Differential distribution of release‐related proteins in the hippocampal CA3 area as revealed by freeze‐fracture replica labeling

131

145

G-protein-coupled inwardly rectifying Kϩ channels (Kir3 channels) coupled to metabotropic GABA B receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABA B receptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABA B1 . Consistent with this observation, a high degree of coclustering of Kir3.2 and GABA B1 was revealed around excitatory synapses by the highly sensitive SDS-digested freeze-fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K ϩ channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites.

Section: Enrichment Of Kir32 Around Excitatory Synapses On Dendriticmentioning

confidence: 67%

Compartment-Dependent Colocalization of Kir3.2-Containing K⁺Channels and GABA_BReceptors in Hippocampal Pyramidal Cells

Kulik¹,

Vida²,

Fukazawa³

et al. 2006

131

145

“…By performing SDS-digested freeze-fracture replica immunolabeling (Hagiwara et al, 2005;Kulik et al, 2006), we found ␤1-integrin in presynaptic compartments of CA1 synapses. Anti-SNAP25 labeling was used to identify presynaptic compartments (Fig.…”

Section: Presynaptic Localization Of ␤1-integrin In Ca1 Synapsesmentioning

confidence: 93%

Role for Reelin in Neurotransmitter Release

Hellwig¹,

Hack²,

Kowalski³

et al. 2011

The extracellular matrix molecule Reelin is known to control neuronal migration during development. Recent evidence suggests that it also plays a role in the maturation of postsynaptic dendrites and spines as well as in synaptic plasticity. Here, we aimed to address the question whether Reelin plays a role in presynaptic structural organization and function. Quantitative electron microscopic analysis of the number of presynaptic boutons in the stratum radiatum of hippocampal region CA1 did not reveal differences between wild-type animals and Reelin-deficient reeler mutant mice. However, additional detailed analysis showed that the number of presynaptic vesicles was significantly increased in CA1 synapses of reeler mutants. To test the hypothesis that vesicle fusion is altered in reeler, we studied proteins known to control transmitter release. SNAP25, a protein of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, was found to be significantly reduced in reeler mutants, whereas other SNARE complex proteins remained unaltered. Addition of recombinant Reelin to organotypic slice cultures of reeler hippocampi substantially rescued not only SNAP25 protein expression levels but also the number of vesicles per bouton area indicating a role for Reelin in presynaptic functions. Next, we analyzed paired-pulse facilitation, a presynaptic mechanism associated with transmitter release, and observed a significant decrease at CA1 synapses of reeler mutants when compared with wild-type animals. Together, these novel findings suggest a role for Reelin in modulating presynaptic release mechanisms.

“…An antibody against vesicular glutamate transporter (vGluT) 2 raised in a guinea pig was used to mark the RG fibers with anti-guinea pig secondary antibody conjugated with 10 nm gold particles (BBI). Although vGluTs are vesicular proteins, they are often detected on presynaptic plasma membrane and thus have been used to identify the origin of presynaptic profiles (Hagiwara et al, 2005;Masugi-Tokita et al, 2007). For labeling of glial glutamate transporters GLT-1 and GLAST, rabbit antibodies against GLT-1 and GLAST, respectively, were used in combination with the vGluT2 antibody.…”

Section: Methodsmentioning

confidence: 99%

Mechanisms Underlying Signal Filtering at a Multisynapse Contact

Budisantoso¹,

Matsui²,

Kamasawa³

et al. 2012

111

Visual information must be relayed through the lateral geniculate nucleus before it reaches the visual cortex. However, not all spikes created in the retina lead to postsynaptic spikes and properties of the retinogeniculate synapse contribute to this filtering. To understand the mechanisms underlying this filtering process, we conducted electrophysiology to assess the properties of signal transmission in the Long-Evans rat. We also performed SDS-digested freeze-fracture replica labeling to quantify the receptor and transporter distribution, as well as EM reconstruction to describe the 3D structure. To analyze the impact of transmitter diffusion on the activity of the receptors, simulations were integrated. We identified that a large contributor to the filtering is the marked paired-pulse depression at this synapse, which was intensified by the morphological characteristics of the contacts. The broad presynaptic and postsynaptic contact area restricts transmitter diffusion two dimensionally. Additionally, the presence of multiple closely arranged release sites invites intersynaptic spillover, which causes desensitization of AMPA receptors. The presence of AMPA receptors that slowly recover from desensitization along with the high presynaptic release probability and multivesicular release at each synapse also contribute to the depression. These features contrast with many other synapses where spatiotemporal spread of transmitter is limited by rapid transmitter clearance allowing synapses to operate more independently. We propose that the micrometer-order structure can ultimately affect the visual information processing.