The activation of mAChR on subplate neurons has been shown to trigger oscillatory glutamatergic network activity in early postnatal neocortex. Here, we explored the long-term consequences of subplate activation on the differentiation of cortical neurons. Stimulating newborn somatosensory and visual cortex ex vivo and organotypic cortex cultures (OTCs) of the visual cortex with carbachol (CCH) led to an upregulation of Bdnf and Zif-268. In OTCs, the stimulation increased the amplitude and frequency of calcium events in the gray matter layers at DIV 3-5. At DIV 5, an acute stimulation rapidly activates p21ras; increases ERK phosphorylation; induces Bdnf, c-Fos, and Zif-268 expression; and increases GluN1, GluN2A, GluN2B, PSD-95, GAD-65, and synaptophysin protein expression. The NMDA receptors were functional, as shown by enhanced ligand-induced dendritic injury. Repeated DIV 1-5 mAChR activation increased, at DIV 10, the expression of GluN2A, of an essential protein of the inhibitory presynapse, VGAT, and the basket-cell-specific presynaptic calcium sensor synaptotagmin-2. Furthermore, it increased the frequency of interneuronal calcium events at DIV 10-15, dendritic length of basket cells, and accelerated the local arborization of basket cell axons at DIV 15. The results suggest that temporally limited mAChR-mediated activation of subplate neurons evokes glutamatergic network activity, which, gradually over the following weeks, promotes cortical circuit development. Specifically, it enhances the expression of excitatory and inhibitory synaptic proteins and accelerates the morphological and functional maturation of basket cells, highlighting the cholinergic input to subplate neurons as a critical regulator of interneuron development.

Electrical activity is considered a key driver for the neurochemical and morphological maturation of neurons and the formation of neuronal networks. Designer receptors exclusively activated by designer drugs (DREADDs) are tools for controlling neuronal activity at the single cell level by triggering specific G protein signaling. Our objective was to investigate if prolonged silencing of differentiating cortical neurons can influence dendritic and axonal maturation. The DREADD hM4Di couples to G(i/o) signaling and evokes hyperpolarization via GIRK channels. HM4Di was biolistically transfected into neurons in organotypic slice cultures of rat visual cortex, and activated by clozapine-N-oxide (CNO) dissolved in H2O; controls expressed hM4Di, but were mock-stimulated with H2O. Neurons were analyzed after treatment for two postnatal time periods, DIV 5-10 and 10-20. We found that CNO treatment delays the maturation of apical dendrites of L2/3 pyramidal cells. Further, the number of collaterals arising from the main axon was significantly lower, as was the number of bouton terminaux along pyramidal cell and basket cell axons. The dendritic maturation of L5/6 pyramidal cells and of multipolar interneurons (basket cells and bitufted cells) was not altered by CNO treatment. Returning CNO-treated cultures to CNO-free medium for 7 days was sufficient to recover dendritic and axonal complexity. Our findings add to the view that activity is a key driver in particular of postnatal L2/3 pyramidal cell maturation. Our results further suggest that inhibitory G protein signaling may represent a factor balancing the strong driving force of neurotrophic factors, electrical activity and calcium signaling.

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-XC were quite "neuron-friendly." Since growth-impaired 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.

During neuronal development, AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are important for neuronal differentiation. Kainate receptors (KARs) are closely related to AMPARs and involved in the regulation of cortical network activity. However, their role for neurite growth and differentiation of cortical neurons is unclear. Here, we used KAR agonists and overexpression of selected KAR subunits and their auxiliary neuropilin and tolloid-like proteins, NETOs, to investigate their influence on dendritic growth and network activity in organotypic cultures of rat visual cortex. Kainate at 500 nM enhanced network activity and promoted development of dendrites in layer II/III pyramidal cells, but not interneurons. GluK2 overexpression promoted dendritic growth in pyramidal cells and interneurons. GluK2 transfectants were highly active and acted as drivers for network activity. GluK1 and NETO1 specifically promoted dendritic growth of interneurons. Our study provides new insights for the roles of KARs and NETOs in the morphological and physiological development of the visual cortex.

The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Visualizing traffic-dependent GPCR signals in neurons is difficult, but important to understand the contribution of GPCRs to synaptic plasticity. We engineered CaMello (Ca2+-melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic-dependent Ca2+ signals in 5-HT2A-R domains. These constructs consist of the light-activated Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca2+ signals and receptor trafficking, and the 5-HT2A C-terminus for targeting into 5-HT2A-R domains. We show that the specific localization of the GPCR to its receptor domain drastically alters the dynamics and localization of the intracellular Ca2+ signals in different neuronal populations in vitro and in vivo. The CaMello method may be extended to every GPCR coupling to the Gq/11 pathway to help unravel new receptor-specific functions in respect to synaptic plasticity and GPCR localization.

The 30-amino acid peptide Y-P30 corresponds to the N-terminus of the primate-specific, sweat gland-derived dermcidin prepropeptide. Previous work has revealed that Y-P30 enhances the interaction of pleiotrophin and syndecans-2/3, and thus represents a natural ligand to study this signaling pathway. In immature neurons, Y-P30 activates the c-Src and p42/44 ERK kinase pathway, increases the amount of F-actin in axonal growth cones, and promotes neuronal survival, cell migration and axonal elongation. The action of Y-P30 on axonal growth requires syndecan-3 and heparan sulfate side chains. Whether Y-P30 has the potential to influence dendrites and dendritic protrusions has not been explored. The latter is suggested by the observations that syndecan-2 expression increases during postnatal development, that syndecan-2 becomes enriched in dendritic spines, and that overexpression of syndecan-2 in immature neurons results in a premature morphological maturation of dendritic spines. Here, analysing rat cortical pyramidal and non-pyramidal neurons in organotypic cultures, we show that Y-P30 does not alter the development of the dendritic arborization patterns. However, Y-P30 treatment decreases the density of apical, but not basal dendritic protrusions at the expense of the filopodia. Analysis of spine morphology revealed an unchanged mushroom/stubby-to-thin spine ratio and a shortening of the longest decile of dendritic protrusions. Whole-cell recordings from cortical principal neurons in dissociated cultures grown in the presence of Y-P30 demonstrated a decrease in the frequency of glutamatergic mEPSCs. Despite these differences in protrusion morphology and synaptic transmission, the latter likely attributable to presynaptic effects, calcium event rate and amplitude recorded in pyramidal neurons in organotypic cultures were not altered by Y-P30 treatment. Together, our data suggest that Y-P30 has the capacity to decelerate spinogenesis and to promote morphological, but not synaptic, maturation of dendritic protrusions.

Bei der Entwicklung und Differenzierung von Neuronen sind AMPA- und NMDA-Rezeptoren wichtig. Kainatrezeptoren sind mit AMPA-Rezeptoren verwandt und an der Regulation von neuronalen Netzwerken beteiligt. Ihre Rolle fur die morphologische und physiologische Reifung kortikale Neurone ist unklar. In diesem Projekt wurde die Rolle der Kainatrezeptoren und ihrer Hilfsproteine NETOs (neuropilin and tolloid-like proteins) untersucht. Eine geringe Dosis von 500 nM Kainat erhohte die Netzwerkaktivitat und das Wachstum von Dendriten der Pyramidalzellen. Die Uberexpression von GluK2 beschleunigte das Dendritenwachstum von Pyramidalzellen und Interneuronen. GluK2-transfizierte Neurone waren deutlich aktiver und konnten als Treiber fur Netzwerkaktivitat fungieren. GluK1 und NETO1 erhohten das Dendritenwachstum spezifisch in Interneuronen. Dieses Projekt liefert neue Erkenntnisse fur die Rolle der Kainatrezeptoren und NETOs bei der morphologischen und physiologischen Reifung der Neurone im visuellen Kortex.

Pro-inflammatory cytokines of the IL-6 family have been linked to the etiology of epilepsy and mental disorders. After infusion of the IL-6 family member leukemia inhibitory factor (LIF) into postnatal day 12–20 rat visual cortex (period of synaptogenesis; equals early childhood in human) and equivalent ages in organotypic cultures (OTC), somatic growth of cortical and subcortical neurons is delayed, expression of interneuron markers reduced and expression of neurotrophins transiently reduced. Further, LIF prevents NT4 from promoting soma growth of GABA-ergic interneurons. We now show that LIF-treatment from DIV 12–20 in OTC compromises the differentiation of fast-spiking GABA-ergic basket neurons: GAD-65/67, Kv3.2, and synaptotagmin-2 proteins are reduced, dendrites are shorter, and axonal varicosities are smaller. Bitufted and Martinotti interneurons are barely affected. Pyramidal cells display lower dendritic spine densities, more filopodia, and shorter axon initial segments. βIV-spectrin, Cav3.1, GABAA receptor subunits α1 and α2, and the phosphorylation of GluN2B at Y1472 are reduced. The frequency of calcium events in pyramidal cells and interneurons is increased, and the antiepileptic neuropeptide Y is upregulated, suggesting a hyperexcitability of the network. In the presence of LIF, neurotrophins fail to activate MAP kinase phosphorylation and c-fos expression, and exogenous NT4 fails to promote the maturation of pyramidal cells and interneurons as it normally would. After discontinuing LIF treatment, bouton size and expression levels of affected proteins normalize except for synaptotagmin-2; moreover, hyperexcitability persists. The results suggest that elevated levels of inflammatory cytokines during this developmental period cause a lasting maldevelopment in particular of basket cells.

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.

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.

An inflammatory episode during brain development is discussed as first hit which could trigger a higher susceptibility for epilepsy or development of mental disorders. We addressed the early developmental actions of an IL-6 type pro-inflammatory cytokine, leukemia inhibitory factor (LIF) on cortical maturation both in vivo and in slice cultures. A rather modest enhancement of LIF signaling was used as a proxy for an early postnatal inflammation. It resulted in a dramatic interneuron synaptopathy. Our results indicate that LIF impairs development of cortical interneuron subsets and causes a lasting imbalance of excitation and inhibition. In particular, LIF not only downregulates the expression of the TrkB receptor ligand NT4, but concurrently prevents the neurotrophins from activating the MAP kinase pathway. As a consequence, strictly TrkB-dependent cortical inhibitory GABA-ergic interneurons, in particular fast-spiking basket (B) and chandelier (C) neurons are severely affected. They display an underdevelopment of the somatodendritic domain, less and smaller presynaptic boutons, a reduced expression of the calcium buffer parvalbumin, the voltage-gated potassium channel Kv3.2, the ultrafast vesicular calcium sensor synaptotagmin-2, and glutamate decarboxylase isoform GAD-65 which synthesized GABA for phasic inhibition. These presynaptic proteins are essential for the fast-spiking properties and a highly synchronous GABA release, both being critical determinants for neuronal oscillations in the cognitive gamma frequency band. Also pyramidal cells are altered: dendritic spines are lower in density and less mature, GABAARα1 at basket cell terminals and α2 at chandelier cell terminals are reduced in expression, the axon initial segment is shorter and its core scaffolding protein ßIV-spectrin is reduced. The presynaptic deficits (outlined in the graphical abstract with left: healthy; right: LIF / inflammation) suggest a serious impairment of inhibition, and calcium imaging indeed reveals a hyperexcitable network. A set of recovery experiments reveals a surprisingly slow recovery of the affected proteins, and intriguingly, synaptotagmin-2 entirely fails to recover. In summary, our study suggests LIF as a negative upstream modulator of TrkB signaling. The impaired trophic support results in a maldevelopment in particular of fast-spiking interneurons, a deficit of inhibition and cortical hyperexcitability.

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.