Mifepristone Prevents Stress-Induced Apoptosis in Newborn Neurons and Increases AMPA Receptor Expression in the Dentate Gyrus of C57/BL6 Mice

Chronic stress produces sustained elevation of corticosteroid levels, which is why it is considered one of the most potent negative regulators of adult hippocampal neurogenesis (AHN). Several mood disorders are accompanied by elevated glucocorticoid levels and have been linked to alterations in AHN, such as major depression (MD). Nevertheless, the mechanism by which acute stress affects the maturation of neural precursors in the dentate gyrus is poorly understood. We analyzed the survival and differentiation of 1 to 8 week-old cells in the dentate gyrus of female C57/BL6 mice following exposure to an acute stressor (the Porsolt or forced swimming test). Furthermore, we evaluated the effects of the glucocorticoid receptor (GR) antagonist mifepristone on the cell death induced by the Porsolt test. Forced swimming induced selective apoptotic cell death in 1 week-old cells, an effect that was abolished by pretreatment with mifepristone. Independent of its antagonism of GR, mifepristone also induced an increase in the percentage of 1 week-old cells that were AMPA+. We propose that the induction of AMPA receptor expression in immature cells may mediate the neuroprotective effects of mifepristone, in line with the proposed antidepressant effects of AMPA receptor potentiators

Selective Cholinergic Depletion in Medial Septum Leads to Impaired Long Term Potentiation and Glutamatergic Synaptic Currents in the Hippocampus

Cholinergic depletion in the medial septum (MS) is associated with impaired hippocampal-dependent learning and memory. Here we investigated whether long term potentiation (LTP) and synaptic currents, mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the CA1 hippocampal region, are affected following cholinergic lesions of the MS. Stereotaxic intra-medioseptal infusions of a selective immunotoxin, 192-saporin, against cholinergic neurons or sterile saline were made in adult rats. Four days after infusions, hippocampal slices were made and LTP, whole cell, and single channel (AMPA or NMDA receptor) currents were recorded. Results demonstrated impairment in the induction and expression of LTP in lesioned rats. Lesioned rats also showed decreases in synaptic currents from CA1 pyramidal cells and synaptosomal single channels of AMPA and NMDA receptors. Our results suggest that MS cholinergic afferents modulate LTP and glutamatergic currents in the CA1 region of the hippocampus, providing a potential synaptic mechanism for the learning and memory deficits observed in the rodent model of selective MS cholinergic lesioning

Single Channel Recordings From Synaptosomal AMPA Receptors

Synaptic glutamate receptors play a prominent role in the excitatory neurotransmission in the vertebrate central nervous system. Although elucidation of the functional properties of glutamate receptors using electrophysiologic analyses has yielded important information, methodological and technological limitations have prevented direct measurement of single channel properties of synaptic receptors. Here, we have isolated murine mossy fiber synaptosomes and reconstituted them into small artificial lipid bilayers to characterize the single-channel properties of synaptic α amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. The reconstituted synaptosomal receptors were activated by nanomolar concentrations of AMPA and blocked by a potent AMPA receptor antagonist. The synaptosomal AMPA receptors exhibited channel conductances of 14–56 pS and linear current-voltage relationship. The open and closed dwell time distributions of single channel currents were best described by three exponentials. These channels frequently exhibited burst behavior with long burst duration of approx 60 ms. Experiments with multichannel recordings revealed that steady state probabilities could not be fitted using a binomial distribution, indicating a cooperative channel gating behavior that would account for larger membrane currents. Our findings suggest that isolation, reconstitution into lipid bilayers, and subsequent single channel analysis of synaptosomal receptors is a useful method for investigation of synaptic AMPA receptors

Impaired ILK Function Is Associated with Deficits in Hippocampal Based Memory and Synaptic Plasticity in a FASD Rat Model.

Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term that encompasses a wide range of anatomical and behavioral problems in children who are exposed to alcohol during the prenatal period. There is no effective treatment for FASD, because of lack of complete characterization of the cellular and molecular mechanisms underlying this condition. Alcohol has been previously characterized to affect integrins and growth factor signaling receptors. Integrin Linked Kinase (ILK) is an effector of integrin and growth-factor signaling which regulates various signaling processes. In FASD, a downstream effector of ILK, Glycogen Synthase Kinase 3β (GSK3β) remains highly active (reduced Ser9 phosphorylation). GSK3β has been known to modulate glutamate receptor trafficking and channel properties. Therefore, we hypothesize that the cognitive deficits accompanying FASD are associated with impairments in the ILK signaling pathway. Pregnant Sprague Dawley rats consumed a "moderate" amount of alcohol throughout gestation, or a calorie-equivalent sucrose solution. Contextual fear conditioning was used to evaluate memory performance in 32-33-day-old pups. Synaptic plasticity was assessed in the Schaffer Collateral pathway, and hippocampal protein lysates were used to evaluate ILK signaling. Alcohol exposed pups showed impaired contextual fear conditioning, as compared to control pups. This reduced memory performance was consistent with decrease in LTP as compared to controls. Hippocampal ILK activity and GSK3β Ser21/9 phosphorylation were significantly lower in alcohol-exposed pups than controls. Increased synaptic expression of GluR2 AMPA receptors was observed with immunoprecipitation of post-synaptic density protein 95 (PSD95). Furthermore, immunoprecipitation of ILK revealed a decreased interaction with GluR2. The ILK pathway appears to play a significant role in memory and synaptic plasticity impairments in FASD rats. These impairments appear to be mediated by reduced GSK3β regulation and increased synaptic stabilization of the calcium-impermeable GluR2 AMPA receptors

Role of dimer interface in activation and desensitization in AMPA receptors

The conversion of chemical to electrical signals by the AMPA receptors is the key step by which these proteins control cognitive and motor responses. Here, we have used luminescence resonance energy transfer (LRET) to gain insight into the conformational changes induced by glutamate binding in the agonist-binding domain in functional AMPA receptors expressed in oocytes and HEK-293 cells. The LRET-based distances indicate that the interface between the upper lobes of the agonist-binding domain within a dimer is in a decoupled state in the unligated Apo state of the receptor. Agonist binding results in the formation of the dimer interface in the open-channel form of the receptor. In the continued presence of glutamate when the receptor is primarily in the desensitized state, the dimer interface is decoupled, confirming that the decoupling of the dimer interface leads to channel closure. The LRET distances also indicate that the dimer interface is preformed before activation in the L484Y mutation and also is formed in the antagonist (ZK200775)-bound form of the AMPA receptor. These results suggests that, although the preformation of the interface is not sufficient to drive channel activation, it could play a role in the energetics of activation and hence modulation of the receptor by auxiliary proteins or small molecules

Prenatal alcohol may control GluR2 protein at the synapse through ILK.

<p>(a). Immunoprecipitation (IP) with anti–PSD-95 from pooled hippocampal protein lysates of rats prenatally-exposed to alcohol and nonexposed controls (<i>n</i> = 5). In exposed rats, precipitate of GluR2 increased as compared to controls, while precipitates of GluR1and ILK did not change. The same blotting membrane was reprobed with anti–PSD-95 as a control for PSD95 pull down. The quantitation is shown adjacent to the blot image (<i>p</i> < 0.05). (b) Immunoprecipitation (IP) with anti–ILK coprecipitates GluR2 from hippocampal protein lysates of exposed and nonexposed animals (<i>n</i> = 5). There was reduced interaction in the prenatal exposed rats as compared to the nonexposed controls. The western blot analysis of immunoprecipitated ILK with anti–ILK antibody was used to confirm equal ILK immunoprecipitation. The quantitation is shown adjacent to the blot image (<i>p</i> < 0.05).</p

Prenatal alcohol impairs synaptic plasticity.

<p>(a) Input-output curves showing hippocampal basal synaptic transmission did not change in rats prenatally exposed to alcohol compared to controls. The graph shows f-EPSP amplitudes (mean ± SEM) as a function of stimulus intensity in the CA1 stratum radiatum. (b) TBS-induced LTP was recorded in hippocampal brain slices from prenatal alcohol exposed and nonexposed control rats (<i>n = 5</i>). The figure presents the time-course of percentage change in field EPSP slopes (mv/ms) with representative traces. The arrow indicates the time at which TBS protocol was delivered and 5 minute baseline is shown for clarity. Inset, representative fEPSP traces taken before the TBS (1,3) and after stabilization of LTP expression (2,4) for control and alcohol groups accordingly. (c) LTP was reduced in animals exposed to prenatal alcohol as compared to the nonexposed controls; [average for exposed animals was 162.2 ± 19.0%, and for nonexposed animals was 129.6 ± 20%, <i>F</i> = 6.217; <i>p</i> < 0.05].</p

Prenatal alcohol impairs ILK activity.

<p>(a) Western blot analysis of total GSK3β and its Ser⁹ phosphorylation state was performed in hippocampal lysates from rats prenatally exposed to alcohol and nonexposed controls (<i>n</i> = 4). There was a significant decrease in the pGSK3β/GSK3β ratio in exposed rats, suggesting increased GSK3β activity as a result of alcohol exposure (<i>p</i> < 0.05). (b) Western blot analysis of expression of ILK in brain hippocampal protein lysates from exposed and nonexposed rats (<i>n</i> = 4). There were no differences in densitometric evaluation shows in expression of ILK as a result of alcohol exposure. (c) ILK activity assay was performed with pooled hippocampal protein lysates from control and alcohol rats (<i>n</i> = 4) and Akt ser⁴⁷³ phosphorylation assessed with western blot analysis. The quantitation of band density analysis shows reduced ILK activity in alcohol-exposed animals (<i>p</i> < 0.05).</p

Schematic diagram showing the probable mechanism through which prenatal alcohol modulates ILK and affects plasticity.

<p>ILK may affect an unknown amino acid phosphorylation which may help in GluR2 receptor internalization. Reduced ILK activity may reduce the target phosphorylation and stabilize GluR2 at the surface thereby increase GluR2 at the synapse. Increased GluR2 and downstream active GSK3β may reduce LTP induction and maintenance. The probable cause of reduced ILK activity could be less mature BDNF availability in the brain of animals prenatally exposed to alcohol.</p

Prenatal alcohol exposure reduces mature BDNF expression.

<p>Western blot analysis of expression of mature BDNF and proBDNF proteins in hippocampal lysates from prenatal alcohol exposed and nonexposed rats (<i>n</i> = 4). Densitometry analysis shows that BDNF to proBDNF ratio decreased in prenatal alcohol exposed as compared to nonexposed rats (<i>p</i> < 0.05).</p