Following the administration of tamoxifen (TAM), ΔG-rabies-mCherry virus particles (red) were injected into the dDG region of the NGI TVA/G+/+ mice for labeling presynaptic cells (red) of dNGIs (yellow). e Monosynaptic retrograde tracing strategy shows that NGI TVA/G+/+ mice were generated by crossing Nestin-Cre ER mice with TVA/G lox P/lox P mice. d The mCherry-expressing cells in the dDG region are co-labeled with anti-DCX (blue). The mCherry-expressing cells (red) in the sub-granular zone of the dDG region stained with DAPI (blue, bottom). c GFP-expressing ChATs (green) in the section stained with anti-ChAT (red) in the vDB region (top). 3 days after the injection of HSV-DIO-mCherry virus (0.2 μl), mCherry-expressing cells were detected in both the vDB (top) and dDG (red, middle and bottom) regions of ChAT-Cre GFP+/+ mice. b A brain section (top) from a ChATs-Cre GFP+/+ mouse shows GFP expression (green, top). a Monosynaptic anterograde tracing strategy shows the application of HSV-DIO-mCherry virus in ChAT-Cre GFP+/+ mice for labeling postsynaptic cells (red) of vChATs (yellow). DCX is widely established as a marker of immature neurons 18, and mCherry +DCX + cells were therefore classified as newly generated immature neurons (NGIs) in the dDG region (dNGIs). Most of these mCherry + cells expressed doublecortin (DCX, mCherry +DCX +, Fig. In the dDG, mCherry was exclusively expressed in a group of granule cells that were predominately located in the inner one-third of the granule cell layer (Fig. A bright red fluorescent signal (mCherry) was detected in the GFP-positive vChATs (GFP +mCherry +) and their direct targeting (postsynaptic) neurons in the dorsal dentate gyrus (dDG) of the adult mice (Fig. At 3 days after the injection, the brain sections were processed. A high titer (0.2 μl, 6 × 10 10 genomic particles/ml) of monosynaptic anterograde herpes simplex virus (HSV) vector that encoded a double-floxed inverted open reading frame mCherry (HSV-DIO-mCherry virus) was subsequently injected into the vDB region of the ChATs-Cre GFP+/+ mice. Staining the sections with an antibody against ChAT confirmed that Cre-GFP was expressed in ChAT neurons (Fig. We used ChATs-Cre GFP+/+ mice, in which Cre-enhanced green fluorescence protein (GFP) is expressed under the control of the ChAT promoter (Fig. In AD mice, cholinergic synaptic transmission is impaired and this impairment contributes to the loss of pattern separation-dependent spatial memory. We demonstrated that vChATs directly innervate newly generated immature neurons (NGIs) in the dorsal zone of the hippocampus (dNGIs) of adult mice. To map the specific neuronal cells that develop synaptic connections with vChATs in adult mice, we used a genetically modified Cre-dependent anterograde monosynaptic tracing system. However the hippocampus consists of diverse types of neuronal cells, including excitatory neurons and GABAergic inhibitory neurons, which of these cell types establish a direct synaptic connection with vChATs remain unknown and a role of this direct cholinergic synaptic connection in spatial learning and memory has not been previously investigated. In the hippocampus, ACh is released from axon terminals of choline acetyltransferase neurons (ChATs) in the vertical diagonal band of Broca (vDB) (vChATs) and plays a role in a range of cognitive activities, such as attention, learning and memory and consciousness 14– 17. However it is still unknown which of many thousands of synapses in the brain undergo degeneration in the early stage of AD and whether this selective degeneration contributes directly to spatial memory loss.Īcetylcholine (ACh) modifies neuronal excitability, alters pre-synaptic neurotransmitter release and coordinates the firing of groups of neurons 11– 13. In Tg2576-APPswe mice (AD mice) that carry a transgene encoding the 695-amino-acid isoform of the human Aβ precursor protein with the Swedish mutation and exhibit plaque pathologies similar to those in AD patients 8, synaptic loss in the CA1 hippocampus reduces the capability of spatial information acquisition 9, 10. In AD patients, the impairments of spatial memory correlate with a reduction of excitatory glutamatergic terminals 6, 7. However, recent studies indicate that spatial memory loss that is known as an early clinical sign of AD is due to synaptic dysfunction rather than neuronal death. Deposition of senile plaques that primarily consist of amyloid-β (Aβ) is a major pathological hallmark in the brains of Alzheimer’s disease (AD) and has long been considered to be associated with a progressive loss of central neurons 1– 5.
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