Scott A. Heldt

Effect of 7,8-Dihydroxyflavone, a Small-Molecule TrkB Agonist, on Emotional Learning

OBJECTIVE Despite increasing awareness of the many important roles played by brain-derived neurotrophic factor (BDNF) activation of TrkB, a fuller understanding of this system and the use of potential TrkB-acting therapeutic agents has been limited by the lack of any identified small-molecule TrkB agonists that fully mimic the actions of BDNF at brain TrkB receptors in vivo. However, 7,8-dihydroxyflavone (7,8-DHF) has recently been identified as a specific TrkB agonist that crosses the blood-brain barrier after oral or intraperitoneal administration. The authors combined pharmacological, biochemical, and behavioral approaches in a preclinical study examining the role of 7,8-DHF in modulating emotional memory in mice. METHOD The authors first examined the ability of systemic 7,8-DHF to activate TrkB receptors in the amygdala. They then examined the effects of systemic 7,8-DHF on acquisition and extinction of conditioned fear, using specific and well-characterized BDNF-dependent learning paradigms in several models using naive mice and mice with prior traumatic stress exposure. RESULTS Amygdala TrkB receptors, which have previously been shown to be required for emotional learning, were activated by systemic 7,8-DHF (at 5 mg/kg i.p.). 7,8-DHF enhanced both the acquisition of fear and its extinction. It also appeared to rescue an extinction deficit in mice with a history of immobilization stress. CONCLUSIONS These data suggest that 7,8-DHF may be an excellent agent for use in understanding the effects of TrkB activation in learning and memory paradigms and may be attractive for use in reversing learning and extinction deficits associated with psychopathology.

Training-induced changes in the expression of GABAA-associated genes in the amygdala after the acquisition and extinction of Pavlovian fear

Previous work suggests the γ‐aminobutyric acid (GABA)ergic system may be dynamically regulated during emotional learning. In the current study we examined training‐induced changes in the expression of GABAA‐related genes and the binding of GABA receptor radioligands in the amygdala after the acquisition and extinction of Pavlovian fear. Using in situ hybridization, we examined the expression pattern changes of mRNAs for GABAergic markers in the lateral, basolateral and central subdivisions of the amygdala in C57Bl/6J mice. These markers included GABA‐synthesizing enzymes (GAD67 and GAD65), major GABAA receptor subunits (α1, α2, α3, α5, β2 and γ2) and the expression of mRNAs that are involved in a variety of GABA‐related intracellular processes, including GABA transporter‐1 (GAT1), GABAA receptor‐associated protein and the GABAA clustering protein, gephyrin. With fear conditioning, we found decreased mRNA levels of α1, α5 and GAD67, as well as deceased benzodiazepine binding in the amygdala. Fear extinction induced an increase in mRNA levels of α2, β2, GAD67 and gephyrin, as well as a decrease in GAT1. Together, these findings indicate that the acquisition of fear induced a downregulation of mRNA markers related to a decrease in amygdala GABAergic function, whereas the acquisition of fear extinction produced an upregulation of GABAergic markers related to enhanced GABAergic transmission.

RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)–CA3–CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2–7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG–CA3–CA1 circuit.

Prepulse Inhibition Deficits in GAD65 Knockout Mice and the Effect of Antipsychotic Treatment

Recent postmortem studies in humans suggest that defects in GABAergic neurotransmission might contribute to the neuropathology associated with schizophrenia. Disturbances in GABAergic systems may also contribute to the sensorimotor gating deficits classically observed in schizophrenic patients, including deficits in prepulse inhibition (PPI). To explore the relationship, the current study examined the integrity of PPI and startle habituation in knockout (KO) mice that lack the GABA synthesizing enzyme glutamic acid decarboxylase 65 (GAD 65). GAD65 KO mice displayed normal baseline and habituated startle responses, which did not differ from GAD65 wild-type (WT) or heterozygous (HET) mice. However, GAD65 KO mice showed robust deficits in PPI which were reversed by the atypical antipsychotic agent clozapine. These results lend support to the view that abnormalities in GABAergic systems might contribute to the basic pathophysiological mechanisms in schizophrenia.

Forebrain and midbrain distribution of major benzodiazepine-sensitive GABAA receptor subunits in the adult C57 mouse as assessed with in situ hybridization.

In the adult brain, GABA is the major inhibitory neurotransmitter. Understanding of the behavioral and pharmacological functions of GABA has been advanced by recent studies of mouse lines that possess mutations in various GABA receptor subtypes and associated proteins. Genetically altered mice have become important tools for discerning GABAergic function. Thus detailed knowledge of the anatomical distribution of different GABA(A) subtype receptors in mice is a prerequisite for understanding the neural circuitry underlying changes in normal and drug-induced behaviors seen in mutated mice. In the current study, we used in situ hybridization histochemistry with [(35)S]UTP-labeled riboprobes to examine the regional expression pattern of mRNA transcripts for seven major GABA(A) receptor subunits in adjacent coronal brain sections (alpha 1, alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2). Our results indicate that many of these GABAergic genes are co-expressed in much of the adult brain including the neocortex, hippocampus, amygdala, thalamus and striatum. However, each gene also shows a unique region-specific distribution pattern, indicative of distinct neuronal circuits that may serve specific physiological and pharmacological functions.

Lesions of the Perirhinal Cortex Interfere With Conditioned Excitation but Not With Conditioned Inhibition of Fear

Posttraining lesions of the perirhinal cortex (Prh) have been shown to interfere with the expression of fear. This study assessed whether Prh lesions would also disrupt the inhibition of fear as measured with conditioned inhibition of fear-potentiated startle. Following light + shock, noise-->light-no shock conditioned-inhibition training, rats were given Prh lesions. The lesions interfered with the expression of fear-potentiated startle to the light. To assess whether conditioned inhibition was affected, the rats were given light + retraining without additional noise-->light-training. The noise-conditioned inhibitor retained its ability to inhibit fear-potentiated startle to the retrained light. These results suggest that the areas of the Prh that are essential for the initial expression of conditioned fear are not important for the expression of conditioned inhibition of fear.

Olfactory-Mediated Fear Conditioning in Mice: Simultaneous Measurements of Fear-Potentiated Startle and Freezing

This study demonstrates that mice display olfactory-cued fear as measured with both freezing and fear-potentiated startle. Following a preconditioning test to measure any unconditioned responses to odor, mice received 5 pairings of a 10-s odor with a 0.25-s, 0.4-mA footshock. The next day, startle and freezing were measured in the presence and absence of the odor. Both fear measures increased after training with amyl acetate (Experiment 1) and acetophenone (Experiment 2). The enhancement of startle did not occur when the same number of odors and shocks were presented in an unpaired fashion (Experiment 3). Furthermore, mice were able to discriminate between an odor paired with shock and a nonreinforced odor (Experiment 4).

Potentiation of GluN2C/D NMDA Receptor Subtypes in the Amygdala Facilitates the Retention of Fear and Extinction Learning in Mice

NMDA receptors are glutamate receptor ion channels that contribute to synaptic plasticity and are important for many forms of learning and memory. In the amygdala, NMDA receptors are critical for the acquisition, retention, and extinction of classically conditioned fear responses. Although the GluN2B subunit has been implicated in both the acquisition and extinction of conditioned fear, GluN2C-knockout mice show reduced conditioned fear responses. Moreover, D-cycloserine (DCS), which facilitates fear extinction, selectively enhances the activity of GluN2C-containing NMDA receptors. To further define the contribution of GluN2C receptors to fear learning, we infused the GluN2C/GluN2D-selective potentiator CIQ bilaterally into the basolateral amygdala (3, 10, or 30 μg/side) following either fear conditioning or fear extinction training. CIQ both increased the expression of conditioned fear 24 h later and enhanced the extinction of the previously conditioned fear response. These results support a critical role for GluN2C receptors in the amygdala in the consolidation of learned fear responses and suggest that increased activity of GluN2C receptors may underlie the therapeutic actions of DCS.

Nogo-A inhibition induces recovery from neglect in rats

Neglect is a complex human cognitive spatial disorder typically induced by damage to prefrontal or posterior parietal association cortices. Behavioral treatments for neglect rarely generalize outside of the therapeutic context or across tasks within the same therapeutic context. Recovery, when it occurs, is spontaneous over the course of weeks to months, but often it is incomplete. A number of studies have indicated that anti-Nogo-A antibodies can be used to enhance plasticity and behavioral recovery following damage to motor cortex, and spinal cord. In the present studies the anti-Nogo-A antibodies IN-1, 7B12, or 11C7 were applied intraventricularly to adult rats demonstrating severe neglect produced by unilateral medial agranular cortex lesions in rats. The three separate anti-Nogo-A antibody groups were treated immediately following the medial agranular cortex lesions. Each of the three antibodies induced dramatic significant behavioral recovery from neglect relative to controls. Severing the corpus callosum to destroy inputs from the contralesional hemisphere resulted in reinstatement of severe neglect, pointing to a possible role of interhemispheric mechanisms in behavioral recovery from neglect.

Effects of light deprivation on recovery from neglect and extinction induced by unilateral lesions of the medial agranular cortex and dorsocentral striatum.

A number of previous studies have indicated that an environmental manipulation, 48 h of light deprivation (LD), produces virtually complete and permanent behavioral recovery of function from neglect induced by medial agranular cortex (AGm) lesions. LD-induced behavioral recovery from neglect is correlated with physiological changes in the dorsolateral striatum, an area that contains the projection zone of AGm efferents in the dorsocentral striatum (DCS). In this study, the behavioral effects of 48 h of LD on subjects with either unilateral DCS, AGm, or combined AGm/DCS lesions were investigated to examine whether the integrity of the DCS is crucial for behavioral recovery from neglect and whether LD will have a therapeutic effect on extinction deficits. Subjects were tested for extinction to bilateral simultaneous stimulation of the forepaws, and visual, auditory and tactile neglect. Forty-eight hours of LD failed to produce behavioral recovery from neglect in rats with DCS lesions, or a therapeutic affect on extinction deficits in any of the groups. The results of this study further support the crucial role of the DCS in recovery from neglect induced by AGm lesions and suggests that the DCS may be the crucial site for the mechanisms leading to LD-induced recovery. Further, the ineffectiveness of LD on extinction suggests that components of the neglect syndrome are dissociable and may require different therapeutic interventions.