Changes in neuronal excitability and synaptic transmission in nucleus accumbens in a transgenic Alzheimer’s disease mouse model

Several previous studies showed that hippocampus and cortex are affected in Alzheimer’s disease (AD). However, other brain regions have also been found to be affected and could contribute with new critical information to the pathophysiological basis of AD. For example, volumetric studies in humans have shown a significant atrophy of the striatum, particularly in the nucleus Accumbens (nAc). The nAc is a key component of the limbic reward system and it is involved in cognition and emotional behaviors such as pleasure, fear, aggression and motivations, all of which are affected in neurodegenerative diseases such as AD. However, its role in AD has not been extensively studied. Therefore, using an AD mouse model, we investigated if the nAc was affected in 6 months old transgenic 2xTg (APP/PS1) mice. Immunohistochemistry (IHC) analysis in 2xTg mice showed increased intraneuronal Aβ accumulation, as well as occasional extracellular amyloid deposits detected through Thioflavin-S staining. Interestingly, the intracellular Aβ pathology was associated to an increase in membrane excitability in dissociated medium spiny neurons (MSNs) of the nAc. IHC and western blot analyses showed a decrease in glycine receptors (GlyR) together with a reduction in the pre- and post-synaptic markers SV2 and gephyrin, respectively, which correlated with a decrease in glycinergic miniature synaptic currents in nAc brain slices. Additionally, voltage-clamp recordings in dissociated MSNs showed a decrease in AMPA- and Gly-evoked currents. Overall, these results showed intracellular Aβ accumulation together with an increase in excitability and synaptic alterations in this mouse model. These findings provide new information that might help to explain changes in motivation, anhedonia, and learning in the onset of AD pathogenesis.

1.2. AP parameters, input resistance and rheobase calculations. Threshold was numerically estimated from first derivative in a V' versus V phase space projection.
From this value, amplitude was calculated to the maximum value reach by the AP.
Finally, we obtained the half width of the AP peak expressed as duration. Input resistance was obtained from the slopes in V/I curves in hyperpolarizing current steps. Rheobase was extrapolated from spikes vs. injected current curves using Origin 2019b (Origin Lab, USA). Spontaneous spike firing frequency was obtained using pClamp10 software (Molecular Devices, USA).
1.3. Protein extraction. The nAc was dissected from 300 μm thick slices, and the tissue was triturated in lysis buffer 1x (3.15 mM Ditiotreitol, EDTA 1 mM, protease inhibitor cocktail 1X). The solution was sonicated 3 times for 3 minutes and centrifuged for 10 minutes at 8,000 g. Then the supernatant was stored at -80 °C.

Western Blot (WB).
Tissue homogenate (50 μg protein) were subjected to electrophoresis on 10% SDS PAGE gels. Proteins were blotted onto PVDF membranes (Bio-Rad) and blocked with 5% milk in 1X TBS-0.1% Tween 20 for 1 hour with stirring. Membranes were cut according to the expected molecular weight for each of the analyzed proteins. Subsequently, each membrane was separately incubated with primary antibodies for αGlyR (1:1000, rabbit, SySys), GlyR (1:200, rabbit, Alomone), Gephyrin (1:500, mouse, SySys) and anti Gβ (1:1000, rabbit, Santa Cruz) overnight. After washes with 1X TBS and 0.1% Tween 20, membranes were incubated for 2 hours with anti-rabbit and anti-mouse secondary antibodies conjugated to HRP (1:5000, Santa Cruz). The immunoreactivity of the proteins was detected using a chemiluminescence reagent (Promega). Levels of Gβ were used as a loading control. Band intensities were analyzed and compared using 'ImageJ' 1.8.0_112 (NIH), https://imagej.nih.gov/ij. Gβ was used as loading control (b and d). From left to right lines are labeled as follows: 1-2 for WT 2 months, 3-4 for WT 12 months, 5-6 for WT 6 months and 7-8 for 2xTg 6 months (2xTg 6M). Lines 6 and 7 from a and b were used in Figure 7 and lines 6-7 from blots in c and d were used in Figure 8 (indicated in a red box). Edges of the used blots were outlined with solid black lines.