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Development of a novel culture method for cholinergic-enriched septo-hippocampal primary neurons aimed to investigate the early, AD-relevant molecular events

Valentina Latina1, Silvia Caioli2, Cristina Zona2, 3, Maria Teresa Ciotti4, Giuseppina Amadoro1, 4* and Pietro Calissano4
  • 1 National Research Council (CNR-IFT), Italy
  • 2 IRCSS Santa Lucia Foundation, Italy
  • 3 University of Rome "Tor Vergata", Italy
  • 4 European Brain research Institute (EBRI), Italy

Introduction The basal forebrain system -including the septal region- plays an important role in memory/learning regulating the excitability of the hippocampus and neocortex and is preferentially affected in the initial stages of Alzheimer’s Disease (AD) neuropathology (1). The phenotypic downregulation of cholinergic neurons occurring at early-stages of AD and, in turn, the associated decline of their projections to cortical areas are seminal events for the cognitive dysfunction of this illness. Consistently, cholinergic neurons -which strictly depend on their survival, neuritic outgrowth and phenotype differentiation from target-derived nerve growth factor (NGF) (2,3)- are progressively reduced in affected patients in correlation with cognitive impairment (4,5,6,7). An unbalanced signaling of NGF/TrKA is causally linked to selective and extensive loss of central cholinergic functions (8,9) and NGF replacement therapy has recently emerged as a new promising disease-modifying treatment for cognitive recovery in patients with mild AD (10,11,12). However, as cholinergic neurons represent only a minor part of distinct neuronal subpopulations located in the septo-hippocampal circuit (13,14,15,16), basic and pharmacological AD research will benefit from their selective and robust enrichment in order to be studied in the absence of any other neuronal and non-neuronal contaminants. Materials and Methods Preparation of septal neuron primary cultures; Western blotting; immunofluorescence; survival assays; whole cell patch-clamp recording (mEPSCs). Results We found that the chronic treatment (10-12D.I.V.) with NGF(100ng/ml) under in vitro conditions of low 0.2%B27 supplementation selectively enriches the basal forebrain cholinergic neurons (+36.36%) at the expense of other non-cholinergic, mainly GABAergic (-38.45%) and glutamatergic (-56.25%) populations of septum. A detailed biochemical, morphological and electrophysiological characterization demonstrates that this newly-developed AD cellular model acquires properties that closely resemble those of mature NGF-responsive cholinergic neurons of central nervous system (CNS). Studies on the earliest molecular events underlying the AD onset carried out on this neuronal paradigm are under current investigation and will be submitted for future publication. Conclusions We provide an inexpensive and valuable in vitro method to obtain a consistent septo-hippocampal cholinergic population. This newly-developed culture protocol provides an unique cellular model to investigate the survival/disease changes of cholinergic projections to cortex occurring at prodromal stages of AD pathology and caused by dysfunction in NGF/TrKA signaling.

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Keywords: NGF (Nerve Growth Factor), Basal forebrain neurons, cholinergic septo-hippocampal primary cultures, Biochemistry, immunocitochemistry, Electrophysiology, Alzheimer Disease

Conference: The Cerebellum inside out: cells, circuits and functions , ERICE (Trapani), Italy, 1 Dec - 5 Dec, 2016.

Presentation Type: poster

Topic: Cellular & Molecular Neuroscience

Citation: Latina V, Caioli S, Zona C, Ciotti M, Amadoro G and Calissano P (2019). Development of a novel culture method for cholinergic-enriched septo-hippocampal primary neurons aimed to investigate the early, AD-relevant molecular events. Conference Abstract: The Cerebellum inside out: cells, circuits and functions . doi: 10.3389/conf.fncel.2017.37.000024

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Received: 16 Nov 2016; Published Online: 25 Jan 2019.

* Correspondence: Dr. Giuseppina Amadoro, National Research Council (CNR-IFT), Rome, Italy, g.amadoro@inmm.cnr.it