The hippocampus plays key roles in learning and memory and is a main target of Alzheimer's disease (AD), which causes progressive memory impairments. Despite numerous investigations about the processes required for the normal hippocampal functions, the neurotransmitter receptors involved in the synaptic deficits by which AD disables the hippocampus are not yet characterized. By combining histoblots, western blots, immunohistochemistry and high‐resolution immunoelectron microscopic methods for GABA_Breceptors, this study provides a quantitative description of the expression and the subcellular localization of GABA_B1in the hippocampus in a mouse model of AD at 1, 6 and 12 months of age. Western blots and histoblots showed that the total amount of protein and the laminar expression pattern of GABA_B1were similar in APP/PS1 mice and in age‐matched wild‐type mice. In contrast, immunoelectron microscopic techniques showed that the subcellular localization of GABA_B1subunit did not change significantly in APP/PS1 mice at 1 month of age, was significantly reduced in thestratum lacunosum‐moleculareof CA1 pyramidal cells at 6 months of age and significantly reduced at the membrane surface of CA1 pyramidal cells at 12 months of age. This reduction of plasma membrane GABA_B1was paralleled by a significant increase of the subunit at the intracellular sites. We further observed a decrease of membrane‐targeted GABA_Breceptors in axon terminals contacting CA1 pyramidal cells. Our data demonstrate compartment‐ and age‐dependent reduction of plasma membrane‐targeted GABA_Breceptors in the CA1 region of the hippocampus, suggesting that this decrease might be enough to alter the GABA_B‐mediated synaptic transmission taking place in AD.

The Kv4 family of voltage-gated K⁺ channels underlie the fast transient (A-type) outward K⁺ current. Although A-type currents are critical to determine somato-dendritic integration in central neurons, relatively little is known about the precise subcellular localisation of the underlying channels in hippocampal circuits. Using histoblot and immunoelectron microscopic techniques, we investigated the expression, regional distribution and subcellular localisation of Kv4.2 and Kv4.3 in the adult brain, as well as the ontogeny of their expression during postnatal development. Histoblot demonstrated that Kv4.2 and Kv4.3 proteins were widely expressed in the brain, with mostly non-overlapping patterns. During development, levels of Kv4.2 and Kv4.3 increased with age but showed marked region- and developmental stage-specific differences. Immunoelectron microscopy showed that labelling for Kv4.2 and Kv4.3 was differentially present in somato-dendritic domains of hippocampal principal cells and interneurons, including the synaptic specialisation. Quantitative analyses indicated that most immunoparticles for Kv4.2 and Kv4.3 were associated with the plasma membrane in dendritic spines and shafts, and that the two channels showed very similar distribution patterns in spines of principal cells and along the surface of granule cells. Our data shed new light on the subcellular localisation of Kv4 channels and provide evidence for their non-uniform distribution over the plasma membrane of hippocampal neurons.

Metabotropic γ-aminobutyric acid (GABA_B) receptors contribute to the control of network activity and information processing in hippocampal circuits by regulating neuronal excitability and synaptic transmission. The dysfunction in the dentate gyrus (DG) has been implicated in Alzheimer´s disease (AD). Given the involvement of GABA_B receptors in AD, to determine their subcellular localisation and possible alteration in granule cells of the DG in a mouse model of AD at 12 months of age, we used high-resolution immunoelectron microscopic analysis. Immunohistochemistry at the light microscopic level showed that the regional and cellular expression pattern of GABA_B1 was similar in an AD model mouse expressing mutated human amyloid precursor protein and presenilin1 (APP/PS1) and in age-matched wild type mice. High-resolution immunoelectron microscopy revealed a distance-dependent gradient of immunolabelling for GABA_B receptors, increasing from proximal to distal dendrites in both wild type and APP/PS1 mice. However, the overall density of GABA_B receptors at the neuronal surface of these postsynaptic compartments of granule cells was significantly reduced in APP/PS1 mice. Parallel to this reduction in surface receptors, we found a significant increase in GABA_B1 at cytoplasmic sites. GABA_B receptors were also detected at presynaptic sites in the molecular layer of the DG. We also found a decrease in plasma membrane GABA_B receptors in axon terminals contacting dendritic spines of granule cells, which was more pronounced in the outer than in the inner molecular layer. Altogether, our data showing post- and presynaptic reduction in surface GABA_B receptors in the DG suggest the alteration of the GABA_B-mediated modulation of excitability and synaptic transmission in granule cells, which may contribute to the cognitive dysfunctions in the APP/PS1 model of AD.

G protein-gated inwardly rectifying K⁺ (GIRK) channels are the main targets controlling excitability and synaptic plasticity on hippocampal neurons. Consequently, dysfunction of GIRK-mediated signalling has been implicated in the pathophysiology of Alzheimer´s disease (AD). Here, we provide a quantitative description on the expression and localisation patterns of GIRK2 in two transgenic mice models of AD (P301S and APP/PS1 mice), combining histoblots and immunoelectron microscopic approaches. The histoblot technique revealed differences in the expression of GIRK2 in the two transgenic mice models. The expression of GIRK2 was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered in APP/PS1 mice at 12 months compared to age-matched wild type mice. Ultrastructural approaches using the pre-embedding immunogold technique, demonstrated that the subcellular localisation of GIRK2 was significantly reduced along the neuronal surface of CA1 pyramidal cells, but increased in its frequency at cytoplasmic sites, in both P301S and APP/PS1 mice. We also found a decrease in plasma membrane GIRK2 channels in axon terminals contacting dendritic spines of CA1 pyramidal cells in P301S and APP/PS1 mice. These data demonstrate for the first time a redistribution of GIRK channels from the plasma membrane to intracellular sites in different compartments of CA1 pyramidal cells. Altogether, the pre- and post-synaptic reduction of GIRK2 channels suggest that GIRK-mediated alteration of the excitability in pyramidal cells could contribute to the cognitive dysfunctions as described in the two AD animal models.

The small-conductance, Ca²⁺-activated K⁺ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca²⁺ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca²⁺: Ca_V2.1 channels and mGlu_1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for Ca_V2.1 channels and mGlu_1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to Ca_V2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca²⁺, and suggest that the ultrastructural association of SK2 with Ca_V2.1 and mGlu_1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca²⁺ transients in PCs.

Alzheimer’s disease (AD) is characterized by a reorganization of brain activity determining network hyperexcitability and loss of synaptic plasticity. Precisely, a dysfunction in metabotropic GABA_B receptor signalling through G protein-gated inwardly rectifying K⁺ (GIRK or Kir3) channels on the hippocampus has been postulated. Thus, we determined the impact of amyloid-β (Aβ) pathology in GIRK channel density, subcellular distribution, and its association with GABA_B receptors in hippocampal CA1 pyramidal neurons from the APP/PS1 mouse model using quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL) and proximity ligation in situ assay (P-LISA). In wild type mice, single SDS-FRL detection revealed a similar dendritic gradient for GIRK1 and GIRK2 in CA1 pyramidal cells, with higher densities in spines, and GIRK3 showed a lower and uniform distribution. Double SDS-FRL showed a co-clustering of GIRK2 and GIRK1 in post- and presynaptic compartments, but not for GIRK2 and GIRK3. Likewise, double GABA_B1 and GIRK2 SDS-FRL detection displayed a high degree of co-clustering in nanodomains (40–50 nm) mostly in spines and axon terminals. In APP/PS1 mice, the density of GIRK2 and GIRK1, but not for GIRK3, was significantly reduced along the neuronal surface of CA1 pyramidal cells and in axon terminals contacting them. Importantly, GABA_B1 and GIRK2 co-clustering was not present in APP/PS1 mice. Similarly, P-LISA experiments revealed a significant reduction in GABA_B1 and GIRK2 interaction on the hippocampus of this animal model. Overall, our results provide compelling evidence showing a significant reduction on the cell surface density of pre- and postsynaptic GIRK1 and GIRK2, but not GIRK3, and a decline in GABA_B receptors and GIRK2 channels co-clustering in hippocampal pyramidal neurons from APP/PS1 mice, thus suggesting that a disruption in the GABA_B receptor–GIRK channel membrane assembly causes dysregulation in the GABA_B signalling via GIRK channels in this AD animal model.