Obesity as a Potential Risk Factor for Vincristine-induced Peripheral Neuropathy.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Vincristine is a core chemotherapeutic agent for patients with ALL; unfortunately, ∼78% will develop vincristine-induced peripheral neuropathy (VIPN). VIPN can result in vincristine dose reductions that decrease therapeutic efficacy: making it important to understand which children are at highest risk for VIPN. We hypothesized that pediatric ALL patients who were obese at diagnosis would develop worse VIPN than healthy weight children with ALL within the first year. Our results confirmed that obese pediatric patients have significantly (P=0.03) worse VIPN than patients of healthy weight.

CYP3A5 genotype and its impact on vincristine pharmacokinetics and development of neuropathy in Kenyan children with cancer.

BACKGROUND: Vincristine (VCR) is a critical part of treatment in pediatric malignancies and is associated with dose-dependent peripheral neuropathy (vincristine-induced peripheral neuropathy [VIPN]). Our previous findings show VCR metabolism is regulated by the CYP3A5 gene. Individuals who are low CYP3A5 expressers metabolize VCR slower and experience more severe VIPN as compared to high expressers. Preliminary observations suggest that Caucasians experience more severe VIPN as compared to nonCaucasians. PROCEDURE: Kenyan children with cancer who were undergoing treatment including VCR were recruited for a prospective cohort study. Patients received IV VCR 2 mg/m2 /dose with a maximum dose of 2.5 mg as part of standard treatment protocols. VCR pharmacokinetics (PK) sampling was collected via dried blood spot cards and genotyping was conducted for common functional variants in CYP3A5, multi-drug resistance 1 (MDR1), and microtubule-associated protein tau (MAPT). VIPN was assessed using five neuropathy tools. RESULTS: The majority of subjects (91%) were CYP3A5 high-expresser genotype. CYP3A5 low-expresser genotype subjects had a significantly higher dose and body surface area normalized area under the curve than CYP3A5 high-expresser genotype subjects (0.28 ± 0.15 hr·m2 /l vs. 0.15 ± 0.011 hr·m2 /l, P = 0.027). Regardless of which assessment tool was utilized, minimal neuropathy was detected in this cohort. There was no difference in the presence or severity of neuropathy assessed between CYP3A5 high- and low-expresser genotype groups. CONCLUSION: Genetic factors are associated with VCR PK. Due to the minimal neuropathy observed in this cohort, there was no demonstrable association between genetic factors or VCR PK with development of VIPN. Further studies are needed to determine the role of genetic factors in optimizing dosing of VCR for maximal benefit.

Characteristics and Challenges of Providing Acupuncture and Chinese Herbal Medicine in Oncology Treatment: Report of Survey Data and Experience of Five Unique Clinical Settings.

Acupuncture is an integrative therapy with strong evidence to support its use in the oncology setting, yet barriers exist for implementation into conventional medical clinics. Though acupuncture is recommended in clinical practice guidelines for oncology, there is little data in the literature showing how acupuncture and other related therapies, including herbal medicine are successfully implemented in some oncology clinics, while others experience barriers to care. To characterize the current use of acupuncture (ACU) and herbal medicine (HM) in oncology clinics, we collected general demographic and usage data from 5 example clinics. In addition, to better understand the barriers faced by ACU and HM clinics in implementing acupuncture as a treatment modality, a survey was deployed to 2320 members of the Society for Integrative Oncology. This article examines the characteristics of oncology settings around the world, and shares data from the survey on the use of these therapies in the field of oncology. The primary barrier to acupuncture care, as reported by providers, was cost. With just under 70% of the oncologists reporting it as the most important obstacle. Additional barriers to implementation included concerns about competency and training, accessibility and safety of herbal medicine during treatment. Though acupuncture is being incorporated into more conventional oncology settings, organized strategies for implementation involving payers and policymakers is needed.

An adolescent rat model of vincristine-induced peripheral neuropathy.

Childhood acute lymphoblastic leukemia (ALL) is a significant clinical problem that can be effectively treated with vincristine, a vinca alkaloid-based chemotherapeutic agent. However, nearly all children receiving vincristine treatment develop vincristine-induced peripheral neuropathy (VIPN). The impact of adolescent vincristine treatment across the lifespan remains poorly understood. We, consequently, developed an adolescent rodent model of VIPN which can be utilized to study possible long term consequences of vincristine treatment in the developing rat. We also evaluated the therapeutic efficacy of voluntary exercise and potential impact of obesity as a genetic risk factor in this model on the development and maintenance of VIPN. Out of all the dosing regimens we evaluated, the most potent VIPN was produced by fifteen consecutive daily intraperitoneal (i.p.) vincristine injections at 100 µg/kg/day, throughout the critical period of adolescence from postnatal day 35 to 49. With this treatment, vincristine-treated animals developed hypersensitivity to mechanical and cold stimulation of the plantar hind paw surface, which outlasted the period of vincristine treatment and resolved within two weeks following the cessation of vincristine injection. By contrast, impairment in grip strength gain was delayed by vincristine treatment, emerging shortly following the termination of vincristine dosing, and persisted into early adulthood without diminishing. Interestingly, voluntary wheel running exercise prevented the development of vincristine-induced hypersensitivities to mechanical and cold stimulation. However, Zucker fa/fa obese animals did not exhibit higher risk of developing VIPN compared to lean rats. Our studies identify sensory and motor impairments produced by vincristine in adolescent animals and support the therapeutic efficacy of voluntary exercise for suppressing VIPN in developing rats.

Role of Basolateral Amygdalar Somatostatin 2 Receptors in a Rat Model of Chronic Anxiety.

Repeated exposure to stress has been implicated in inducing chronic anxiety states. Stress related increases in anxiety responses are likely mediated by activation of corticotropin-releasing factor receptors (CRFR) in the amygdala, particularly the basolateral amygdala (BLA). Within the BLA, acute injections of the CRFR agonist urocortin 1 (Ucn1) leads to acute anxiety, whereas repeated daily injections of subthreshold-doses of Ucn1 produces a long-lasting, persistent anxiety-like phenotype, a phenomenon referred to as Ucn1-priming. Relative gene expressions from the BLA of vehicle and Ucn1-primed rats were analyzed with quantitative RT-PCR using a predesigned panel of 82 neuroscience-related genes. Compared to vehicle-primed rats, only expression of the somatostatin receptor 2 gene (Sstr2) was significantly reduced in the BLA of Ucn1-primed rats. The contribution of Sstr2 on an anxiety phenotype was tested by injecting a Sstr2 antagonist into the BLA in un-primed rats. The Sstr2 antagonist increased anxiety-like behavior. Notably, pretreatment with Sstr2 agonist injected into the BLA blocked anxiety-inducing effects of acute Ucn1 BLA-injections and delayed anxiety expression during Ucn1-priming. However, concomitant Sstr2 agonist pretreatment during Ucn-1 priming did not prevent either the development of a chronic anxiety state or a reduction of BLA Sstr2 expression induced by priming. The data demonstrate that the persistent anxiety-like phenotype observed with Ucn1-priming in the BLA is associated with a selective reduction of Sstr2 gene expression. Although Sstr2 activation in the BLA blocks acute anxiogenic effects of stress and down-regulation of BLA Sstr2, it does not suppress the long-term consequences of prolonged exposure to stress-related challenges.

A Metabolomics Approach for Early Prediction of Vincristine-Induced Peripheral Neuropathy.

Vincristine is a core chemotherapeutic drug administered to pediatric acute lymphoblastic leukemia patients. Despite its efficacy in treating leukemia, it can lead to severe peripheral neuropathy in a subgroup of the patients. Peripheral neuropathy is a debilitating and painful side-effect that can severely impact an individual's quality of life. Currently, there are no established predictors of peripheral neuropathy incidence during the early stage of chemotherapeutic treatment. As a result, patients who are not susceptible to peripheral neuropathy may receive sub-therapeutic treatment due to an empirical upper cap on the dose, while others may experience severe neuropathy at the same dose. Contrary to previous genomics based approaches, we employed a metabolomics approach to identify small sets of metabolites that can be used to predict a patient's susceptibility to peripheral neuropathy at different time points during the treatment. Using those identified metabolites, we developed a novel strategy to predict peripheral neuropathy and subsequently adjust the vincristine dose accordingly. In accordance with this novel strategy, we created a free user-friendly tool, VIPNp, for physicians to easily implement our prediction strategy. Our results showed that focusing on metabolites, which encompasses both genotypic and phenotypic variations, can enable early prediction of peripheral neuropathy in pediatric leukemia patients.

Neuropeptide Y in the amygdala induces long-term resilience to stress-induced reductions in social responses but not hypothalamic-adrenal-pituitary axis activity or hyperthermia.

Resilience to mental and physical stress is a key determinant for the survival and functioning of mammals. Although the importance of stress resilience has been recognized, the underlying neural mediators have not yet been identified. Neuropeptide Y (NPY) is a peptide known for its anti-anxiety-like effects mediated via the amygdala. The results of our current study demonstrate, for the first time that repeated administration of NPY directly into the basolateral nucleus of the amygdala (BLA) produces selective stress-resilient behavioral responses to an acute restraint challenge as measured in the social interaction test, but has no effect on hypothalamic-adrenal-pituitary axis activity or stress-induced hyperthermia. More importantly, the resilient behaviors observed in the NPY-treated animals were present for up to 8 weeks. Antagonizing the activity of calcineurin, a protein phosphatase involved in neuronal remodeling and present in NPY receptor containing neurons within the BLA, blocked the development of long-term, but not the acute increases in social interaction responses induced by NPY administration. This suggests that the NPY-induced long-term behavioral resilience to restraint stress may occur via mechanisms involving neuronal plasticity. These studies suggest one putative physiologic mechanism underlying stress resilience and could identify novel targets for development of therapies that can augment the ability to cope with stress.

Genetic Variants Associated With Vincristine-Induced Peripheral Neuropathy in Two Populations of Children With Acute Lymphoblastic Leukemia.

Vincristine is one of the core chemotherapy agents used in the treatment of pediatric acute lymphoblastic leukemia (ALL). However, one of the major toxicities resulting from vincristine exposure is vincristine-induced peripheral neuropathy (VIPN). When VIPN results in significant morbidity, the vincristine dose may need to be reduced, thus potentially decreasing the effectiveness of treatment. To date, there are no robust biomarkers used clinically to determine which patients will be at risk for worse neuropathy. The current study included genomewide association study (GWAS) in two independent cohorts: Pediatric Oncology Group (POG) ALL trials and a multicenter study based at Indiana University in children with ALL. A meta-analysis of the cohorts identified two single-nucleotide polymorphisms (SNPs), rs1045644 and rs7963521, as being significantly (P value threshold 0.05/4749 = 1.05E-05) associated with neuropathy. Subsequently these SNPs may be effective biomarkers of VIPN in children with ALL.

Angiotensin-II is a putative neurotransmitter in lactate-induced panic-like responses in rats with disruption of GABAergic inhibition in the dorsomedial hypothalamus.

Intravenous sodium lactate infusions or the noradrenergic agent yohimbine reliably induce panic attacks in humans with panic disorder but not in healthy controls. However, the exact mechanism of lactate eliciting a panic attack is still unknown. In rats with chronic disruption of GABA-mediated inhibition in the dorsomedial hypothalamus (DMH), achieved by chronic microinfusion of the glutamic acid decarboxylase inhibitor L-allylglycine, sodium lactate infusions or yohimbine elicits panic-like responses (i.e., anxiety, tachycardia, hypertension, and tachypnea). In the present study, previous injections of the angiotensin-II (A-II) type 1 receptor antagonist losartan and the nonspecific A-II receptor antagonist saralasin into the DMH of "panic-prone" rats blocked the anxiety-like and physiological components of lactate-induced panic-like responses. In addition, direct injections of A-II into the DMH of these panic-prone rats also elicited panic-like responses that were blocked by pretreatment with saralasin. Microinjections of saralasin into the DMH did not block the panic-like responses elicited by intravenous infusions of the noradrenergic agent yohimbine or by direct injections of NMDA into the DMH. The presence of the A-II type 1 receptors in the region of the DMH was demonstrated using immunohistochemistry. Thus, these results implicate A-II pathways and the A-II receptors in the hypothalamus as putative substrates for sodium lactate-induced panic-like responses in vulnerable subjects.

Neonatal amygdala lesions: co-occurring impact on social/fear-related behavior and cocaine sensitization in adult rats.

Neurodevelopmental abnormalities of temporal-limbic structures may underlie both adult psychiatric syndromes and increased addiction vulnerability, leading to high frequencies of "dual diagnosis" disorders. Although the amygdala is implicated in various mental disorders and drug addiction, no studies have explored the impact of early developmental damage to the amygdala on phenotypes relating to mental illness and addictions as co-occurring processes. We tested rats with neonatal amygdala lesions (NAML) vs. SHAM-operated controls in a battery of tests--novel field activity, elevated plus maze (EPM), and social interaction (SI) at baseline and after odor and restraint stress--followed by measures of cocaine sensitization (15 mg/kg vs. saline x 5 days + challenge session 2 weeks later) and remeasurement of SI. NAMLs showed increased novelty-related locomotion, less fear responding in the EPM, and resistance to predator-odor- but not to restraint-induced suppression of SI. NAMLs also had elevated cocaine sensitization profiles, and cocaine history differentially affected subsequent SI in NAMLs compared with SHAMs. NAMLs may provide models for understanding a shared neurobiological basis for and complex interactions among psychiatric symptoms, drug exposure history, and addiction vulnerability.

From anxiety to autism: spectrum of abnormal social behaviors modeled by progressive disruption of inhibitory neuronal function in the basolateral amygdala in Wistar rats.

RATIONALE: Social behaviors are disrupted in several psychiatric disorders. The amygdala is a key brain region involved in social behaviors, and amygdala pathology has been implicated in disease states ranging from social anxiety disorder to autism. OBJECTIVE: To test the effects of progressive disruption of the inhibitory function within the basolateral nucleus of the amygdala (BLA) on conspecific social interaction in rats and investigate functional networks from the ventral medial prefrontal cortex (mPFCv) to the BLA. MATERIALS AND METHODS: BLA inhibitory tone was disrupted by priming it with the stress-peptide corticotrophin releasing factor (CRF) receptor agonist urocortin 1 (Ucn 1, 6 fmol), or by selective lesioning of a subset of BLA-GABAergic interneurons containing neurokinin 1 receptors using the targeted toxin SSP-Saporin. The effects of the disruption of GABAergic tone in the BLA were examined using a repeated exposure and habituation paradigm of social interaction (SI/h). Lesions and selectivity of lesions were confirmed postmortem. Additionally, effects of stimulating mPFCv on cFos activity in interneurons of the BLA were examined. RESULTS: Rats primed with Ucn 1 showed persistent social inhibition, which could be overcome with habituation, putatively modeling social anxiety. Rats with a selective lesioning of a subset of GABAergic interneurons in the BLA exhibited persistent social inhibition that was not reversed by SI/h paradigm. We also demonstrate selective functional inputs to this subset of interneurons when mPFCv was activated. CONCLUSIONS: These models with different gradations of disrupted BLA inhibition could help to study social dysfunction in disorders ranging from social anxiety to autism spectrum disorders.

Corticotrophin releasing factor-induced synaptic plasticity in the amygdala translates stress into emotional disorders.

The amygdala is involved in the associative processes for both appetitive and aversive emotions, and its function is modulated by stress hormones. The neuropeptide corticotrophin releasing factor (CRF) is released during stress and has been linked to many stress-related behavioral, autonomic, and endocrine responses. In the present study, nonanxiety-inducing doses of a potent CRF type 1 and 2 receptor agonist, urocortin (Ucn), was infused locally into the basolateral amygdala (BLA) of rats. After 5 daily injections of Ucn, the animals developed anxiety-like responses in behavioral tests. Intravenous administration of the anxiogenic agent sodium lactate elicited robust increases in blood pressure, respiratory rate, and heart rate. Furthermore, in the absence of any additional Ucn treatment, these behavioral and autonomic responses persisted for >30 d. Whole-cell patch-clamp recordings from BLA neurons of these hyper-reactive animals revealed a pronounced reduction in both spontaneous and stimulation-evoked IPSPs, leading to a hyperexcitability of the BLA network. This Ucn-induced plasticity appears to be dependent on NMDA receptor and subsequent calcium-calmodulin-dependent protein kinase II (CaMKII) activation, because it is blocked by pretreatment with NMDA receptor antagonists and by coadministration of CaMKII inhibitors. Our results show for the first time a stress peptide-induced behavioral syndrome that can be correlated with cellular mechanisms of neural plasticity, a novel mechanism that may explain the etiological role of stress in several chronic psychiatric and medical disorders.

Stress and central Urocortin increase anxiety-like behavior in the social interaction test via the CRF1 receptor.

Corticotropin releasing factor (CRF) and Urocortin are important neurotransmitters in the regulation of physiological and behavioral responses to stress. Centrally administered CRF or Urocortin produces anxiety-like responses in numerous animal models of anxiety disorders. Previous studies in our lab have shown that Urocortin infused into the basolateral nucleus of the amygdala produces anxiety-like responses in the social interaction test. Subsequently, in the current study we prepared a specific CRF1 receptor antagonist (N-Cyclopropylmethyl-2,5-dimethyl-N-propyl-N'-(2,4,6-trichloro-phenyl)-pyrimidine-4,6-diamine, NBI3b1996) to examine in this paradigm. This CRF1 receptor antagonist inhibited the ex vivo binding of 125I-sauvagine to rat cerebellum with an ED50 of 6 mg/kg, i.p. NBI3b1996 produced a dose-dependent antagonism of Urocortin-induced anxiety-like behavior in Social Interaction test with an ED50 of 6 mg/kg, i.p. The compound had no effect on baseline social interaction. In addition, the CRF1 receptor antagonist prevented the stress-induced decrease in social interaction. These results provide further support for the CRF1 receptor in anxiety-like behavior and suggest this pathway is quiescent in unstressed animals.

Project development teams: a novel mechanism for accelerating translational research.

The trend in conducting successful biomedical research is shifting from individual academic labs to coordinated collaborative research teams. Teams of experienced investigators with a wide variety of expertise are now critical for developing and maintaining a successful, productive research program. However, assembling a team whose members have the right expertise requires a great deal of time and many resources. To assist investigators seeking such resources, the Indiana Clinical and Translational Sciences Institute (Indiana CTSI) created the Project Development Teams (PDTs) program to support translational research on and across the Indiana University-Purdue University Indianapolis, Indiana University, Purdue University, and University of Notre Dame campuses. PDTs are multidisciplinary committees of seasoned researchers who assist investigators, at any stage of research, in transforming ideas/hypotheses into well-designed translational research projects. The teams help investigators capitalize on Indiana CTSI resources by providing investigators with, as needed, mentoring and career development; protocol development; pilot funding; institutional review board, regulatory, and/or nursing support; intellectual property support; access to institutional technology; and assistance with biostatistics, bioethics, recruiting participants, data mining, engaging community health, and collaborating with other investigators.Indiana CTSI leaders have analyzed metrics, collected since the inception of the PDT program in 2008 from both investigators and team members, and found evidence strongly suggesting that the highly responsive teams have become an important one-stop venue for facilitating productive interactions between basic and clinical scientists across four campuses, have aided in advancing the careers of junior faculty, and have helped investigators successfully obtain external funds.

The amygdala, panic disorder, and cardiovascular responses.

The amygdala is implicated in a number of emotional responses including conditioned fear and anxiety, and it appears to regulate the behavioral and autonomic responses associated with such emotional responses. The basolateral nucleus of the amygdala (BLA) is under tonic GABAergic inhibition, and acutely blocking this inhibition results in increased anxiety-like behavior, conditioned avoidance, and sympathetically mediated cardiovascular activation. By contrast, activation of the BLA with the stress-related neuropeptide corticotropin-releasing factor results in anxiety-like behavior, but not cardiovascular activation. Furthermore, repeated activation of this region with subthreshold GABA blockade or corticotropin-releasing factor-mediated excitation (priming) results in a chronic anxiety-like state, with susceptibility to panic-like arousal following intravenous lactate infusions. The chronic anxiety state appears to result from a loss of basal inhibitory drive in the BLA as a result of NMDA-dependent synaptic plasticity involving cyclic AMP and calcium calmodulin kinase II (CAM KII)-mediated changes. The lactate-induced panic-like response appears to involve angiotensin-II mediated activation of the BLA. These results suggest that the BLA has a significant role in regulating anxiety, autonomic responses, and the development of anxiety disorders.

Interactions between NPY and CRF in the amygdala to regulate emotionality.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system and currently there are four known receptor subtypes Y1, Y2, Y4 and Y5. Central NPY and its receptors have been implicated in a variety of physiological processes such as epilepsy, sleep, obesity, learning and memory, gastrointestinal regulation, alcoholism, depression and anxiety. The localization of these receptors within the brain is consistent with the roles mentioned, as they are found in varying density within the limbic structures, such as the hippocampal formation, amygdala, hypothalamus and septum. It is well understood that NPY produces anxiolytic responses following central administration under stressful or anxiety-provoking situations. In contrast, central administration of the neuropeptide corticotropin-releasing factor (CRF) produces anxiogenic behaviors. It has been proposed that NPY counteracts the effects of CRF to maintain no net change in emotional state, e.g., emotional homeostasis. In this article, we review the scientific literature describing the NPY-CRF relationship, specifically as it relates to the modulation of the CRF-mediated stress responses via the amygdala, a key forebrain structure involved in the regulation of emotional states.

Neuropeptide Y-Y2 receptors mediate anxiety in the amygdala.

The behavioral effects of direct injection of the neuropeptide Y (NPY) Y2 receptor agonist C2-NPY into the basolateral nucleus of the amygdala (BLA) was assessed in rats utilizing the social interaction test (SI). C2-NPY decreased SI time in a dose-dependent manner with a significant change observed at a dose of 80 pmol/100 nl. The anxiogenic effects produced by intra-amygdalar C2-NPY injections were reversed with intraperitoneal administration of alprazolam (1 mg/kg), a known anxiolytic. These findings support the hypothesis that Y2 receptors are involved in the regulation of the anxiety response.

NPY Y1 receptors differentially modulate GABAA and NMDA receptors via divergent signal-transduction pathways to reduce excitability of amygdala neurons.

Neuropeptide Y (NPY) administration into the basolateral amygdala (BLA) decreases anxiety-like behavior, mediated in part through the Y1 receptor (Y1R) isoform. Activation of Y1Rs results in G-protein-mediated reduction of cAMP levels, which results in reduced excitability of amygdala projection neurons. Understanding the mechanisms linking decreased cAMP levels to reduced excitability in amygdala neurons is important for identifying novel anxiolytic targets. We studied the intracellular mechanisms of activation of Y1Rs on synaptic transmission in the BLA. Activating Y1Rs by [Leu(31),Pro(34)]-NPY (L-P NPY) reduced the amplitude of evoked NMDA-mediated excitatory postsynaptic currents (eEPSCs), without affecting AMPA-mediated eEPSCs, but conversely increased the amplitude of GABAA-mediated evoked inhibitory postsynaptic currents (eIPSCs). Both effects were abolished by the Y1R antagonist, PD160170. Intracellular GDP-ß-S, or pre-treatment with either forskolin or 8Br-cAMP, eliminated the effects of L-P NPY on both NMDA- and GABAA-mediated currents. Thus, both the NMDA and GABAA effects of Y1R activation in the BLA are G-protein-mediated and cAMP-dependent. Pipette inclusion of protein kinase A (PKA) catalytic subunit blocked the effect of L-P NPY on GABAA-mediated eIPSCs, but not on NMDA-mediated eEPSCs. Conversely, activating the exchange protein activated by cAMP (Epac) with 8CPT-2Me-cAMP blocked the effect of L-P NPY on NMDA-mediated eEPSCs, but not on GABAA-mediated eIPSCs. Thus, NPY regulates amygdala excitability via two signal-transduction events, with reduced PKA activity enhancing GABAA-mediated eIPSCs and Epac deactivation reducing NMDA-mediated eEPSCs. This multipathway regulation of NMDA- and GABAA-mediated currents may be important for NPY plasticity and stress resilience in the amygdala.

Angiotensin II's role in sodium lactate-induced panic-like responses in rats with repeated urocortin 1 injections into the basolateral amygdala: amygdalar angiotensin receptors and panic.

Rats treated with three daily urocortin 1 (UCN) injections into the basolateral amygdala (BLA; i.e., UCN/BLA-primed rats) develop prolonged anxiety-associated behavior and vulnerability to panic-like physiological responses (i.e., tachycardia, hypertension and tachypnea) following intravenous infusions of 0.5 M sodium lactate (NaLac, an ordinarily mild interoceptive stressor). In these UCN-primed rats, the osmosensitive subfornical organ (SFO) may be a potential site that detects increases in plasma NaLac and mobilizes panic pathways since inhibiting the SFO blocks panic following NaLac in this model. Furthermore, since SFO neurons synthesize angiotensin II (A-II), we hypothesized that the SFO projects to the BLA and releases A-II to mobilizing panic responses in UCN/BLA-primed rats following NaLac infusions. To test this hypothesis, rats received daily bilateral injections of UCN or vehicle into the BLA daily for 3 days. Five to seven days following the intra-BLA injections, we microinjected either the nonspecific A-II type 1 (AT1r) and 2 (AT2r) receptor antagonist saralasin, or the AT2r-selective antagonist PD123319 into the BLA prior to the NaLac challenge. The UCN/BLA-primed rats pre-injected with saralasin, but not PD123319 or vehicle, had reduced NaLac-induced anxiety-associated behavior and panic-associated tachycardia and tachypnea responses. We then confirmed the presence of AT1rs in the BLA using immunohistochemistry which, combined with the previous data, suggest that A-II's panicogenic effects in the BLA is AT1r dependent. Surprisingly, the SFO had almost no neurons that directly innervate the BLA, which suggests an indirect pathway for relaying the NaLac signal. Overall these results are the first to implicate A-II and AT1rs as putative neurotransmitter-receptors in NaLac induced panic-like responses in UCN/BLA-primed rats.

Cell-specific expression of calcineurin immunoreactivity within the rat basolateral amygdala complex and colocalization with the neuropeptide Y Y1 receptor.

Neuropeptide Y (NPY) produces potent anxiolytic effects via activation of NPY Y1 receptors (Y1r) within the basolateral amygdaloid complex (BLA). The role of NPY in the BLA was recently expanded to include the ability to produce stress resilience and long-lasting reductions in anxiety-like behavior. These persistent behavioral effects are dependent upon activity of the protein phosphatase, calcineurin (CaN), which has long been associated with shaping long-term synaptic signaling. Furthermore, NPY-induced reductions in anxiety-like behavior persist months after intra-BLA delivery, which together indicate a form of neuronal plasticity had likely occurred. To define a site of action for NPY-induced CaN signaling within the BLA, we employed multi-label immunohistochemistry to determine which cell types express CaN and if CaN colocalizes with the Y1r. We have previously reported that both major neuronal cell populations in the BLA, pyramidal projection neurons and GABAergic interneurons, express the Y1r. Therefore, this current study evaluated CaN immunoreactivity in these cell types, along with Y1r immunoreactivity. Antibodies against calcium-calmodulin kinase II (CaMKII) and GABA were used to identify pyramidal neurons and GABAergic interneurons, respectively. A large population of CaN immunoreactive cells displayed Y1r immunoreactivity (90%). Nearly all (98%) pyramidal neurons displayed CaN immunoreactivity, while only a small percentage of interneurons (10%) contained CaN immunoreactivity. Overall, these anatomical findings provide a model whereby NPY could directly regulate CaN activity in the BLA via activation of the Y1r on CaN-expressing, pyramidal neurons. Importantly, they support BLA pyramidal neurons as prime targets for neuronal plasticity associated with the long-term reductions in anxiety-like behavior produced by NPY injections into the BLA.