Dorsal Periaqueductal Gray Research Articles

Dopamine-mediated formation of a memory module in the nucleus accumbens for goal-directed navigation.

Spatial memories guide navigation efficiently toward desired destinations. However, the neuronal and circuit mechanisms underlying the encoding of goal locations and its translation into goal-directed navigation remain unclear. Here we demonstrate that mice rapidly form a spatial memory of a shelter during shelter experiences, guiding escape behavior toward the goal location-a shelter-when under threat. Dopaminergic neurons in the ventral tegmental area and their projection to the nucleus accumbens (NAc) encode safety signals associated with the shelter. Optogenetically induced phasic dopamine signals are sufficient to create a place memory that directs escape navigation. Converging dopaminergic and hippocampal glutamatergic inputs to the NAc mediate the formation of a goal-related memory within a subpopulation of NAc neurons during shelter experiences. Artificial co-activation of this goal-related NAc ensemble with neurons in the dorsal periaqueductal gray was sufficient to trigger memory-guided, rather than random, escape behavior. These findings provide causal evidence of cognitive circuit modules linking memory with goal-directed action.

Different coding characteristics between flight and freezing in dorsal periaqueductal gray of mice during exposure to innate threats.

Flight and freezing are two vital defensive behaviors that mice display to avoid natural enemies. When they are exposed to innate threats, visual cues are processed and transmitted by the visual system into the emotional nuclei and finally transmitted to the periaqueductal gray (PAG) to induce defensive behaviors. However, how the dorsal PAG (dPAG) encodes the two defensive behaviors is unclear. Multi-array electrodes were implanted in the dPAG nuclei of C57BL/6 mice. Two kinds of visual stimuli (looming and sweeping) were used to induce defensive behaviors in mice. Neural signals under different defense behaviors were recorded, and the encoding characteristics of the two behaviors were extracted and analyzed from spike firing and frequency oscillations. Finally, synchronization of neural activity during the defense process was analyzed. The neural activity between flight and freezing behaviors showed different firing patterns, and the differences in the inter-spike interval distribution were mainly reflected in the 2-10ms period. The frequency band activities under both defensive behaviors were concentrated in the theta band; the active frequency of flight was ~8 to 10Hz, whereas that of freezing behavior was ~6 to 8Hz. The network connection density under both defense behaviors was significantly higher than the period before and after defensive behavior occurred, indicating that there was a high synchronization of neural activity during the defense process. The dPAG nuclei of mice have different coding features between flight and freezing behaviors; during strong looming stimulation, fast neuro-instinctive decision making is required while encountering weak sweeping stimulation, and computable planning late behavior is predicted in the early stage. The frequency band activities under both defensive behaviors were concentrated in the theta band. There was a high synchronization of neural activity during the defense process, which may be a key factor triggering different defensive behaviors.

Imbalance of flight–freeze responses and their cellular correlates in the Nlgn3−/y rat model of autism

BackgroundMutations in the postsynaptic transmembrane protein neuroligin-3 are highly correlative with autism spectrum disorders (ASDs) and intellectual disabilities (IDs). Fear learning is well studied in models of these disorders, however differences in fear response behaviours are often overlooked. We aim to examine fear behaviour and its cellular underpinnings in a rat model of ASD/ID lacking Nlgn3.MethodsThis study uses a range of behavioural tests to understand differences in fear response behaviour in Nlgn3−/y rats. Following this, we examined the physiological underpinnings of this in neurons of the periaqueductal grey (PAG), a midbrain area involved in flight-or-freeze responses. We used whole-cell patch-clamp recordings from ex vivo PAG slices, in addition to in vivo local-field potential recordings and electrical stimulation of the PAG in wildtype and Nlgn3−/y rats. We analysed behavioural data with two- and three-way ANOVAS and electrophysiological data with generalised linear mixed modelling (GLMM).ResultsWe observed that, unlike the wildtype, Nlgn3−/y rats are more likely to response with flight rather than freezing in threatening situations. Electrophysiological findings were in agreement with these behavioural outcomes. We found in ex vivo slices from Nlgn3−/y rats that neurons in dorsal PAG (dPAG) showed intrinsic hyperexcitability compared to wildtype. Similarly, stimulating dPAG in vivo revealed that lower magnitudes sufficed to evoke flight behaviour in Nlgn3−/y than wildtype rats, indicating the functional impact of the increased cellular excitability.LimitationsOur findings do not examine what specific cell type in the PAG is likely responsible for these phenotypes. Furthermore, we have focussed on phenotypes in young adult animals, whilst the human condition associated with NLGN3 mutations appears during the first few years of life.ConclusionsWe describe altered fear responses in Nlgn3−/y rats and provide evidence that this is the result of a circuit bias that predisposes flight over freeze responses. Additionally, we demonstrate the first link between PAG dysfunction and ASD/ID. This study provides new insight into potential pathophysiologies leading to anxiety disorders and changes to fear responses in individuals with ASD.

Examination of the periaqueductal gray as a site for controlling arterial pressure in the conscious spontaneously hypertensive rat.

Our understanding of central nervous system regulation of the set-point of arterial pressure remains incomplete, especially in conditions of hypertension. The ventrolateral periaqueductal gray (vlPAG) is of particular interest given that its acute activation induces hypotension and sympatho-inhibition in anaesthetised, normotensive animals, and recent preliminary studies have shown that vlPAG stimulation can reduce blood pressure in refractory hypertensive patients. To assist our mechanistic understanding, we investigated whether electrical stimulation of the vlPAG had depressor actions in a model of neurogenic hypertension, the spontaneously hypertensive (SH) rat. We found that electrical stimulation of the lateral and vlPAG (2-6V, 20-40Hz, 0.18-0.2ms pulse width) decreased arterial pressure (-19±4mmHg, n=8) and heart rate (median-18bpm) in anaesthetised SH rats. In contrast, in conscious freely-moving SH rats fitted with blood pressure telemetry, stimulation of this same region produced failed to evoked a hypotensive response (n=13; either no change, n=9; or an increase in arterial pressure of 23±4mmHg, n=4). The hypotensive action of the vlPAG observed in anaesthetised animals has been attributed to inhibition of pre-sympathetic neurones originating in the rostral ventrolateral medulla. We therefore used an un-anaesthetised, decerebrate SH rat preparation to investigate whether activation of vlPAG neurons produced sympatho-inhibition that might be below the threshold at which a peripheral vascular response could be observed. Only sympatho-excitatory responses to electrical and excitatory amino acid microinjections were observed, and these were evoked from both the dorsal and ventral PAG; no responses were evoked from the vlPAG. We conclude that the vlPAG is not a reliable antihypertensive locus in the awake SH rat. We discuss the potential importance of the state-dependency of the hypotensive response that can be evoked from the vlPAG, which has important implications for translating to humans.

S-Ketamine Pretreatment Alleviates Anxiety-Like Behaviors and Mechanical Allodynia and Blocks the Pro-inflammatory Response in Striatum and Periaqueductal Gray From a Post-traumatic Stress Disorder Model.

This study aims to explore the regulatory effect of S-ketamine on the mechanical allodynia, anxiety-like behaviors and microglia activation in adult male rats exposed to an animal model of post-traumatic stress disorder (PTSD). The rat PTSD model was established by the exposure to single-prolonged stress (SPS), and 1 day later, rats were intraperitoneally injected with 5 mg/kg S-ketamine or normal saline, respectively. Paw withdrawal mechanical threshold was measured 2 days before, and 1, 3, 5, 7, 10, 14, 21 and 28 days after injection to assess mechanical allodynia in the SPS-exposed rats. For anxiety-like behaviors, the open field test and elevated plus maze test were performed at 7 and 14 days after S-ketamine treatment in the SPS-exposed rats, respectively. SPS-induced rats presented pronounced mechanical allodynia and anxiety-like behaviors, which were alleviated by S-ketamine treatment. After behavioral tests, rats were sacrificed for collecting the anterior cingulate cortex (ACC), prefrontal cortex (PFC), dorsal striatum, and periaqueductal gray (PAG). Protein levels of TNF-α, IL-1β, p-NF-κB, and NF-κB in brain regions were examined by Western blot. In addition, microglia activation in each brain region was determined by immunofluorescence staining of the microglia-specific biomarker Iba-1. Interestingly, pro-inflammatory cytokines were significantly upregulated in the dorsal striatum and PAG, rather than ACC and PFC. Activated microglia was observed in the dorsal striatum and PAG as well, and upregulated p-NF-κB was detected in the dorsal striatum. Inflammatory response, phosphorylation of NF-κB and microglia activation in certain brain regions were significantly alleviated by S-ketamine treatment. Collectively, S-ketamine is a promising drug in alleviating mechanical allodynia, anxiety-like behaviors, and pro-inflammatory responses in discrete brain regions in a model of PTSD.

Neural correlates of distinct levels of predatory threat in dorsal periaqueductal grey neurons.

The dorsal periaqueductal grey (PAG) is an important site for integrating predatory threats. However, it remains unclear whether predator-related activation in PAG primarily reflects threat itself and thus can distinguish between various degrees of threat, or rather reflects threat-oriented behaviours, with the PAG potentially orchestrating different types of defensive repertoire. To address this issue, we performed extracellular recording of dorsal PAG neurons in freely behaving rats and examined neuronal and behavioural responses to stimulus conditions with distinct levels of predatory threat. Animals were sequentially exposed to a nonthreatening stimulus familiar environment (exposure to habituated environment) and to a novel nonthreatening stimulus (i.e., a toy animal-plush) and to conditions with high (exposure to a live cat), intermediate (exposure to the environment just visited by the cat, with remnant predator scent), and low (exposure on the following day to the predatory context) levels of predatory threat. To test for contributions of both threat stimuli and behaviour to changes in firing rate, we applied a Poisson generalized linear model regression, using the different predator stimulus conditions and defensive repertoires as predictor variables. Analysis revealed that the different predator stimulus conditions were more predictive of changes in firing rate (primarily threat-induced increases) than the different defensive repertoires. Thus, the dorsal PAG may code for different levels of predatory threat, more than it directly orchestrates distinct threat-oriented behaviours. The present results open interesting perspectives to investigate the role of the dorsal PAG in mediating primal emotional and cognitive responses to fear-inducing stimuli.

Enhanced responsiveness to hypoxic panicogenic challenge in female rats in late diestrus is suppressed by short-term, low-dose fluoxetine: Involvement of the dorsal raphe nucleus and the dorsal periaqueductal gray.

Acute hypoxia, which is panicogenic in humans, also evokes panic-like behavior in male rats. Panic disorder is more common in women and susceptibility increases during the premenstrual phase of the cycle. We here investigated for the first time the impact of hypoxia on the expression of panic-like escape behavior by female rats and its relationship with the estrous cycle. We also evaluated functional activation of the midbrain panic circuitry in response to this panicogenic stimulus and whether short-term, low-dose fluoxetine treatment inhibits the hyper-responsiveness of females in late diestrus. Male and female Sprague Dawley rats were exposed to 7% O2. Females in late diestrus were also tested after short-term treatment with fluoxetine (1.75 or 10 mg/kg, i.p.). Brains were harvested and processed for c-Fos and tryptophan hydroxylase immunoreactivity in the periaqueductal gray matter (PAG) and dorsal raphe nucleus (DR). Acute hypoxia evoked escape in both sexes. Overall, females were more responsive than males and this is clearer in late diestrus phase. In both sexes, hypoxia induced functional activation (c-Fos expression) in non-serotonergic cells in the lateral wings of the DR and dorsomedial PAG, which was greater in late diestrus than proestrus (lowest behavioral response to hypoxia). Increased responding in late diestrus (behavioral and cellular levels) was prevented by 1.75, but not 10 mg/kg fluoxetine. The response of female rats to acute hypoxia models panic behavior in women. Low-dose fluoxetine administered in the premenstrual phase deserves further attention for management of panic disorders in women.

Blockade of GPR55 in dorsal periaqueductal gray produces anxiety-like behaviors and evocates defensive aggressive responses in alcohol-pre-exposed rats

GPR55 is a receptor expressed in several central nervous system areas, including the periaqueductal gray (PAG). Current knowledge of GPR55 physiology in PAG only covers pain integration, but it is involved in other actions such as anxiety, panic, motivated behaviors, and alcohol intake. In the present study, juvenile male Wistar rats were unexposed (alcohol-naïve group; A-naïve) or exposed to alcohol for 5 weeks (alcohol-pre-exposed group; A-pre-exposed). Posteriorly, animals received intra dorsal-PAG (D-PAG) injections of vehicle (10% DMSO), LPI (1 nmol/0.5 µl) and ML-193 (1 nmol/0.5 µl, a selective GPR55 antagonist). Finally, defensive burying behavior (DBB) paradigm and alcohol preference were evaluated. Compared to the A-naïve group, the A-pre-exposed vehicle group had higher (p < 0.05): (i) time of immobility; (ii) latency to and duration of burying; and (iii) alcohol consumption. In both groups (i.e., A-naïve and A-pre-exposed) treatment with LPI: (i) decreased duration of burying (p < 0.05); (ii) suppressed time of immobility; and (iii) increased alcohol intake (p < 0.05). On the other hand, treatment with ML-193: (i) decreased duration of immobility in A-pre-exposed (but not in A-naïve rats); (ii) promoted an aggressive response against the shock-probe in A-pre-exposed rats (p < 0.05); and (iii) increased alcohol intake (p < 0.05). Our results suggest that blockade of GPR55 in D-PAG is associated with anxiety-like behaviors, defensive aggressive behaviors, and higher alcohol intake, whereas LPI in D-PAG produced anxiolytic-like effects (probably GPR55-mediated), but not prevention of alcohol intake.

NPY-Y1 receptors in dorsal periaqueductal gray modulate anxiety, alcohol intake, and relapse in Wistar rats

Neuropeptide Y (NPY) is likely the main endogenous anxiolytic neuromodulator involved in alcohol intake. NPY-Y1, a receptor for NPY, is highly expressed in the periaqueductal gray (PAG), a mesencephalic structure involved in integrating nervous activity to the performance of active and passive defensive behaviors related to fear and anxiety. Interestingly, anxiety and fear are some of the prevailing emotional negative states during alcohol abstinence. Moreover, an inverse relationship between NPY activity and alcohol consumption has been frequently reported, mainly in the extended amygdala. Nevertheless, both the roles of NPY and that of the receptor involved in these actions have been scarcely studied. Thus, the aim of this study was to analyze the pharmacological effect of NPY and NPY-Y1 receptor blockade into the dorsal periaqueductal gray (D-PAG) in an alcohol consumption and relapse paradigm in adult male Wistar rats.Ninety-six rats at postnatal day 42 (PND-42) were classified as having low and high anxiety (LA and HA), respectively, through the elevated plus maze test (EPM). Then, those animals were randomly divided into alcohol naïve (AN) and forced alcohol consumption (FAC) groups. A cannula was implanted in D-PAG to microinject vehicle (VEH), NPY, or BIBP-3226 (a selective NPY-Y1 receptor antagonist). A defensive burying behavior test (DBB) was performed to assess the anxiety-like state during withdrawal, followed by a 24-hour free choice voluntary alcohol intake test. Under our experimental conditions, NPY microinjection decreased alcohol consumption in HA rats, whereas NPY-Y1 receptor blockade in D-PAG produced a notably anxiogenic effect and higher alcohol intake and relapse. In conclusion, NPY in the D-PAG, most likely acting on NPY-Y1 receptors, induced a significant anxiolytic effect and prominently inhibited alcohol consumption and relapse in Wistar rats.

Fatty Acid Amide Hydrolase (FAAH) Inhibition in the Dorsal Periaqueductal Gray Improves Chronic Neuropathic Pain in Rats

Neuropathic pain is a critical treatment challenge that accompanies many injury and disease states in which peripheral nerves are damaged. The periaqueductal gray (PAG) in the midbrain is an integrative region that receives sensory input and, in turn, mediates descending sensory inhibition producing antinociception. Increased endocannabinoid concentrations in the dorsal PAG (dPAG) are associated with analgesic effects in neuropathy, and recent data from our lab reveal reduced content of the endocannabinoid anandamide and upregulation of its catabolic enzyme fatty acid amide hydrolase (FAAH) in the dPAG of rats with chronic neuropathic pain, suggesting that maladaptive changes in endocannabinoid signaling in the dPAG could contribute to chronic pain. Therefore, the present study was undertaken to determine the therapeutic potential of FAAH inhibitors in the dPAG for the treatment of chronic neuropathic pain.Male and female rats (230–300 gm) underwent ligation of the tibial and peroneal branches of the sciatic nerve, leaving the sural nerve intact. Guide cannulae were implanted bilaterally targeting the dPAG in a sub‐group of injured rats. Hyperalgesia (sustained lifting/shaking/grooming after plantar pin touch) was tested prior to surgery, and on days 1,3,7,10,18 and 21 post injury. Twothirds of injured rats demonstrated a hyperalgesia response rate &gt;20% to noxious mechanical stimulation of the hindpaw on day 21 and were considered to have developed chronic hyperalgesia. In these rats, hyperalgesia testing was performed prior to, and 3hr after, administration of a FAAH inhibitor (PF‐3845 as either 2mg/kg i.p., or 0.6ng intra‐dPAG) or vehicle on days 21, 22 and 23. Values for hyperalgesia response rate at 21 days were considered as baseline prior to drug administration. Systemic administration of the FAAH inhibitor reduced the hyperalgesia response rate in both male and female rats. In males, a reduction of 50% was maintained after PF‐3845 administered i.p., while female rats demonstrated a similar consistent response with a 40% reduction in hyperalgesia behavior. A progressive decrease in hyperalgesia response rate up to 80% in males and 50% in females was observed after direct administration of PF‐3845 into the dPAG over the 3d test period. However, the analgesia subsided in the 24hr period between drug administrations for both sexes. These data suggest that A) dysregulation of the endocannabinoid system in the dPAG may contribute to chronic neuropathic pain, and B) that inhibition of the catabolic enzyme FAAH in the dPAG could have therapeutic potential by enhancing endocannabinoid signaling.Support or Funding InformationSupported by Merit Review Award # RX002747 from the U.S. Department of Veterans Affairs Rehabilitation, Research and Development Program.

Amygdala Arginine Vasopressin Modulates Chronic Ethanol Withdrawal Anxiety‐Like Behavior in the Social Interaction Task

Chronic ethanol (EtOH) exposure induces neurobehavioral maladaptations in the brain though the precise changes have not been fully explored. The centralnucleus of the amygdala (CEA) regulates anxiety-like behavior induced by withdrawal from chronic intermittent EtOH (CIE) exposure, and the arginine vasopressin (AVP) system within the CEA regulates many anxiety-like behaviors. Thus, adaptations occur in the CEA AVP system due to chronic EtOH exposure, which lead to anxiety-like behaviors in rats. Chronic exposure to a low-dose EtOH (4.5% wt/vol) induces anxiety-like behavior in rats. Wistar or Sprague Dawley rats were exposed to a modified CIE or CIE, while intra-CEA microinjections of AVP or a V1b receptor antagonist were used to elicit or block withdrawal-induced anxiety. Additionally, AVP microinjections into the CEA were given 24hours following 15days of continuous high-dose EtOH (7% wt/vol), a time period when rats no longer express anxiety. Chemogenetics was also used to activate the basolateral amygdala (BLA) or deactivate the dorsal periaqueductal gray=(dm/dlPAG) therefore PAG=periaqueductal gray to elicit or block withdrawal-induced anxiety. AVP microinjected into the CEA in lieu of exposure to the first 2 cycles of CIE was sufficient to induce anxiety-like behavior in these commonly used rat strains. The V1b receptor antagonist, but not an oxytocin receptor agonist, into the CEA during the first 2 withdrawal cycles suppressed anxiety. However, activation of the BLA in lieu of exposure to the first 2 cycles of CIE was insufficient to induce anxiety-like behavior. AVP microinjection into the CEA 24hours into withdrawal reelicited anxiety-like behavior, and deactivation of the dm/dlPAG reduced this effect of CEA AVP. Taken together, this study demonstrates a role of CEA AVP and a CEA-dm/dlPAG circuit in the development of anxiety induced by CIE. Such information is valuable for identifying novel therapeutic targets for alcohol- and anxiety-associated disorders.

The rostrodorsal periaqueductal gray influences both innate fear responses and acquisition of fear memory in animals exposed to a live predator.

A few studies have evaluated the behavioral roles of the periaqueductal gray (PAG) in animals facing ethologically relevant threats. Exposure to a live cat induces striking activation in the rostrodorsal and caudal ventral PAG. In the present investigation, we first showed that cytotoxic lesions of the rostrodorsal and caudal ventral PAG had similar effects on innate fear responses during cat exposure, practically abolishing freezing and increasing risk assessment responses. Conversely, rostrodorsal PAG lesions but not caudal ventral lesions disrupted learned contextual fear responses to cat exposure. Next, we examined how muscimol inactivation of the rostrodorsal PAG at different times (i.e., during, immediately after and 20min after cat exposure) influences learned contextual fear responses, and we found that inactivation of the rostrodorsal PAG during or immediately after cat exposure but not 20min later impaired contextual fear learning. Thus, suggesting that the rostrodorsal PAG is involved in the acquisition, but not the consolidation, of contextual fear memory to predatory threat. Notably, the dosolateral PAG contains a distinct population of neurons containing the neuronal nitric oxide synthase (nNOS) enzyme, and in the last experiment, we investigated how nitric oxide released in rostrodorsal PAG influences contextual fear memory processing. Accordingly, injection of a selective nNOS inhibitor into the rostrodorsal PAG immediately after cat exposure disrupted learned contextual responses. Overall, the present findings suggest that the acquisition of contextual fear learning is influenced by an optimum level of dorsal PAG activation, which extends from during to shortly after predator exposure and depends on local NO release.

Restricted lesions of the ventrolateral or dorsal columns of the periaqueductal gray promotes distinct effects on tonic immobility and defensive analgesia in guinea pigs.

Tonic immobility (TI) is an innate defensive response exhibited by prey when physical contact with a predator is prolonged and inescapable. This defensive response is able to activate analgesia mechanisms; this activation has adaptive value because, during an attack by a predator, the manifestation of recuperative behaviors can affect the appropriate behavioral defense strategy. Some studies have suggested that similar structures of the central nervous system can regulate the response of both TI and nociception. Thus, this study evaluated the effect of chemical lesion through the administration of ibotenic acid in restricted brain areas of the periaqueductal gray matter (PAG) in guinea pig on the TI response and nociception evaluated in the hot plate test before and after emission of TI. The data showed that an irreversible chemical lesion in the ventrolateral PAG reduced of the TI response as well as defensive antinociception. However, a lesion in the dorsal PAG blocked the defensive antinociception induced by TI but did not alter TI duration. In summary, one could hypothesize that the neural substrates responsible for defensive behavior and antinociception represent similar systems that are distinct in modulation. Thus, the ventrolateral PAG has been associated with the modulation of TI and the defensive antinociception induced by TI. In contrast, the integrity of the dorsal PAG should be necessary for defensive antinociception to occur but not to elicit TI behavior in guinea pigs.

Anatomical projections of the dorsomedial hypothalamus to the periaqueductal grey and their role in thermoregulation: a cautionary note.

The DMH is known to regulate brown adipose tissue (BAT) thermogenesis via projections to sympathetic premotor neurons in the raphe pallidus, but there is evidence that the periaqueductal gray (PAG) is also an important relay in the descending pathways regulating thermogenesis. The anatomical projections from the DMH to the PAG subdivisions and their function are largely elusive, and may differ per anterior–posterior level from bregma. We here aimed to investigate the anatomical projections from the DMH to the PAG along the entire anterior–posterior axis of the PAG, and to study the role of these projections in thermogenesis in Wistar rats. Anterograde channel rhodopsin viral tracing showed that the DMH projects especially to the dorsal and lateral PAG. Retrograde rabies viral tracing confirmed this, but also indicated that the PAG receives a diffuse input from the DMH and adjacent hypothalamic subregions. We aimed to study the role of the identified DMH to PAG projections in thermogenesis in conscious rats by specifically activating them using a combination of canine adenovirus‐2 (CAV2Cre) and Cre‐dependent designer receptor exclusively activated by designer drugs (DREADD) technology. Chemogenetic activation of DMH to PAG projections increased BAT temperature and core body temperature, but we cannot exclude the possibility that at least some thermogenic effects were mediated by adjacent hypothalamic subregions due to difficulties in specifically targeting the DMH and distinct subdivisions of the PAG because of diffuse virus expression. To conclude, our study shows the complexity of the anatomical and functional connection between the hypothalamus and the PAG, and some technical challenges in studying their connection.

Mechanism of nitric oxide and acid-sensing ion channel 1a modulation of panic-like behaviour in the dorsal periaqueductal grey of the mouse

Predators induce defensive responses and fear behaviours in prey. The rat exposure test (RET) is frequently used as an animal model of panic. Nitric oxide (NO) which has been reported to be activated by the NMDA receptor, in turn mediates calcium/calmodulin-dependent protein kinase II (CaMKII) signalling pathways in defensive responses. ACCN2, the orthologous human gene of acid-sensing ion channel 1a (ASIC1a), is also associated with panic disorder; however, few studies have focused on the role of ASIC1a in the modulation of panic and calcium/CaMKII signalling by NO. In the present study, NG-nitro-L-arginine-methyl-ester (L-NAME; non-selective NOS inhibitor), S-nitroso-N-acetyl-D,L-penicillamine (SNAP; NO donor), and psalmotoxin (PcTx-1; selective ASIC1a blocker) were administered to the dorsal periaqueductal grey (dPAG) before the predator stimulus, and the roles of NO in the expression of ASIC1a, phosphorylation of CaMKIIα (p-CaMKIIα) and expression of calmodulin (CaM) were investigated. The effects of ASIC1a, p-CaMKIIα and CaM regulation were also examined. Our results showed that intra-dPAG infusion of L-NAME weakened panic-like behaviour and decreased ASIC1a, p-CaMKIIα and CaM expression levels, whereas intra-dPAG infusion of SNAP enhanced panic-like behaviour and increased ASIC1a, p-CaMKIIα and CaM levels. Intra-dPAG infusion of PcTx-1 also weakened panic-like behaviour and decreased p-CaMKIIα expression level. Taken together, these results indicate that NO and ASIC1a are involved in the modulation of RET-induced panic-like behaviour in the dPAG. NO regulates the calcium/CaMKII signalling pathways, and ASIC1a participates in this regulation.

Daily maternal separations during stress hyporesponsive period decrease the thresholds of panic-like behaviors to electrical stimulation of the dorsal periaqueductal gray of the adult rat

The present study examined whether early life maternal separation (MS), a model of childhood separation anxiety, predisposes to panic at adulthood. For this purpose, male pups were submitted to 3-h daily maternal separations along postnatal (PN) days of either the ‘stress hyporesponsive period’ (SHRP) from PN4 to PN14 (MS11) or throughout lactation from PN2 to PN21 (MS20). Pups were further reunited to conscious (CM) or anesthetized (AM) mothers to assess the effect of mother-pup interaction upon reunion. Controls were subjected to brief handling (15 s) once a day throughout lactation (BH20). As adults (PN60), rats were tested for the thresholds to evoke panic-like behaviors upon electrical stimulation of dorsal periaqueductal gray matter and exposed to an elevated plus-maze, an open-field, a forced swim and a sucrose preference test. A factor analysis was also performed to gain insight into the meaning of behavioral tests. MS11-CM rather than MS20-CM rats showed enhanced panic responses and reductions in both swimming and sucrose preference. Panic facilitations were less intense in mother-neglected rats. Although MS did not affect anxiety, MS11-AM showed robust reductions of defecation in an open-field. Factor analysis singled out anxiety, hedonia, exploration, coping and gut activity. Although sucrose preference and coping loaded on separate factors, appetite (adult weight) correlated with active coping in both forced swim and open-field (central area exploration). Concluding, whereas 3h-daily maternal separations during SHRP increased rat’s susceptibility to experimental panic attacks, separations throughout lactation had no effects on panic and enhanced active coping.

Kindling epileptogenesis and panic-like behavior: Their bidirectional connection and contribution to epilepsy-associated depression

Anxiety is one of the most common comorbidities of epilepsy, which has major detrimental effects on the quality of life. Generalized anxiety disorder (GAD) associated with epilepsy has been receiving most attention. However, several other forms of anxiety reportedly present in patients with epilepsy, including panic disorder (PD). In this study, using an animal model of limbic epilepsy, we examined the interplay between epilepsy and panic-like behavior (PLB). Further, considering the high degree of comorbidity between depression on the one hand, and both epilepsy and PD on the other hand, we studied whether and how the presence of PLB in animals with epilepsy would affect their performance in depression-relevant tests. Fifty-day-old male Wistar rats were subjected to repeated alternating electrical stimulations of the basolateral amygdala (BLA) to induce kindling of limbic seizures, and the dorsal periaqueductal gray (DPAG) to induce panic-like episodes. Seizure susceptibility and panic reaction threshold were examined before the first and 24h after the last stimulation. At the end of the stimulations, the rats were examined in depression-relevant tests: saccharin preference test (SPT) for anhedonia and forced swimming test (FST) for despair/hopelessness. With regard to kindling, BLA+DPAG stimulation induced more profound increase of seizure susceptibility than BLA stimulation alone (evident as the reduction of the afterdischarge threshold and the increase of the afterdischarge duration). With regard to PLB, the BLA+DPAG stimulation exacerbated the severity of panic-like episodes, as compared with the DPAG stimulation alone. Basolateral amygdala stimulation alone had no effects on panic-like reactions, and DPAG stimulation alone did not modify kindling epileptogenesis. Combined stimulation of BLA and DPAG induced depressive-like behavioral impairments. This is the first experimental study showing bidirectional, mutually exacerbating effect of epilepsy and PLB, and the precipitation of depressive-like state by the epilepsy–PLB comorbidity.

B2-kinin receptors in the dorsal periaqueductal gray are implicated in the panicolytic-like effect of opiorphin

Reported results have shown that the pentapeptide opiorphin inhibits oligopeptidases that degrade brain neuropeptides, and has analgesic and antidepressant effects in experimental animals, without either tolerance or dependency after chronic administration. In a previous study we showed that opiorphin has a panicolytic-like effect in the dorsal periaqueductal gray (dPAG) electrical stimulation test (EST), mediated by the μ-opioid receptor (MOR). This study further analyzes the mechanism of opiorphin panicolytic action, using the EST and drug injection inside the dPAG. The obtained results showed that blockade of the 5-HT1A receptors with WAY-100635 did not change the escape-impairing effect of opiorphin, and combined injection of sub-effective doses of opiorphin and the 5-HT1A-agonist 8-OH-DPAT did not have a significant anti-escape effect. In contrast, the anti-escape effect of opiorphin was antagonized by pretreatment with the kinin B2 receptor blocker HOE-140, and association of sub-effective doses of opiorphin and bradykinin caused a significant anti-escape effect. The anti-escape effect of bradykinin was not affected by previous administration of WAY-100635. Therefore, the anti-escape effect of opiorphin in the dPAG seems to be mediated by endogenous bradykinin, acting on kinin B2 receptors, which previous results have shown to interact synergistically with MOR in the dPAG to restrain escape in two animal models of panic.Chemical compounds: Opiorphin (PubChem CID: 25195667); WAY100635 maleate salt (PubChem CID: 11957721); 8-OH-DPAT hydrobromide (PubChem CID: 6917794); Bradykinin (PubChem CID: 439201); HOE-140 (Icatibant) (PubChem CID: 6918173).

Panicolytic-like effect of tramadol is mediated by opioid receptors in the dorsal periaqueductal grey

Tramadol is a synthetic opioid prescribed for the treatment of moderate to severe pain, acting as agonist of μ-opioid receptors and serotonin (5-HT) and noradrenaline (NE) reuptake inhibitor. This study evaluated the effects of tramadol in rats submitted to the elevated T-maze (ETM), an animal model that evaluates behavioural parameters such as anxiety and panic. Male Wistar rats were intraperitoneally (i.p.) treated acutely with tramadol (16 and 32mg/kg) and were submitted to the ETM. Tramadol (32mg/kg) promoted a panicolytic-like effect. Considering that dorsal periaqueductal grey (dPAG) is the main brain structure related to the pathophysiology of panic disorder (PD), this study also evaluated the participation of 5-HT and opioid receptors located in the dPAG in the panicolytic-like effect of tramadol. Seven days after stereotaxic surgery for implantation of a cannula in the dPAG, the animals were submitted to the test. To assess the involvement of 5-HT1A receptors on the effect of tramadol, we combined the 5-HT1A receptor antagonist, WAY100635 (0.37nmol), microinjected intra-dPAG, 10min prior to the administration of tramadol (32mg/kg, i.p.). WAY100635 did not block the panicolytic-like effect of tramadol. We also associated the non-selective opioid receptor antagonist, naloxone, systemically (1mg/kg, i.p.) or intra-dPAG (0.5nmol) administered 10min prior to tramadol (32mg/kg, i.p.). Naloxone blocked the panicolytic-like effect of tramadol in both routes of administrations, showing that tramadol modulates acute panic defensive behaviours through its interaction with opioid receptors located in the dPAG.

μ-Opioid and 5-HT1A receptors in the dorsomedial hypothalamus interact for the regulation of panic-related defensive responses.

The dorsomedial hypothalamus (DMH) and the dorsal periaqueductal gray (DPAG) have been implicated in the genesis and regulation of panic-related defensive behaviors, such as escape. Previous results point to an interaction between serotonergic and opioidergic systems within the DPAG to inhibit escape, involving µ-opioid and 5-HT1A receptors (5-HT1AR). In the present study we explore this interaction in the DMH, using escape elicited by electrical stimulation of this area as a panic attack index. The obtained results show that intra-DMH administration of the non-selective opioid receptor antagonist naloxone (0.5 nmol) prevented the panicolytic-like effect of a local injection of serotonin (20 nmol). Pretreatment with the selective μ-opioid receptor (MOR) antagonist CTOP (1 nmol) blocked the panicolytic-like effect of the 5-HT1AR agonist 8-OHDPAT (8 nmol). Intra-DMH injection of the selective MOR agonist DAMGO (0.3 nmol) also inhibited escape behavior, and a previous injection of the 5-HT1AR antagonist WAY-100635 (0.37 nmol) counteracted this panicolytic-like effect. These results offer the first evidence that serotonergic and opioidergic systems work together within the DMH to inhibit panic-like behavior through an interaction between µ-opioid and 5-HT1A receptors, as previously described in the DPAG.