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.