electrophysiological-morphological-characterization-neuronal

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Research Centre Jülich

Cajal-Retzius (CR) cells are among the earliest generated neurons and are thought to play a role in corticogenesis and early neuronal migration. However, the role of CR cells in an early cortical microcircuit is still rather unclear. We therefore have investigated the morphology and physiology of CR cells by using whole-cell patch-clamp recordings combined with intracellular biocytin filling in acute brain slices of postnatal day 5-11 rats. CR cells are characterized by a long horizontally oriented dendrite; the axonal collaterals form a dense horizontally oriented plexus in layer 1 and to a certain extent in layer 2/3, projecting over >2 mm of cortical surface. The bouton density is relatively high, and synaptic contacts are established preferentially with dendritic spines or shafts of excitatory neurons, presumably terminal tuft dendrites of pyramidal neurons. In turn, CR cells receive dense GABAergic and non-GABAergic input on somata, dendritic shafts, and spine-like appendages. Extracellular stimulation in layer 1 could activate both GABAergic and glutamatergic synaptic inputs. The GABAergic response was blocked by the GABA(A) receptor antagonist bicuculline. The glutamatergic response was mediated solely by NMDA receptors and was highly sensitive to ifenprodil, indicating that it was mediated mainly via NR1/NR2B subunit-containing receptors. NMDA EPSPs were apparent in 1 mm extracellular Mg2+, suggesting that this pure NMDA synapse is not silent functionally. Together, the long-range horizontal projection of the axon, the high density of synaptic boutons, and the functional synaptic input of CR cells suggest that they are an integral part of an early cortical network.

Axonal projection, input and output synapses, and synaptic physiology of Cajal-Retzius cells in the developing rat neocortex.

In the neocortex, most excitatory, glutamatergic synapses are established during the first 4-5 weeks after birth. During this period profound changes in the properties of synaptic transmission occur. Excitatory postsynaptic potentials (EPSPs) at immature synaptic connections are profoundly and progressively reduced in response to moderate to high frequency (5-100 Hz) stimulation. With maturation, this frequency-dependent depression becomes progressively weaker and may eventually transform into a weak to moderate EPSP facilitation. In parallel to changes in the short-term plasticity, a reduction in the synaptic reliability occurs at most glutamatergic neocortical synapses: immature synapses show a high probability of neurotransmitter release as indicated by their low failure rate and small EPSP amplitude variation. This high reliability is reduced in mature synapses, which show considerably higher failure rates and more variable EPSP amplitudes. During early neocortical development synaptic vesicle pools are not yet fully differentiated and their replenishment may be slow, thus resulting in EPSP amplitude depression. The decrease in the probability of neurotransmitter release may be the result of an altered Ca(2+) control in the presynaptic terminal with a reduced Ca(2+) influx and/or a higher Ca(2+) buffering capacity. This may lead to a lower synaptic reliability and a weaker short-term synaptic depression with maturation.

Developmental alterations in the functional properties of excitatory neocortical synapses.

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.

Article	Year
Axonal projection, input and output synapses, and synaptic physiology of Cajal-Retzius cells in the developing rat neocortex. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 12177189	2002
Developmental alterations in the functional properties of excitatory neocortical synapses. The Journal of physiology\| PubMed ID: 19273572	2009
Improved biocytin labeling and neuronal 3D reconstruction. Nature protocols\| PubMed ID: 22301777	2012

Gabriele Radnikow

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