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

BACKGROUND: Chronic ethanol (EtOH) exposure induces neurobehavioral maladaptations in the brain though the precise changes have not been fully explored. The central nucleus 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. METHODS: 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 24 hours following 15 days 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. RESULTS: 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 24 hours into withdrawal reelicited anxiety-like behavior, and deactivation of the dm/dlPAG reduced this effect of CEA AVP. CONCLUSIONS: 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.

Effects of thermoneutrality on food intake, body weight, and body composition in a Prader-Willi syndrome mouse model.

OBJECTIVE: Prader-Willi syndrome (PWS) is a multisystem genetic disorder. Unfortunately, none of several mouse models carrying PWS mutations emulates the entirety of the human PWS phenotype, including hyperphagia plus obesity. METHODS: To determine whether housing at thermoneutrality (TN, 30 °C) permits the development of hyperphagia and obesity in the Snord116del PWS mouse model, the effects of housing three different ages of Snord116del and wild-type (WT) littermates at TN versus room temperature (RT, 22-24 °C) for 8 weeks were compared. RESULTS: Snord116del mice born and maintained at TN exhibited lower body weight curves, lower percentage fat mass, and lower food intake than WT mice at RT. In 4- to 6-month-old high-fat diet-fed female mice, TN raised the Snord116del body weight curve closer to that of RT-housed WT mice although the TN-housed Snord116del mice did not gain more adiposity or exhibit greater food intake. In 6- to 8-month-old high-fat diet-fed male mice, body weight, adiposity, and food intake of TN-housed Snord116del mice remained far below levels in RT-housed WT mice. TN elicited hypotonia in Snord116del adults and exacerbated mortality of Snord116del newborns. CONCLUSIONS: In none of three tested TN protocols were greater food intake, body weight, or adiposity induced in Snord116del mice compared with RT-housed WT mice.

Hemispheric differences in the number of parvalbumin-positive neurons in subdivisions of the rat basolateral amygdala complex.

The amygdala is a bilateral temporal lobe brain region which plays an important role in emotional processing. Past studies on the amygdala have shown hemispheric differences in amygdalar processes and responses associated with specific pain and fear behaviors. Despite the functional differences in the amygdala, few studies have been performed to characterize whether anatomical differences exist between the left and right amygdala. Parvalbumin (PV) is a phenotypic marker for an inhibitory interneuronal population in cortical brain structures such as the basolateral amygdala complex (BLC). This study examined the number of PV-positive neurons in the left and right BLC of adult, male Long-Evans rats using unbiased stereology. Coronal sections through the rostral-caudal extent of the BLC were immunohistochemically-stained for PV and the optical fractionator method was used to obtain an unbiased estimate of the number of PV-positive neurons in subdivisions through the BLC. The lateral and basolateral amygdala divisions of the BLC were analyzed, were subdivided into the dorsolateral, ventrolateral and ventromedial and the posterior, anterior and ventral subdivisions, respectively. The results indicate that there are significantly more PV-positive neurons in the left basolateral amygdala compared to the right, with a significant difference specifically in the posterior subdivision. This difference in PV neuronal number could help explain the distinct hemispheric roles of the BLC in the behavioral processing following exposure to painful and fearful stimuli.

A mouse model for chronic pain-induced increase in ethanol consumption.

Chronic pain conditions are often comorbid with alcohol abuse. "Self-medication" with alcohol introduces a host of problems associated with the abuse of alcohol which over time has the potential of exacerbating the painful condition. Despite the prevalence of chronic pain being associated with alcohol abuse, rodent models which mimic the comorbid conditions are lacking. In this study, we model osteoarthritis (OA) in C57BL/6J mice by surgically destabilizing the medial meniscus (DMM). Sham-operated mice served as controls. Thirteen weeks after surgery, DMM but not sham-operated mice exhibited pronounced incapacitance of the surgically manipulated hind limb compared with the nonsurgically manipulated hind limb. At this time, the mice were exposed to the 2-bottle ethanol choice, beginning with 2.5% with a gradual increasing to 20%. Compared with sham controls, DMM mice consumed more EtOH and preferred EtOH over water at the 20% EtOH concentration. Histological analysis verified that the DMM mice exhibited significant damage to the articular cartilage and osteophyte growth compared with sham controls and these measures of the severity of OA correlated with the amount of ethanol intake. Thus, the combination of the DMM model of OA with the enhanced two-bottle ethanol choice is a potential preclinical approach in mice by which the basis of the comorbid association of alcohol abuse and chronic pain conditions can be explored.

Distinct neuronal populations in the basolateral and central amygdala are activated with acute pain, conditioned fear, and fear-conditioned analgesia.

Fear-conditioned analgesia (FCA) is modulated by brain areas involved in the descending inhibitory pain pathway such as the basolateral (BLA) and central amygdala (CEA). The BLA contains Ca2+/calmodulin-dependent protein kinase II (CaMKII) and parvalbumin (PV) neurons. CEA neurons are primarily inhibitory (GABAergic) that comprise enkephalin (ENK) interneurons and corticotropin-releasing factor (CRF) - neurons that project to the periaqueductal grey. The purpose of our experiment was to determine the pattern of activation of CaMKII/PV and ENK/CRF neurons following the expression of acute pain, conditioned fear, and FCA. A significant reduction was observed in nociceptive behaviors in mice re-exposed to a contextually-aversive environment. Using NeuN and cFos as markers for activated neurons, CaMKII, PV, ENK, or CRF were used to identify neuronal subtypes. We find that mice expressing conditioned fear displayed an increase in c-Fos/CaMKII co-localization in the lateral amygdala and BLA compared to controls. Additionally a significant increase in cFos/CRF co-localization was observed in mice expressing FCA. These results show that amygdala processing of conditioned contextual aversive, nociceptive, and FCA behaviors involve different neuronal phenotypes and neural circuits between, within, and from various amygdala nuclei. This information will be important in developing novel therapies for treating pain and emotive disorders in humans.

Activation of corticotropin releasing factor-containing neurons in the rat central amygdala and bed nucleus of the stria terminalis following exposure to two different anxiogenic stressors.

Rats exposed to the odor of a predator or to the elevated plus maze (EPM) express unique unconditioned fear behaviors. The extended amygdala has previously been demonstrated to mediate the response to both predator odor and the EPM. We seek to determine if divergent amygdalar microcircuits are associated with the different behavioral responses. The current experiments compared activation of corticotropin-releasing factor (CRF)-containing neuronal populations in the central amygdala and bed nucleus of the stria terminalis (BNST) of rats exposed to either the EPM (5 min) versus home cage controls, or predator (ferret) odor versus butyric acid, or no odor (30 min). Sections of the brains were prepared for dual-labeled immunohistochemistry and counts of c-Fos co-localized with CRF were made in the centrolateral and centromedial amygdala (CLA and CMA) as well as the dorsolateral (dl), dorsomedial (dm), and ventral (v) BNST. Ferret odor-exposed rats displayed an increase in duration and a decrease in latency of defensive burying versus control rats. Exposure to both predator stress and EPM induced neuronal activation in the BNST, but not the central amygdala, and similar levels of neuronal activation were seen in both the high and low anxiety groups in the BNST after EPM exposure. Dual-labeled immunohistochemistry showed a significant increase in the percentage of CRF/c-Fos co-localization in the vBNST of ferret odor-exposed rats compared to control and butyric acid-exposed groups as well as EPM-exposed rats compared to home cage controls. In addition, an increase in the percentage of CRF-containing neurons co-localized with c-Fos was observed in the dmBNST after EPM exposure. No changes in co-localization of CRF with c-Fos was observed with these treatments in either the CLA or CMA. These results suggest that predator odor and EPM exposure activates CRF neurons in the BNST to a much greater extent than CRF neurons of the central amygdala, and indicates unconditioned anxiogenic stimuli may activate unique anatomical circuits in the extended amygdala.

Comparison of the activation of somatostatin- and neuropeptide Y-containing neuronal populations of the rat amygdala following two different anxiogenic stressors.

Rats exposed to the odor of a predator or to the elevated plus maze express fear behaviors without a prior exposure to either stimulus. The expression of innate fear provides for an ideal model of anxiety which can aid in the elucidation of brain circuits involved in anxiety-related behaviors. The current experiments compared activation of neuropeptide-containing neuronal populations in the amygdala of rats exposed to either the elevated plus maze (EPM; 5 min) versus home cage controls, or predator ferret odor versus butyric acid, or no odor (30 min). Sections of the brains were prepared for dual-labeled immunohistochemistry and counts of c-Fos co-localized with somatostatin (SOM) or neuropeptide Y (NPY) were made in the basolateral (BLA), central (CEA), medial (MEA) nuclei of the amygdala. Ferret odor and butyric acid exposure significantly decreased the percentage of SOM-positive neurons also immunoreactive for c-Fos in the anterior BLA compared to controls, whereas EPM exposure yielded a significant increase in the activation of SOM-positive neurons versus home cage controls. In the CEA, ferret odor and butyric exposure significantly decreased the percentage of SOM-positive neurons also immunoreactive for c-Fos compared to no-odor controls whereas EPM exposure yielded no change versus controls. In the MEA, both ferret odor exposure and EPM exposure resulted in increased SOM co-localized with c-Fos compared to control groups whereas NPY co-localized with c-Fos occurred following ferret odor exposure, but not EPM exposure. These results indicate that phenotypically distinct neuronal populations of the amygdala are differentially activated following exposure to different anxiogenic stimuli. These studies further elucidate the fundamental neurocircuitry of anxiety and could possibly explain the differential behavioral effects of predator versus novelty-induced stress.

Fear-induced suppression of nociceptive behaviour and activation of Akt signalling in the rat periaqueductal grey: role of fatty acid amide hydrolase.

The endocannabinoid system regulates nociception and aversion and mediates fear-conditioned analgesia (FCA). We investigated the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which inhibits the catabolism of the endocannabinoid anandamide and related N-acylethanolamines, on expression of FCA and fear and pain related behaviour per se in rats. We also examined associated alterations in the expression of the signal transduction molecule phospho-Akt in the periaqueductal grey (PAG) by immunoblotting. FCA was modelled by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. URB597 (0.3 mg/kg, i.p.) enhanced FCA and increased fear-related behaviour in formalin-treated rats. Conditioned fear per se in non-formalin-treated rats was associated with increased expression of phospho-Akt in the PAG. URB597 reduced the expression of fear-related behaviour in the early part of the trial, an effect that was accompanied by attenuation of the fear-induced increase in phospho-Akt expression in the PAG. Intra-plantar injection of formalin also reduced the fear-induced increase in phospho-Akt expression. These data provide evidence for a role of FAAH in FCA, fear responding in the presence or absence of nociceptive tone, and fear-evoked increases in PAG phospho-Akt expression. In addition, the results suggest that fear-evoked activation of Akt signalling in the PAG is abolished in the presence of nociceptive tone.

Molecular and electrophysiological changes in the prefrontal cortex-amygdala-dorsal periaqueductal grey pathway during persistent pain state and fear-conditioned analgesia.

Fear-conditioned analgesia (FCA) is the reduction in pain responding which is expressed upon re-exposure to a context previously paired with an aversive stimulus. Projections along the prefrontal cortex (PFC)-amygdala-dorsal periaqueductal grey (dPAG) pathway may mediate FCA. However, there is a paucity of studies measuring both molecular and electrophysiological changes in this pathway in rats expressing persistent pain-related behaviour or FCA. Male Lister-hooded rats, with stimulating and recording electrodes implanted in the amygdala and dPAG, respectively, either received or did not receive footshock (0.4 mA) paired with context, followed 23.5 h later by an intraplantar injection of saline or formalin (50 µL, 2.5%) into the right hindpaw. Thirty minutes post-formalin/saline, rats were re-exposed to the context for 15 min, during which pain-related behaviours were assessed in addition to evoked field potential recordings in the amygdala-dPAG pathway. Immediately after the 15-minute trial, PFC tissue was isolated for measurement of total and phosphorylated extracellular-signal regulated kinase (ERK) by western blotting. Formalin-evoked nociceptive behaviour in non-fear-conditioned rats was associated with increased field potential amplitude in the dPAG and increased relative expression of phospho-ERK in the PFC. These effects were abolished in rats expressing FCA. Fear conditioning in non-formalin treated rats was associated with increased phospho-ERK in the PFC but no change in field potential amplitude in the dPAG. Together, these data suggest differential, state-dependent alterations in electrophysiological activity and ERK phosphorylation along the PFC-amygdala-dPAG pathway during pain, conditioned fear, and FCA.

Stress-induced analgesia.

For over 30 years, scientists have been investigating the phenomenon of pain suppression upon exposure to unconditioned or conditioned stressful stimuli, commonly known as stress-induced analgesia. These studies have revealed that individual sensitivity to stress-induced analgesia can vary greatly and that this sensitivity is coupled to many different phenotypes including the degree of opioid sensitivity and startle response. Furthermore, stress-induced analgesia is influenced by age, gender, and prior experience to stressful, painful, or other environmental stimuli. Stress-induced analgesia is mediated by activation of the descending inhibitory pain pathway. Pharmacological and neurochemical studies have demonstrated involvement of a large number of neurotransmitters and neuropeptides. In particular, there are key roles for the endogenous opioid, monoamine, cannabinoid, gamma-aminobutyric acid and glutamate systems. The study of stress-induced analgesia has enhanced our understanding of the fundamental physiology of pain and stress and can be a useful approach for uncovering new therapeutic targets for the treatment of pain and stress-related disorders.

Endocannabinoid-mediated enhancement of fear-conditioned analgesia in rats: opioid receptor dependency and molecular correlates.

The opioid and endocannabinoid systems mediate analgesia expressed upon re-exposure to a contextually aversive stimulus (fear-conditioned analgesia; FCA), and modulate the mitogen-activated protein kinase (MAPK) pathway. However, an interaction between the opioid and endocannabinoid systems during FCA has not been investigated at the behavioural or molecular level. FCA was modeled in male Lister-hooded rats by assessing formalin-evoked nociceptive behaviour in an arena previously paired with footshock. Administration of the fatty acid amide hydrolase and endocannabinoid catabolism inhibitor, URB597 (0.3 mg/kg, i.p.), enhanced expression of FCA. The opioid receptor antagonist, naloxone, attenuated FCA and attenuated the URB597-induced enhancement of FCA. SR141716A (CB(1) antagonist) and SR144528 (CB(2) antagonist) also attenuated the URB597-mediated enhancement of FCA. Expression of FCA was associated with increased relative phospho-ERK2 expression in the amygdala, an effect blocked by naloxone, SR141716A, and SR144528. Furthermore, URB597-mediated enhancement of FCA was associated with reduced phospho-ERK1 and phospho-ERK2 in the amygdala. Phospho-ERK1/2 expression in the hippocampus, prefrontal cortex, and thalamus was unchanged following FCA and drug treatment. None of the drugs affected formalin-evoked nociceptive behaviour or phospho-ERK1/2 expression in non-fear-conditioned rats. These data suggest that endocannabinoid-mediated enhancement of FCA is abolished by pharmacological blockade of opioid receptors as well as CB(1) or CB(2) receptors. Both pharmacological enhancement (with URB597) and attenuation (with naloxone) of this form of endogenous analgesia were associated with reduced expression of phospho-ERK1/2 in the amygdaloid complex arguing against a causal role for ERK1/2 signaling in the amygdala during expression of FCA or its modulation by opioids or cannabinoids.

Disruption of COX-2 modulates gene expression and the cardiac injury response to doxorubicin.

To determine the role of cyclooxygenase (COX)-2 in anthracycline-induced cardiac toxicity, we administered doxorubicin (Dox) to mice with genetic disruption of COX-2 (COX-2-/-). After treatment with Dox, COX-2-/- mice had increased cardiac dysfunction and cardiac cell apoptosis compared with Dox-treated wild-type mice. The expression of the death-associated protein kinase-related apoptosis-inducing protein kinase-2 was also increased in Dox-treated COX-2-/- animals. The altered gene expression, cardiac injury, and dysfunction after Dox treatment in COX-2-/- mice was attenuated by a stable prostacyclin analog, iloprost. Wild-type mice treated with Dox developed cardiac fibrosis that was absent in COX-2-/- mice and unaffected by iloprost. These results suggest that genetic disruption of COX-2 increases the cardiac dysfunction after treatment with Dox by an increase in cardiac cell apoptosis. This Dox-induced cardiotoxicity in COX-2-/- mice was attenuated by a prostacyclin analog, suggesting a protective role for prostaglandins in this setting.