The microdomain that orchestrates action potential initiation in neurons is the axon initial segment (AIS). It has long been considered to be a rather homogeneous domain at the very proximal axon hillock with relatively stable length, particularly in cortical pyramidal cells. However, studies in other brain regions paint a different picture. In hippocampal CA1, up to 50% of axons emerge from basal dendrites. Further, in about 30% of thick-tufted layer V pyramidal neurons in rat somatosensory cortex, axons have a dendritic origin. Consequently, the AIS is separated from the soma. Recent in vitro and in vivo studies have shown that cellular excitability is a function of AIS length/position and somatodendritic morphology, undermining a potentially significant impact of AIS heterogeneity for neuronal function. We therefore investigated neocortical axon morphology and AIS composition, hypothesizing that the initial observation of seemingly homogeneous AIS is inadequate and needs to take into account neuronal cell types. Here, we biolistically transfected cortical neurons in organotypic cultures to visualize the entire neuron and classify cell types in combination with immunolabeling against AIS markers. Using confocal microscopy and morphometric analysis, we investigated axon origin, AIS position, length, diameter as well as distance to the soma. We find a substantial AIS heterogeneity in visual cortical neurons, classified into three groups: (I) axons with somatic origin with proximal AIS at the axon hillock; (II) axons with somatic origin with distal AIS, with a discernible gap between the AIS and the soma; and (III) axons with dendritic origin (axon-carrying dendrite cell, AcD cell) and an AIS either starting directly at the axon origin or more distal to that point. Pyramidal cells have significantly longer AIS than interneurons. Interneurons with vertical columnar axonal projections have significantly more distal AIS locations than all other cells with their prevailing phenotype as an AcD cell. In contrast, neurons with perisomatic terminations display most often an axon originating from the soma. Our data contribute to the emerging understanding that AIS morphology is highly variable, and potentially a function of the cell type.

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The ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptors (AMPARs) have been implicated in the establishment of dendritic architecture. The transmembrane AMPA receptor regulatory proteins (TARPs) regulate AMPAR function and trafficking into synaptic membranes. In the current study, we employ type I and type II TARPs to modulate expression levels and function of endogenous AMPARs and investigate in organotypic cultures (OTCs) of rat occipital cortex whether this influences neuronal differentiation. Our results show that in early development [5-10 days in vitro (DIV)] only the type I TARP γ-8 promotes pyramidal cell dendritic growth by increasing spontaneous calcium amplitude and GluA2/3 expression in soma and dendrites. Later in development (10-15 DIV), the type I TARPs γ-2, γ-3 and γ-8 promote dendritic growth, whereas γ-4 reduced dendritic growth. The type II TARPs failed to alter dendritic morphology. The TARP-induced dendritic growth was restricted to the apical dendrites of pyramidal cells and it did not affect interneurons. Moreover, we studied the effects of short hairpin RNA-induced knockdown of endogenous γ-8 and showed a reduction of dendritic complexity and amplitudes of spontaneous calcium transients. In addition, the cytoplasmic tail (CT) of γ-8 was required for dendritic growth. Single-cell calcium imaging showed that the γ-8 CT domain increases amplitude but not frequency of calcium transients, suggesting a regulatory mechanism involving the γ-8 CT domain in the postsynaptic compartment. Indeed, the effect of γ-8 overexpression was reversed by APV, indicating a contribution of NMDA receptors. Our results suggest that selected type I TARPs influence activity-dependent dendritogenesis of immature pyramidal neurons.

A multitude of 18 iGluR receptor subunits, many of which are diversified by splicing and RNA editing, localize to >20 excitatory and inhibitory neocortical neuron types defined by physiology, morphology, and transcriptome in addition to various types of glial, endothelial, and blood cells. Here we have compiled the published expression of iGluR subunits in the areas and cell types of developing and adult cortex of rat, mouse, carnivore, bovine, monkey, and human as determined with antibody- and mRNA-based techniques. iGluRs are differentially expressed in the cortical areas and in the species, and all have a unique developmental pattern. Differences are quantitative rather than a mere absence/presence of expression. iGluR are too ubiquitously expressed and of limited use as markers for areas or layers. A focus has been the iGluR profile of cortical interneuron types. For instance, GluK1 and GluN3A are enriched in, but not specific for, interneurons; moreover, the interneurons expressing these subunits belong to different types. Adressing the types is still a major hurdle because type-specific markers are lacking, and the frequently used neuropeptide/CaBP signatures are subject to regulation by age and activity and vary as well between species and areas. RNA-seq reveals almost all subunits in the two morphofunctionally characterized interneuron types of adult cortical layer I, suggesting a fairly broad expression at the RNA level. It remains to be determined whether all proteins are synthesized, to which pre- or postsynaptic subdomains in a given neuron type they localize, and whether all are involved in synaptic transmission. J. Comp. Neurol. 525:976-1033, 2017. © 2016 Wiley Periodicals, Inc.

A battery of genetically encoded calcium indicators (GECIs) with different binding kinetics and calcium affinities was developed over the recent years to permit long-term calcium imaging. GECIs are calcium buffers and therefore, expression of GECIs may interfere with calcium homeostasis and signaling pathways important for neuronal differentiation and survival. Our objective was to investigate if the biolistically induced expression of five commonly used GECIs at two postnatal time points (days 14 and 22-25) could affect the morphological maturation of cortical neurons in organotypic slice cultures of rat visual cortex. Expression of GCaMP3 in both time windows, and of GCaMP5G and TN-XXL in the later time window impaired apical and /or basal dendrite growth of pyramidal neurons. With time, the proportion of GECI transfectants with nuclear filling increased, but an only prolonged expression of TN-XXL caused higher levels of neurodegeneration. In multipolar interneurons, only GCaMP3 evoked a transient growth delay during the early time window. GCaMP6m and GCaMP6m-X C were quite "neuron-friendly." Since growthimpaired neurons might not have the physiological responses typical of age-matched wildtype neurons the results obtained after prolonged developmental expression of certain GECIs might need to be interpreted with caution.