The Acute Effects of Amyloid-Beta1–42 on Glutamatergic Receptor and Transporter Expression in the Mouse Hippocampus

Abstract

Alzheimer's disease (AD) is the leading type of dementia worldwide. Despite an increasing burden of disease due to a rapidly aging population, there is still a lack of complete understanding of the precise pathological mechanisms which drive its progression. Glutamate is the main excitatory neurotransmitter in the brain and plays an essential role in the normal function and excitability of neuronal networks. While previous studies have shown alterations in the function of the glutamatergic system in AD, the underlying etiology of beta amyloid (Aβ 1−42) induced changes has not been explored. Here we have investigated the acute effects of stereotaxic hippocampal Aβ 1−42 injection on specific glutamatergic receptors and transporters in the mouse hippocampus, using immunohistochemistry and confocal microscopy 3 days after Aβ 1−42 injection in aged male C57BL/6 mice, before the onset of neuronal cell death. We show that acute injection of Aβ 1−42 is sufficient to induce cognitive deficits 3 days post-injection. We also report no significant changes in glutamate receptor subunits GluA1, GluA2, VGluT1, and VGluT2 in response to acute injection of Aβ 1−42 when compared with the ACSF-vehicle injected mice. However, we observed increased expression in the DG hilus and ventral stratum (str.) granulosum, CA3 str. radiatum and str. oriens, and CA1 str. radiatum of the GluN1 subunit, and increased expression within the CA3 str. radiatum and decreased expression within the DG str. granulosum of the GluN2A subunit in Aβ 1−42 injected mice compared to NC, and a similar trend observed when compared to ACSF-injected mice. We also observed alterations in expression patterns of glutamatergic receptor subunits and transporters within specific layers of hippocampal subregions in response to a microinjection stimulus. These findings indicate that the pathological alterations in the glutamatergic system observed in AD are likely to be partially a result of both acute and chronic exposure to Aβ 1−42 and implies a much more complex circuit mechanism associated with glutamatergic dysfunction than simply glutamate-mediated excitotoxic neuronal death.

Amyloid-Beta1-42 -Induced Increase in GABAergic Tonic Conductance in Mouse Hippocampal CA1 Pyramidal Cells

“…Aβ 1-42 is routinely produced as a recombinant protein fused with maltose-binding protein (MBP), with a proteolytic cleavage site for Factor X protease between the two segments based on (Wilson C. MSc Thesis, University of Otago, 2007) [34,35]. This strategy utilizes the solubilizing character of the maltose-binding protein, a product of the MalE gene, to ensure expression of the soluble protein at a high concentration in Escherichia coli.…”

“…The optimal incubation time required to produce the highly toxic oligomers is 48-120 h. Western blots of an ageing profile of Aβ were analyzed both on non-dissociating gels, where the monomer decreases, and an oligomer appears by 48 h, and SDS gels where the dimer and trimer of Aβ 1-42 are seen as well as a higher molecular weight oligomer [35]. Only aggregates from misfolded Aβ 1-42 are deduced to be SDS insoluble [36], explaining why SDS gels show lower amounts with the less stably-aggregated species dissociated by the SDS [35]. Furthermore, it is very likely that the aged amyloid beta sample injected that included dimers, trimers and possible tetramers underwent further aggregation in the extracellular space following intracerebral injection.…”

“…Bilateral coordinates for stereotaxic Aβ 1-42 injection at three depths within the CA1 region of the hippocampus were determined relative to bregma (antero-posterior, −2.0 mm; medial-lateral, ±1.3 mm; dorsal-ventral, −1.9, 2.4, and 2.9 mm) according to the Paxinos and Franklin's mouse brain atlas [38]. Stereotaxic bilateral administration of 1 µL of 20 µM neurotoxic Aβ 1-42 [34,35] or ACSF as vehicle or scrAβ 1-42 (AS-25382, AnaSpec) into the CA1 region was performed at a rate of 0.1 µL/min.…”

“…The amount of time spent to explore the new object is considered as an index of recognition memory. The discrimination ratio for a novel over a familiar object was calculated as follows: time near a new object minus the time near the old object, divided by time near the new object plus the time near the old object [34,35].…”

“…Following behavioral testing, for immunohistochemistry experiments mice were deeply anesthetized with 75 mg/kg ketamine and 1 mg/kg domitor (Pfizer; New York, NY, USA) and perfused transcardially with 20 mL of ice-cold 4% paraformaldehyde in phosphate buffer, pH 7.6. Brains were removed and post-fixed in paraformaldehyde solution for 2 h at 21 • C and then incubated in 30% sucrose in Tris-phosphate saline (TBS; pH 7.6, 0.05 M Tris, 0.15 M NaCl) solution overnight at 4 • C. Four serial sets of 30 µm-thick coronal brain sections were cut using a freezing microtome [34,35,39].…”