amygdala and entorhinal cortex. Within the hippocampus, pyramidal cells of the areas CA1 and CA3 and interneurons of the dentate hilus are most vulnerable, whereas granule cells of the dentate gyrus, CA2 pyramidal cells and the subiculum are comparatively preserved. TLE can be initiated by prolonged febrile convulsions in early life or by a status epilepticus. It often takes years until the disease becomes clinically manifest (latent period). In animal models an initial status epilepticus is induced by application of a convulsant drug (e.g., kainic acid, pilocarpine) or by sustained electrical stimulation. Spontaneous seizures start thereafter within 1–3 weeks. The primary molecular mechanism underlying neuronal cell losses after status epilepticus are excitotoxic mechanisms caused by excessive release of

Anxiety disorders are the most prevalent central nervous system diseases imposing a high social burden to our society. Emotional processing is particularly controlled by GABA-ergic transmission in the amygdala. Using in situ hybridization and immunohistochemistry we now investigated changes in the expression of GABA synthesizing enzymes (GAD65 and GAD67), GABAA (α1–5, β1–3, γ1–2) and GABAB receptor subunits (GBBR1, GBBR2) in amygdaloid nuclei of high anxiety-related behavior (HAB) mice in comparison to mice selected for normal anxiety-related behavior (NAB). Levels of GAD65 and GAD67 mRNAs and protein, as well as those of GABA were increased in the amygdala of HAB mice. Relative to NAB controls, mRNA expression of the GABAA receptor subunits β1, β2 and γ2 was specifically increased in the basolateral amygdala of HAB mice while transcription of α5 and γ1 subunits was reduced in the central and medial amygdala. On the protein level, increases in β2 and γ2 subunit immunoreactivities were evident in the basolateral amygdala of HAB mice. No change in GABAB receptor expression was observed. These findings point towards an imbalanced GABA-ergic neurotransmission in the amygdala of HAB mice. On the other hand, FosB, a marker for neuronal activity, was increased in principal neurons of the basolateral amygdala in HAB mice, reflecting activation of excitatory neurons, possibly as a consequence of reduced GABA-ergic tonic inhibition through α5 and γ1 containing receptors. Ultimately these mechanisms may lead to the compensatory activation of GABA transmission, as indicated by the increased expression of GAD65/67 in HAB mice., Graphical Abstract Highlights ▶Mice with increased trait anxiety revealed: ▶Increased activity of principal neurons of the basolateral amygdala. ▶A compensatory increase of glutamate decarboxylase expression. ▶Differential alterations of GABAA receptor subunit expression in the amygdala.

Background Neuropeptide Y (NPY) is a 36-amino-acid peptide that is abundantly expressed in the central nervous system. It is involved in various physiological and pathophysiological processes, including energy homeostasis, pain and epilepsy, but also in anxiety and depression. Consistent findings have demonstrated an anxiolytic effect of NPY. The presence of different NPY receptors in the amygdala and the effects of NPY on anxiety raise the question, whether NPY and its receptors may influence acquisition and extinction of conditioned fear. Therefore, we investigated NPY and NPY receptor knockout mice in Pavlovian fear conditioning.

Background Neuropeptide Y is a highly conserved 36 amino acid peptide that is widely distributed in the peripheral and central nervous systems. Besides its functions in various metabolic processes, NPY has attracted considerable attention in modulating emotional-affective behavior. NPY exerts a solid anxiolytic effect most likely mediated by Y1 receptors, whereas stimulation of predominantly pre-synaptic Y2 receptors results in increased anxiety. However, little is known about an involvement of NPY in processing of fear.