Functional deletion of neuropeptide Y receptors type 2 in local synaptic networks of anteroventral BNST facilitates recall and increases return of fear.

Return of previously extinguished fear memories presents a major hurdle in treatment of fear-related disorders. Neuropeptide Y receptors type 2 (Y2R) in the bed nucleus of stria terminalis (BNST) seem to play a crucial role in modulation of remote fear memories. Here, we targeted Cre-channelrhodopsin-2 to defined subregions of BNST or central amygdala (CeA) in floxed Y2R mice (Y2lox/lox) for functional deletion of Y2R. We combined fear training and behavioral studies in vivo with optogenetic-electrophysiological analysis of BNST synaptic network activity ex vivo, in order to identify regional and cellular specificities of Y2R influence. Deletion of Y2R in the ventral section of anterior BNST (BNSTav) did not affect fear acquisition, but increased conditioned fear during recall and extinction learning, and aggravated remote fear return. By contrast, deletion of Y2R in the dorsal section of anterior BNST (BNSTad) or CeA did not influence acquisition, extinction or return of fear memories. Ex vivo optogenetic-electrophysiological analysis revealed Y2R-expressing local GABAergic inhibitory networks in BNST, both within (intraregional) and in-between (inter-regional) BNST subregions. Stimulation of Y2R resulted in a presynaptically mediated reduction of GABAergic responses, which did not differ between intraregional but predominantly affected inter-regional connections from BNSTav to BNSTad. Moreover, deletion of Y2R decreased the excitation/inhibition balance in BNSTav neurons, suggesting a regulatory influence of endogenous NPY via intraregional GABAergic microcircuits. This study reveals Y2R within local GABAergic networks in BNST as key elements in facilitating extinction and reducing return of remote fear memories, suggesting a potential avenue for translational purposes.

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

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.

Measuring Locomotor Activity and Behavioral Aspects of Rodents Living in the Home-Cage.

Automatization and technological advances have led to a larger number of methods and systems to monitor and measure locomotor activity and more specific behavior of a wide variety of animal species in various environmental conditions in laboratory settings. In rodents, the majority of these systems require the animals to be temporarily taken away from their home-cage into separate observation cage environments which requires manual handling and consequently evokes distress for the animal and may alter behavioral responses. An automated high-throughput approach can overcome this problem. Therefore, this review describes existing automated methods and technologies which enable the measurement of locomotor activity and behavioral aspects of rodents in their most meaningful and stress-free laboratory environment: the home-cage. In line with the Directive 2010/63/EU and the 3R principles (replacement, reduction, refinement), this review furthermore assesses their suitability and potential for group-housed conditions as a refinement strategy, highlighting their current technological and practical limitations. It covers electrical capacitance technology and radio-frequency identification (RFID), which focus mainly on voluntary locomotor activity in both single and multiple rodents, respectively. Infrared beams and force plates expand the detection beyond locomotor activity toward basic behavioral traits but discover their full potential in individually housed rodents only. Despite the great premises of these approaches in terms of behavioral pattern recognition, more sophisticated methods, such as (RFID-assisted) video tracking technology need to be applied to enable the automated analysis of advanced behavioral aspects of individual animals in social housing conditions.

Corrigendum: Measuring Locomotor Activity and Behavioral Aspects of Rodents Living in the Home-Cage.

[This corrects the article DOI: 10.3389/fnbeh.2022.877323.].

Single stimulation of Y2 receptors in BNSTav facilitates extinction and dampens reinstatement of fear.

RATIONALE: 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. OBJECTIVE: Therefore, we investigated reinstatement of remote fear following early Y2R manipulation in BNSTav in relation to concomitant extinction training in mice. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Nimodipine is more effective than nifedipine in attenuating morphine tolerance on chronic co-administration in the rat tail-flick test.

Opioids, when co-administered with L-type calcium channel blockers (L-CCBs) show morphine like higher antinociceptive effect. This antinociceptive effect has been further investigated using a different experimental paradigm. The effect of two different L-CCBs (nifedipine and nimodipine) on morphine-induced antinociception was studied by the tail-flick test (40 min after morphine administration) in adult Wistar rats. A fixed-dose of nimodipine or nifedipine (2 mg/kg, once daily) was combined with a fixed dose of morphine (10 mg/kg, twice daily) for 10 days. Co-administration of L-CCBs significantly increased the antinociceptive effect of morphine, even 12 hr after administration. Also, nimodipine was more effective than nifedipine. Nimodipine was further studied using a higher and escalating doses of morphine (20-30 mg/kg twice daily for 14 days). Nimodipine increased the antinociceptive effect of morphine in the latter part of the study (days nine to fourteen) though significant difference was observed on 11th evening and 12th morning. No obvious adverse effects were observed in the present study. The results show for the first time that nimodipine is more effective than nifedipine and that these L-CCBs continue to be effective, even 12 hr after administration in the tail-flick test.

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

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.

Intrathecal co-administration of morphine and nimodipine produces higher antinociceptive effect by synergistic interaction as evident by injecting different doses of each drug in rats.

Earlier, we reported that morphine-nimodipine combination produces significantly higher antinociception after intrathecal but not after systemic administration in mice. Different doses of morphine and nimodipine (5 microg of morphine, 5 microg of nimodipine, 5 microg each of morphine and nimodipine, 10 microg of morphine, 10 microg of nimodipine, 10 microg morphine with 5 microg nimodipine and 5 microg of morphine with 10 microg of nimodipine) were now injected intrathecally in Wistar rats to further characterise this antinociceptive effect. The acute antinociceptive effect was measured by the tail-flick test between 15 min to 7 h. The onset of maximum antinociception (100% MPE) was earlier (by 15 min) in nimodipine (5 microg) than in morphine (5 microg) treated group (by 30 min). Though earlier in onset, 5 microg nimodipine produced transient antinociception, which was significantly higher than saline treated controls for the initial 30 min only. Morphine (5 microg) produced significantly higher antinociception between 15 min to 3:30 h in comparison to control animals. However, co-administration of both morphine and nimodipine led to significantly higher antinociception than morphine alone at 4:00 h and also between 5:00 to 6:30 h. Interestingly, the combined antinociceptive action of morphine and nimodipine was not significantly different from 10 microg of morphine, which indicated synergistic interaction. Naloxone (5 mg/kg) could reverse this antinociceptive effect of morphine-nimodipine combination though it failed to reverse nimodipine (5 microg)-mediated antinociception at 15 min. Increasing the dose of either morphine or nimodipine to 10 mug did not increase antinociception except between 6:30-7:00 h. No obvious side effect was noted after administration of either morphine or nimodipine or both.

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.

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 microg) and nimodipine (1 microg) co-administered intraspinally in mice was observed using the tail flick test. It was compared to the analgesic effect of these drugs (morphine - 250 microg subcutaneously; nimodipine - 100 microg 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 mu -opioid receptors and L-type calcium channels respectively.

Acute analgesic effect of loperamide as compared to morphine after intrathecal administration in rat.

Loperamide, a mu opioid receptor agonist, which is commonly used as an antidiarrhoeal agent has been reported to possess analgesic activity after intrathecal administration. However, the exact analgesic profile, i.e., onset, duration and intensity of analgesia in relation to morphine is not fully known. In the present study, the acute analgesic effect of loperamide (5 microg) was compared with that of morphine (5 microg) and morphine + loperamide (5 microg of each) using the tail flick method after intrathecal administration. Naloxone (5 mg/kg) reversibility of the analgesic effect was also studied. The analgesic response of loperamide was significantly higher than morphine. Even after 22 hr, maximum possible effect was greater than 49%. Naloxone partially antagonized the analgesic effect of loperamide. This suggested that loperamide may be acting through blockade of Ca2+ channels besides activating mu opioid receptors. Loperamide may prove to be a better substitute for morphine as spinal analgesic.

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.