Joan Yonchek

Altered Hippocampal Circuit Function in C3H α7 Null Mutant Heterozygous Mice

The alpha7 subtype of nicotinic receptor is highly expressed in the hippocampus where it is purported to modulate release of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The alpha7 receptor-mediated release of GABA is thought to contribute to hippocampal inhibition (gating) of response to repetitive auditory stimulation. This hypothesis is supported by observations of hippocampal auditory gating deficits in mouse strains with low levels of hippocampal alpha7 receptors compared to strains with high levels of hippocampal alpha7 receptors. The difficulty with comparisons between mouse strains, however, is that different strains have different genetic backgrounds. Thus, the observed interstrain differences in hippocampal auditory gating might result from factors other than interstrain variations in the density of hippocampal alpha7 receptors. To address this issue, hippocampal binding of the alpha7 receptor-selective antagonist alpha-bungarotoxin as well as hippocampal auditory gating characteristics were compared in C3H wild type and C3H alpha7 receptor null mutant heterozygous mice. The C3H alpha7 heterozygous mice exhibited significant reductions in hippocampal alpha7 receptor levels and abnormal hippocampal auditory gating compared to the C3H wild type mice. In addition, a general increase in CA3 pyramidal neuron responsivity was observed in the heterozygous mice compared to the wild type mice. These data suggest that decreasing hippocampal alpha7 receptor density results in a profound alteration in hippocampal circuit function.

Reduced Chrna7 expression in mice is associated with decreases in hippocampal markers of inhibitory function: implications for neuropsychiatric diseases

The α7* nicotinic acetylcholine receptor encoded by CHRNA7 (human)/Chrna7 (mice) regulates the release of both the inhibitory neurotransmitter GABA and the excitatory neurotransmitter glutamate in the hippocampal formation. A heterozygous (Het) deletion at 15q13.3 containing CHRNA7 is associated with increased risk for schizophrenia, autism, and epilepsy. Each of these diseases are characterized by abnormalities in excitatory and inhibitory hippocampal circuit function. Reduced Chrna7 expression results in decreased hippocampal α7* receptor density, abnormal hippocampal auditory sensory processing, and increased hippocampal CA3 pyramidal neuron activity in C3H mice Het for a null mutation in Chrna7. These abnormalities demonstrate that decreased Chrna7 expression alters hippocampal inhibitory circuit function. The current study examined the specific impact of reduced Chrna7 expression on hippocampal inhibitory circuits by measuring the levels of GABA, GABA(A) receptors, the GABA synthetic enzyme l-glutamic acid decarboxylase-65 (GAD-65), and the vesicular GABA transporter 1 (GAT-1) in wild-type (Chrna7 +/+) and Het (Chrna7 +/-) C3H α7 mice of both genders. GAD-65 levels were significantly decreased in male and female Het C3H α7 mice, whereas GABA(A) receptors were significantly reduced only in male Het C3H α7 mice. No changes in GABA and GAT-1 levels were detected. These data suggest that reduced CHRNA7 expression may contribute to the abnormalities in hippocampal inhibitory circuits observed in schizophrenia, autism, and/or epilepsy.

A novel mouse model of pediatric cardiac arrest and cardiopulmonary resuscitation reveals age-dependent neuronal sensitivities to ischemic injury.

BACKGROUND Pediatric sudden cardiac arrest (CA) is an unfortunate and devastating condition, often leading to poor neurologic outcomes. However, little experimental data on the pathophysiology of pediatric CA is currently available due to the scarcity of animal models. NEW METHOD We developed a novel experimental model of pediatric cardiac arrest and cardiopulmonary resuscitation (CA/CPR) using postnatal day 20-25 mice. Adult (8-12 weeks) and pediatric (P20-25) mice were subjected to 6min CA/CPR. Hippocampal CA1 and striatal neuronal injury were quantified 3 days after resuscitation by hematoxylin and eosin (H&E) and Fluoro-Jade B staining, respectively. RESULTS Pediatric mice exhibited less neuronal injury in both CA1 hippocampal and striatal neurons compared to adult mice. Increasing ischemia time to 8 min CA/CPR resulted in an increase in hippocampal injury in pediatric mice, resulting in similar damage in adult and pediatric brains. In contrast, striatal injury in the pediatric brain following 6 or 8 min CA/CPR remained extremely low. As observed in adult mice, cardiac arrest causes delayed neuronal death in pediatric mice, with hippocampal CA1 neuronal damage maturing at 72 h after insult. Finally, mild therapeutic hypothermia reduced hippocampal CA1 neuronal injury after pediatric CA/CPR. COMPARISON WITH EXISTING METHOD This is the first report of a cardiac arrest and CPR model of global cerebral ischemia in mice. CONCLUSIONS Therefore, the mouse pediatric CA/CPR model we developed is unique and will provide an important new tool to the research community for the study of pediatric brain injury.

Continuous administration of a selective α7 nicotinic partial agonist, DMXBA, improves sensory inhibition without causing tachyphylaxis or receptor upregulation in DBA/2 mice

Stimulation of nicotinic receptors, specifically the alpha7 subtype, improves sensory inhibition and cognitive function in receptor deficient humans and rodents. However, stimulation with a full agonist, such as nicotine, produces rapid tachyphylaxis of the P20N40-measured sensory inhibition process. 3-(2,4-dimethoxybenzylidine) anabaseine (DMXBA, also GTS-21) selectively activates the alpha7 nicotinic receptor, and in acute administration studies, has been shown to improve deficient sensory inhibition in both humans and rodents with repeated dosing. Unlike nicotine, this partial agonist acted without inducing tachyphylaxis. Here, we assessed the ability of DMXBA to improve sensory inhibition in DBA/2 mice after 7 days of continuous administration via a subcutaneously implanted osmotic minipump. When assessed on day 8, mice receiving saline showed the characteristic deficient sensory inhibition seen with untreated DBA/2 mice. The 25- and 50-mg/ml infusion concentrations of DMXBA, but not the 100-mg/ml, produced significantly improved sensory inhibition in the mice, exclusively through a decrease in test amplitude. No concentration significantly upregulated hippocampal alpha7 receptor levels. DMXBA levels in the brain were higher than plasma at 2 of the 3 concentrations infused. These data suggest that continuous exposure to DMXBA does not significantly affect the underlying responsiveness of the sensory inhibition pathway to this partial agonist, nor cause receptor upregulation, at these relatively low brain concentrations. The ability of DMXBA to maintain its effectiveness during constant administration conditions may be due to an ability to activate alpha7 receptors at low concentrations, and consequently low fractional occupancy of the five possible binding sites on this homomeric receptor.

Development of hippocampal α7 nicotinic receptors in C3H and DBA/2 congenic mice

The time course and pattern of development of hippocampal alpha7 nicotinic acetylcholine receptors is discernibly different in C3H and DBA/2 mice. In C3H mice, the alpha7 receptor is initially expressed on embryonic day 13, exhibits an increase in density in area CA1 perinatally and is characterized by a dense, diffuse band of alpha-bungarotoxin binding at the CA3/CA1 border in the adult. In contrast, the alpha7 receptor is initially expressed on embryonic day 16 in DBA/2 mice, does not exhibit a transient perinatal increase in binding density in area CA1 and is characterized by alpha-bungarotoxin binding to numerous Nissl-stained structures in CA1 lacunosum/moleculare in the adult. Currently, it is not known whether these developmental differences occur solely as a result of the different alleles of the alpha7 receptor gene (Chrna7) expressed by the two strains or whether strain-specific background factors also play a role. The present study qualitatively examines this question by comparing alpha7 receptor development in congenic mice in which the DBA/2 allele of Chrna7 has been introgressed onto a C3H genetic background and, conversely, the C3H allele of Chrna7 has been introgressed onto a DBA/2 genetic background. The data suggest that hippocampal alpha7 receptor development is controlled predominantly by a region of mouse chromosome 7 that contains the strain-specific Chrna7 allele.

Region-specific role for GluN2B-containing NMDA receptors in injury to Purkinje cells and CA1 neurons following global cerebral ischemia

Motor deficits are present in cardiac arrest survivors and injury to cerebellar Purkinje cells (PCs) likely contribute to impairments in motor coordination and post-hypoxic myoclonus. N-Methyl-D-aspartic acid (NMDA) receptor-mediated excitotoxicity is a well-established mechanism of cell death in several brain regions, but the role of NMDA receptors in PC injury remains understudied. Emerging data in cortical and hippocampal neurons indicate that the GluN2A-containing NMDA receptors signal to improve cell survival and GluN2B-containing receptors contribute to neuronal injury. This study compared neuronal injury in the hippocampal CA1 region to that in PCs and investigated the role of NMDA receptors in PC injury in our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Analysis of cell density demonstrated a 24% loss of PCs within 24 h after 8 min CA/CPR and injury stabilized to 33% by 7 days. The subunit promiscuous NMDA receptor antagonist MK-801 protected both CA1 neurons and PCs from ischemic injury following CA/CPR, demonstrating a role for NMDA receptor activation in injury to both brain regions. In contrast, the GluN2B antagonist, Co 101244, had no effect on PC loss while protecting against injury in the CA1 region. These data indicate that ischemic injury to cerebellar PCs progresses via different cell death mechanisms compared to hippocampal CA1 neurons.

Endogenous Neuronal Replacement in the Juvenile Brain Following Cerebral Ischemia

Replacement of dead neurons following ischemia, either via enhanced endogenous neurogenesis or stem cell therapy, has long been sought. Unfortunately, while various therapies that enhance neurogenesis or stem cell therapies have proven beneficial in animal models, they have all uniformly failed to truly replace dead neurons in the ischemic core to facilitate long-term recovery. Remarkably, we observe robust repopulation of medium-spiny neurons within the ischemic core of juvenile mice following experimental stroke. Despite extensive neuronal cell death in the injured striatum of both juveniles and adults at acute time points after ischemia (24 h and 7 d), mature newborn neurons replaced lost striatal neurons at 30 d post-ischemia. This neuronal repopulation was found only in juveniles, not adults, and importantly, was accompanied by enhanced post-ischemic behavioral recovery at 30 d. Ablation of neurogenesis using irradiation prevented neuronal replacement and functional recovery in MCAo-injured juvenile mice. In contrast, findings in adults were consistent with previous reports, that newborn neurons failed to mature and died, offering little therapeutic potential. These data provide support for neuronal replacement and consequent functional recovery following ischemic stroke and new targets in the development of novel therapies to treat stroke.

Delayed inhibition of tonic inhibition enhances functional recovery following experimental ischemic stroke

The current study focuses on the ability to improve cognitive function after stroke with interventions administered at delayed/chronic time points. In light of recent studies demonstrating delayed GABA antagonists improve motor function, we utilized electrophysiology, biochemistry and neurobehavioral methods to investigate the role of α5 GABAA receptors on hippocampal plasticity and functional recovery following ischemic stroke. Male C57Bl/6 mice were exposed to 45 min transient middle cerebral artery occlusion and analysis of synaptic and functional deficits performed 7 or 30 days after recovery. Our findings indicate that hippocampal long-term potentiation (LTP) is impaired 7 days after stroke and remain impaired for at least 30 days. We demonstrate that ex vivo administration of L655,708 reversed ischemia-induced plasticity deficits and importantly, in vivo administration at delayed time-points reversed stroke-induced memory deficits. Western blot analysis of hippocampal tissue reveals proteins responsible for GABA synthesis are upregulated (GAD65/67 and MAOB), increasing GABA in hippocampal interneurons 30 days after stroke. Thus, our data indicate that both synaptic plasticity and memory impairments observed after stroke are caused by excessive tonic GABA activity, making inhibition of specific GABA activity at delayed timepoints a potential therapeutic approach to improve functional recovery and reverse cognitive impairments after stroke.