During the past years, we have set up a pipeline to evaluate, in a layer and cell-dependent manner, the intrinsic neuronal operational properties in the iNPH biopsies. Our electrophysiological interrogations have shown that the biopsies are viable and retain the required microcircuit to study network and synaptic function in the human cortex. From all incoming iNPH shunt surgeries, we are re-slicing the biopsy for multxielectorde recordings, neuronal patch clamp, spatial transcriptomics, single cell sequencing, immunohistochemistry and electron microscopy. Moreover, any remaining piece of the tissue is stored for later proteomic/lipidomic analysis.
Since our multilectrode array (MEA) setup is equipped with perfusion system, we are able to carry out pharmacological treatments of the iNPH biopsy slices. Following the baseline recordings, we expose the slices to NMDA to elicit glutamatergic neuronal activity or carbachol to elicit cholinergic signalling. Our analysis of the MEA recordings have revealed a hyperexcitable phenotype in response to NMDA stimulation in L2/L3 neurons in biopsies with AD-related pathology. This hyper excitable phenotype was specific to layers 2/3 and was absent in the deeper layers of the cortex. This functional hyperexcitable phenotype was associated with transcriptional initiation of immediate early gene expression in the excitatory neurone reciding in layers 2/3 of the cortex of the patients that had beta-amyloid pathology in their resected biopsies. These data have been published Gazestani et al., Cell 2023. Since we have continued our collection of the biopsies, we have now generated even a larger dataset of samples. Since within this dataset the number of Tau-positive biopsies is substantial, we are re-analyzed the data to evaluate whether co-occuring tau pathology further contributes to the observed hyperexcitable phenotype upon NDMA exposure. Our pipeline was featured in Alzforum (
https://www.alzforum.org/news/research-news/fresh-brain-every-friday-biopsies-transform-alzheimers-science(se abrirá en una nueva ventana)). We demonstrated that the iNPH patient brain tissue with early AD-related pathology manifest hyperexcitability in neural circuit that is relevant to AD.
We also have established protocols to induce synaptic strengthening in these biopsies enabling us to carry out mechanistic studies on how synaptic plasticity is altered during the course of early AD pathology progression. We established a method induce synaptic strengthening using Theta Burst Stimulation (TBS) protocols on multielectrode array (MEA). Our 60-electrode array (Multichannel Systems) enables us to evaluate the capacity of the slices to exhibit potentiation spatially, which can then be correlated with the immunohistochemical stainings for the pathological protein aggregates. The TBS induced MEA-waveforms underwent preprocessing, unsupervised machine learning for quality control and advanced time series analysis. We employed feature selection and dimensionality reduction to prevent data oversimplification. Time series features, as well as potentiation features were extracted from all the waveforms. To investigate whether the presence of early AD-related pathology present in the subpopulation of the biopsies affects the waveform features, we immunostained the biopsies using antibodies against Aβ (WO2) and tau (AT180). The time-series and potentiation features of each pair of individual waveforms (pre- and post-TBS) were used to train a supervised RF classifier. We used SHapley Additive exPlanations (SHAP) values to measure which features contributed the most to differentiation between the patient groups. Interestingly, our analysis algorithm was capable of classifying correctly samples into their respective pathology groups with very high accuracy. t-SNE atlases representing the different waveforms demonstrated significant differences in the waveforms between the patients with no pathology, Aβ pathology, Aβ and tau or tau pathology only.
