Associative learning is thought to involve different forms of activity-dependent synaptic plasticity. Although previous studies have mostly focused on learning-related changes occurring at excitatory glutamatergic synapses, we found that associative learning, such as fear conditioning, also entails long-lasting functional and structural plasticity of GABAergic synapses onto pyramidal neurons of the murine basal amygdala. Fear conditioning-mediated structural remodeling of GABAergic synapses was associated with a change in mIPSC kinetics and an increase in the fraction of synaptic benzodiazepinesensitive (BZD) GABA A receptors containing the a2 subunit without altering the intrasynaptic distribution and overall amount of BZD-GABA A receptors. These structural and functional synaptic changes were partly reversed by extinction training. These findings provide evidence that associative learning, such as Pavlovian fear conditioning and extinction, sculpts inhibitory synapses to regulate inhibition of active neuronal networks, a process that may tune amygdala circuit responses to threats.

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non-disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute-long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze-substitution, sample rehydration and cryosection-immunolabelling or with freeze-fracture replica-immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry. In vitro grown vertebrate cells are an indispensable tool for cell biological research, and are widely used for the subcellular localisation of macromolecules at the level of electron microscopy (EM). Yet, the fast and efficient harvest of living, adherent cells without affecting cellular morphology and physiology is still a not satisfactorily solved problem. Thus, the potential of recent improvements (1-3) of cryo-immuno-EM (cryo-IEM) has so far not been fully exploited.To begin with, immunogold-labelling of cryosections according to Tokuyasu (4) has been successfully combined with rapid cryofixation (2,3). The new 'hybrid' approach avoids artefacts resulting from conventional chemical fixation. It is based on cryofixation of native, unfixed specimens, followed by chemical stabilisation of cellular ultrastructure and antigenicity at around −90• C by means of freeze-substitution (FS) and, finally, sample rehydration and postfixation prior to cryosectioning (2,3). So far, this modified Tokuyasu-technique was mainly used for tissues and suspension cultures (2,3,5), but not regularly for adherent cell cultures, with very few exceptions, when relatively large, voluminous cells (HepG2) were cultured on gelatine beads (Cytodex™, Sigma: Ø ∼100 μm; refs. (2,6)). Yet, in our previous studies we found that commercially available beads are less suitable for applying this advanced IEMtechnique to flat and/or small cells, such as mouse embryonic fibroblasts (MEF) with a maximal height of ∼4 μm (ref.(7) and our unpublished data). Considering the adverse ratio of bead-diameter versus cell-height it is evident that most of the area of an average 400 × 400 μm-cryosection is occupied by section profiles of the carrier beads, but not by the cells under investigation. The analysis of a fair amount of cells, however, is mandatory for unbiased stereology (8). Furthermore, sampling of cells grown on beads prior to cryofixation requires time-consuming intermediate enrichment-steps, possibly leading to unwanted physiological and ultrastructural alterations.The second cutting-edge IEM-technique to mention here is sodium dodecyl sulphate-digested freeze-fracture replica labelling (SDS-FRL (1)). So far, SDS-FRL was predominantly applied to chemically fixed tissues e.g. (9); see also (10) for review. The few SDS-FRL studies on natively cryofixed monolayers relayed on...

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre-and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

Metabotropic glutamate receptors (mGluRs) couple to G-proteins to modulate slow synaptic transmission via intracellular second messengers. The first cloned mGluR, mGluR1, regulates motor coordination, synaptic plasticity and synapse elimination. mGluR1 undergoes alternative splicing giving rise to four translated variants that differ in their intracellular C-terminal domains. Our current knowledge about mGluR1 relates almost entirely to the long mGluR1α isoform, whereas little is known about the other shorter variants. To study the expression of mGluR1γ, we have generated by means of the CRISPR/Cas9 system a new knock-in (KI) mouse line in which the C-terminus of this variant carries two short tags. Using this mouse line, we could establish that mGluR1γ is either untranslated or in amounts that are undetectable in the mouse cerebellum, indicating that only mGluR1α and mGluR1β are present and active at cerebellar synapses. The trafficking and function of mGluR1 appear strongly influenced by adaptor proteins such as long Homers that bind to the C-terminus of mGluR1α. We generated a second transgenic (Tg) mouse line in which mGluR1α carries a point mutation in its Homer binding domain and studied whether disruption of this interaction influenced mGluR1 subcellular localization at cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses by means of the freeze-fracture replica immunolabeling technique. These Tg animals did not show any overt behavioral phenotype, and despite the typical mGluR1 perisynaptic distribution was not significantly changed, we observed a higher probability of intrasynaptic diffusion suggesting that long Homers regulate the lateral mobility of mGluR1. We extended our ultrastructural analysis to other mouse lines in which only one mGluR1 variant was reintroduced in PC of mGluR1-knock out (KO) mice. This work revealed that mGluR1α preferentially accumulates closer to the edge of the postsynaptic density (PSD), whereas mGluR1β has a less pronounced perijunctional distribution and, in the absence of mGluR1α, its trafficking to the plasma membrane is impaired with an accumulation in intracellular organelles. In conclusion, our study sets several firm points on largely disputed matters, namely expression of mGluR1γ and role of the C-terminal domain of mGluR1 splice variants on their perisynaptic clustering.

No abstract

The amygdala plays a crucial role in attaching emotional significance to environmental cues. Its intercalated cell masses (ITC) are tight clusters of GABAergic neurons, which are distributed around the basolateral amygdala complex. Distinct ITC clusters are involved in the acquisition and extinction of conditioned fear responses. Previously, we have shown that fear memory retrieval reduces the AMPA/NMDA ratio at thalamic afferents to ITC neurons within the dorsal medio-paracapsular cluster. Here, we investigate the molecular mechanisms underlying the fear-mediated reduction in the AMPA/NMDA ratio at these synapses and, in particular, whether specific changes in the synaptic density of AMPA receptors underlie the observed change. To this aim, we used a detergent-digested freeze-fracture replica immunolabeling technique (FRIL) approach that enables to visualize the spatial distribution of intrasynaptic AMPA receptors at high resolution. AMPA receptors were detected using an antibody raised against an epitope common to all AMPA subunits. To visualize thalamic inputs, we virally transduced the posterior thalamic complex with Channelrhodopsin 2-YFP, which is anterogradely transported along axons. Using face-matched replica, we confirmed that the postsynaptic elements were ITC neurons due to their prominent expression of μ-opioid receptors. With this approach, we show that, following auditory fear conditioning in mice, the formation and retrieval of fear memory is linked to a significant reduction in the density of AMPA receptors, particularly at spine synapses formed by inputs of the posterior intralaminar thalamic and medial geniculate nuclei onto identified ITC neurons. Our study is one of the few that has directly linked the regulation of AMPA receptor trafficking to memory processes in identified neuronal networks, by showing that fear-memory induced reduction in AMPA/NMDA ratio at thalamic-ITC synapses is associated with a reduced postsynaptic AMPA receptor density.

Neue Gesetze, neue Normen, technischer Fortschritt, Fachkräftemangel: Dies sind nur einige Punkte, die für eine kontinuierliche Weiterbildung - auch nach einem erfolgreich abgeschlossenen Studium, einer Techniker- oder Meisterqualifizierung oder einer Berufsausbildung - sprechen. Lebenslanges Lernen ist daher kein hohler Anspruch, sondern die Notwendigkeit, eigenes Wissen immer zu aktualisieren sowie weiteres Fachwissen und neue Fähigkeiten zu erwerben. Diese Metadaten wurden zur Verfügung gestellt von der Literaturdatenbank RSWB®plus

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre-and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning. Video LinkThe video component of this article can be found at https://www.jove.com/video/53853/ . This original method had, however, several limitations, i.e., severe fragmentation of replicas, which hampered meaningful correlations of labeled molecules with individually mapped cells in complex tissues such as the brain. Approximately 10 years ago, Shigemoto and Fukazawa progressively improved the technique 2 . This was paralleled by efforts from another group of scientists at the Boulder laboratories of the Colorado State University, who also significantly improved the technique, in particular for the study of gap junctions 3 .