Guanfacine treatment improves ADHD phenotypes of impulsivity and hyperactivity in a neurofibromatosis type 1 mouse model.

BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a mutation in one copy of the neurofibromin gene (NF1+/-). Even though approximately 40-60% of children with NF1 meet the criteria for attention deficit hyperactivity disorder (ADHD), very few preclinical studies, if any, have investigated alterations in impulsivity and risk-taking behavior. Mice with deletion of a single NF1 gene (Nf1+/-) recapitulate many of the phenotypes of NF1 patients. METHODS: We compared wild-type (WT) and Nf1+/- mouse strains to investigate differences in impulsivity and hyperactivity using the delay discounting task (DDT), cliff avoidance reaction (CAR) test, and open field. We also investigated whether treatment with the clinically effective alpha-2A adrenergic receptor agonist, guanfacine (0.3 mg/kg, i.p.), would reverse deficits observed in behavioral inhibition. RESULTS: Nf1+/- mice chose a higher percentage of smaller rewards when both 10- and 20-s delays were administered compared to WT mice, suggesting Nf1+/- mice are more impulsive. When treated with guanfacine (0.3 mg/kg, i.p.), Nf1+/- mice exhibited decreased impulsive choice by waiting for the larger, delayed reward. Nf1+/- mice also exhibited deficits in behavioral inhibition compared to WT mice in the CAR test by repetitively entering the outer edge of the platform where they risk falling. Treatment with guanfacine ameliorated these deficits. In addition, Nf1+/- mice exhibited hyperactivity as increased distance was traveled compared to WT controls in the open field. This hyperactivity in Nf1+/- mice was reduced with guanfacine pre-treatment. CONCLUSIONS: Overall, our study confirms that Nf1+/- mice exhibit deficits in behavioral inhibition in multiple contexts, a key feature of ADHD, and can be used as a model system to identify alterations in neural circuitry associated with symptoms of ADHD in children with NF1.

Panic results in unique molecular and network changes in the amygdala that facilitate fear responses.

Recurrent panic attacks (PAs) are a common feature of panic disorder (PD) and post-traumatic stress disorder (PTSD). Several distinct brain regions are involved in the regulation of panic responses, such as perifornical hypothalamus (PeF), periaqueductal gray, amygdala and frontal cortex. We have previously shown that inhibition of GABA synthesis in the PeF produces panic-vulnerable rats. Here, we investigate the mechanisms by which a panic-vulnerable state could lead to persistent fear. We first show that optogenetic activation of glutamatergic terminals from the PeF to the basolateral amygdala (BLA) enhanced the acquisition, delayed the extinction and induced the persistence of fear responses 3 weeks later, confirming a functional PeF-amygdala pathway involved in fear learning. Similar to optogenetic activation of PeF, panic-prone rats also exhibited delayed extinction. Next, we demonstrate that panic-prone rats had altered inhibitory and enhanced excitatory synaptic transmission of the principal neurons, and reduced protein levels of metabotropic glutamate type 2 receptor (mGluR2) in the BLA. Application of an mGluR2-positive allosteric modulator (PAM) reduced glutamate neurotransmission in the BLA slices from panic-prone rats. Treating panic-prone rats with mGluR2 PAM blocked sodium lactate (NaLac)-induced panic responses and normalized fear extinction deficits. Finally, in a subset of patients with comorbid PD, treatment with mGluR2 PAM resulted in complete remission of panic symptoms. These data demonstrate that a panic-prone state leads to specific reduction in mGluR2 function within the amygdala network and facilitates fear, and mGluR2 PAMs could be a targeted treatment for panic symptoms in PD and PTSD patients.

PSD95 and nNOS interaction as a novel molecular target to modulate conditioned fear: relevance to PTSD.

Stimulation of N-methyl-D-aspartic acid receptors (NMDARs) and the resulting increase of nitric oxide (NO) production are critical for fear memory formation. Following NMDAR activation, efficient production of NO requires linking the 95 kDa postsynaptic density protein (PSD95), a scaffolding protein to neuronal nitric oxide synthase (nNOS). A variety of previously studied NMDAR antagonists and NOS inhibitors can disrupt fear conditioning, but they also affect many other CNS functions such as motor activity, anxiety, and learning. We hypothesized that disrupting nNOS and PSD95 interaction in the amygdala, a critical site for fear memory formation, will reduce conditioned fear. Our results show that systemic treatment with ZL006, a compound that disrupts PSD95/nNOS binding, attenuates fear memory compared to its inactive isomer ZL007. Co-immunoprecipitation after fear conditioning showed a robust increase in the amygdala PSD95/nNOS binding, which was blocked by systemic pre-administration of ZL006. Treatment of amygdala slices with ZL006 also impaired long-term potentiation (LTP), a cellular signature of synaptic plasticity. Direct intra-amygdala infusion of ZL006 also attenuated conditioned fear. Finally, unlike NMDAR antagonist MK-801, ZL006 does not affect locomotion, social interaction, object recognition memory, and spatial memory. These findings support the hypothesis that disrupting the PSD95/nNOS interaction downstream of NMDARs selectively reduces fear memory, and highlights PSD95/nNOS interaction as a novel target for fear-related disorders, such as posttraumatic stress disorder.

Dopamine D1 receptors co-distribute with N-methyl-D-aspartic acid type-1 subunits and modulate synaptically-evoked N-methyl-D-aspartic acid currents in rat basolateral amygdala.

Activation of dopamine D1 or glutamate, N-methyl-d-aspartic acid (NMDA) receptors in the basolateral amygdala (BLA) can potently influence affective behaviors and associative learning. Physical protein-protein interactions also can occur between C-terminal peptides of D1 receptors and the NMDA-receptor subunit-1 (NR1), suggesting intracellular associations of direct relevance to dopaminergic modulation of NMDA currents. We examined this possibility by combining electron microscopic immunolabeling of the D1 and NR1 C-terminal peptides with in vitro patch-clamp recording in the rat BLA. In the in vivo preparations, D1 and NR1 were localized to the surface or endomembranes of many of the same somata and dendrites as well as a few axon terminals, including those forming asymmetric, excitatory-type synapses. In vitro analysis of physiologically characterized projection neurons revealed an excitatory response to bath application of either dopamine or the preferential D1 receptor agonist, dihydrexidine. In these neurons, dopamine also selectively reduced stimulation-evoked isolated NMDA receptor-mediated currents, but not isolated non-NMDA receptor-mediated currents or the response to exogenous NMDA application. The selective reduction of the NMDA receptor-mediated currents suggests that this effect occurs at a postsynaptic locus. Moreover, both D1 and NR1 were localized to postsynaptic surfaces of biocytin-filled and physiologically characterized projection neurons. Our results provide ultrastructural evidence for D1/NR1 endomembrane associations that may dynamically contribute to the attenuation of NMDA receptor-mediated currents following prior activation of D1 receptors in BLA projection neurons. The potential for postsynaptic cross-talk between D1 and NMDA receptors in BLA projection neurons as well as a similar interaction in presynaptic terminals could have important implications for the formation and extinction of affective memories.

Effects of preconditioning on myocardial interstitial levels of ATP and its catabolites during regional ischemia and reperfusion in the rat.

The interstitial accumulation of adenine nucleotide breakdown products (ANBP) in the myocardium during ischemia has been shown to provide a useful index of the ischemic injury, whereas reperfusion ANBP washout rate has been regarded as an index of reperfusion damage. The purpose of this study was, using cardiac microdialysis, to examine in the rat model of regional ischemia/reperfusion the relationship between the duration of ischemia and these indices and to assess the profile of interstitial ATP concentrations and the beneficial effects of ischemic preconditioning (IP). The rats underwent 10, 20, 30 or 40 min of coronary artery occlusion and 50 min of reperfusion. Regional ischemia, with its duration, provoked a progressive increase in dialysate ANBP in the ischemic zone. The rate of purine washout during reperfusion exponentially declined with an increase in duration of the ischemic period. IP, induced by three 5-min episodes of ischemia, each separated by 5 min of reperfusion, significantly reduced the accumulation of ANBP during the 30-min period of sustained ischemia and resulted in a marked acceleration of reperfusion ANBP washout, indicating the improvement of postischemic microcirculation. These effects were suggested to be, at least in part, responsible for the infarct size limitation observed. Using the relationship between the duration of ischemia and ANBP washout rate, it could be demonstrated that IP produced similar facilitation of purine washout as shortening of the ischemic period in nonpreconditioned rats from 30 to approximately 7 min. Regional 20-min ischemia induced an early peak increase in interstitial fluid ATP which correlated with the maximal incidence of ventricular arrhythmias, whereas IP abolished both ATP release and arrhythmias during the sustained ischemia. These findings suggest that ATP may be an important mediator of ischemia-induced ventricular arrhythmias.