Dilip Verma

NPY controls fear conditioning and fear extinction by combined action on Y1 and Y2 receptors

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.

Hunger Promotes Fear Extinction by Activation of an Amygdala Microcircuit

Emotions control evolutionarily-conserved behavior that is central to survival in a natural environment. Imbalance within emotional circuitries, however, may result in malfunction and manifestation of anxiety disorders. Thus, a better understanding of emotional processes and, in particular, the interaction of the networks involved is of considerable clinical relevance. Although neurobiological substrates of emotionally controlled circuitries are increasingly evident, their mutual influences are not. To investigate interactions between hunger and fear, we performed Pavlovian fear conditioning in fasted wild-type mice and in mice with genetic modification of a feeding-related gene. Furthermore, we analyzed in these mice the electrophysiological microcircuits underlying fear extinction. Short-term fasting before fear acquisition specifically impaired long-term fear memory, whereas fasting before fear extinction facilitated extinction learning. Furthermore, genetic deletion of the Y4 receptor reduced appetite and completely impaired fear extinction, a phenomenon that was rescued by fasting. A marked increase in feed-forward inhibition between the basolateral and central amygdala has been proposed as a synaptic correlate of fear extinction and involves activation of the medial intercalated cells. This form of plasticity was lost in Y4KO mice. Fasting before extinction learning, however, resulted in specific activation of the medial intercalated neurons and re-established the enhancement of feed-forward inhibition in this amygdala microcircuit of Y4KO mice. Hence, consolidation of fear and extinction memories is differentially regulated by hunger, suggesting that fasting and modification of feeding-related genes could augment the effectiveness of exposure therapy and provide novel drug targets for treatment of anxiety disorders.

Differential expression of L- and N-type voltage-sensitive calcium channels in the spinal cord of morphine+nimodipine treated rats.

We have earlier reported that nifedipine and nimodipine, both L-type voltage-sensitive calcium channel (L-VSCC) antagonists, attenuate the development of tolerance to chronic administration of morphine in the rat. In the present study, we have investigated the expression of L- and N-type VSCC using immunohisto-chemistry, in the cervical region of the spinal cords from animals treated chronically with morphine alone or in combination with nimodipine. The highest expression of both VSCCs within the spinal cord was detected within the superficial laminae of the dorsal horn, which indicates that these channels play an important role in the spinal processing of pain. After morphine tolerance, the expression of both the channels in the superficial laminae was significantly higher than control animals. However, morphine+nimodipine administration produced a differential effect, that is, the expression of L-VSCC decreased while that of N-VSCC increased. The study shows that the expression of these channels is plastic and subject to change depending upon the drug administered. This in turn can determine overall responsiveness to morphine.

Structure and function of the amygdaloid NPY system: NPY Y2 receptors regulate excitatory and inhibitory synaptic transmission in the centromedial amygdala

The amygdala is essential for generating emotional-affective behaviors. It consists of several nuclei with highly selective, elaborate functions. In particular, the central extended amygdala, consisting of the central amygdala (CEA) and the bed nucleus of the stria terminalis (BNST) is an essential component actively controlling efferent connections to downstream effectors like hypothalamus and brain stem. Both, CEA and BNST contain high amounts of different neuropeptides that significantly contribute to synaptic transmission. Among these, neuropeptide Y (NPY) has emerged as an important anxiolytic and fear-reducing neuromodulator. Here, we characterized the expression, connectivity and electrophysiological function of NPY and Y2 receptors within the CEA. We identified several NPY-expressing neuronal populations, including somatostatin- and calretinin-expressing neurons. Furthermore, in the main intercalated nucleus, NPY is expressed primarily in dopamine D1 receptor-expressing neurons but also in interspersed somatostatin-expressing neurons. Interestingly, NPY neurons did not co-localize with the Y2 receptor. Retrograde tract tracing experiments revealed that NPY neurons reciprocally connect the CEA and BNST. Functionally, the Y2 receptor agonist PYY3-36, reduced both, inhibitory as well as excitatory synaptic transmission in the centromedial amygdala (CEm). However, we also provide evidence that lack of NPY or Y2 receptors results in increased GABA release specifically at inhibitory synapses in the CEm. Taken together, our findings suggest that NPY expressed by distinct populations of neurons can modulate afferent and efferent projections of the CEA via presynaptic Y2 receptors located at inhibitory and excitatory synapses.

NPY Y2 receptors in the central amygdala reduce cued but not contextual fear

The amygdala is fundamental for associative fear and extinction learning. Recently, also the central nucleus of the amygdala (CEA) has emerged as a site of plasticity actively controlling efferent connections to downstream effector brain areas. Although synaptic transmission is primarily mediated by glutamate and GABA, neuropeptides critically influence the overall response. While neuropeptide Y (NPY) acting via postsynaptic Y1 receptors exerts an important anxiolytic and fear-reducing action, the role of the predominantly presynaptic Y2 receptors is less defined. To investigate the role of Y2 receptors in the CEA we employed viral-vector mediated over-expression of the Y2 selective agonist NPY3-36 in fear conditioning and extinction experiments. NPY3-36 over-expression in the CEA resulted in reduced fear expression during fear acquisition and recall. Interestingly, this effect was blocked by intraperitoneal injection of a brain-penetrant Y2 receptor antagonist. Furthermore, over-expression of NPY3-36 in the CEA also reduced fear expression during fear extinction of CS-induced but not context-related fear. Again, fear extinction appeared delayed by peripheral injection of a Y2 receptor antagonist JNJ-31020028. Importantly, mice with over-expression of NPY3-36 in the CEA also displayed reduced spontaneous recovery and reinstatement, suggesting that Y2 receptor activation supports a permanent suppression of fear. Local deletion of Y2 receptors in the CEA, on the other hand, increased the expression of CS-induced freezing during fear recall and fear extinction. Thus, NPY inhibits fear learning and promotes cued extinction by reducing fear expression also via activation of presynaptic Y2 receptors on CEA neurons.

Enhanced analgesic effect of morphine-nimodipine combination after intraspinal administration as compared to systemic administration in mice

Calcium plays an important role in the pathophysiology of pain. A number of studies have investigated the effect of L-type calcium channel blockers on the analgesic response of morphine. However, the results are conflicting. In the present study, the antinociceptive effect of morphine (2–5 Μg) and nimodipine (1 Μg) co-administered intraspinally in mice was observed using the tail flick test. It was compared to the analgesic effect of these drugs (morphine — 250 Μg subcutaneously; nimodipine — 100 Μg intraperitoneally) after systemic administration. Nimodipine is highly lipophilic and readily crosses the blood brain barrier. Addition of nimodipine to morphine potentiated the analgesic response of the latter when administered through the intraspinal route but not when administered through systemic route. It may be due to direct inhibitory effect of morphine and nimodipine on neurons of superficial laminae of the spinal cord after binding to Μ-opioid receptors and L-type calcium channels respectively.

Pancreatic polypeptide and its central Y4 receptors are essential for cued fear extinction and permanent suppression of fear.

Avoiding danger and finding food are closely related behaviours that are essential for surviving in a natural environment. Growing evidence supports an important role of gut‐brain peptides in modulating energy homeostasis and emotional‐affective behaviour. For instance, postprandial release of pancreatic polypeptide (PP) reduced food intake and altered stress‐induced motor activity and anxiety by activating central Y4 receptors.

Neuropeptide Y2 receptors in anteroventral BNST control remote fear memory depending on extinction training

HIGHLIGHTSBoth the anterior BNST and the NPY system are critical for anxiety‐like behavior.Y2 receptors in BNSTav regulate fear extinction and remote fear memory.Y2 blockade prevents, while Y2 stimulation mimics influences of extinction on remote fear.Alterations in c‐Fos expression in BNSTav parallel changes in remote fear memory. ABSTRACT 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.

Reduced fear conditioning after viral vector mediated neuropeptide Y administration into the basolateral 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.

Hippocampal NPY Y2 receptors modulate memory depending on emotional valence and time

ABSTRACT 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. HIGHLIGHTSNPY and Y2 receptors are expressed by complementary neuronal populations of the dorsal hippocampus.Deletion of Y2 receptors increases contextual fear memory, while improving working memory and spatial memory.Re‐expression of Y2 receptors in the dorsal hippocampus of Y2KO mice reduces contextual fear but decreases spatial memory.NPY may suppress contextual fear via Y2 receptors by inhibiting hippocampal memory processing.