Reelin Regulates Developmental Desynchronization Transition of Neocortical Network Activity.

The hyperpolarization-activated cation current, I(h), plays an important role in regulating intrinsic neuronal excitability in the brain. In hippocampal pyramidal neurons, I(h) is mediated by h channels comprised primarily of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits, HCN1 and HCN2. Pyramidal neuron h channels within hippocampal area CA1 are remarkably enriched in distal apical dendrites, and this unique distribution pattern is critical for regulating dendritic excitability. We utilized biochemical and immunohistochemical approaches in organotypic slice cultures to explore factors that control h channel localization in dendrites. We found that distal dendritic enrichment of HCN1 is first detectable at postnatal day 13, reaching maximal enrichment by the 3rd postnatal week. Interestingly we found that an intact entorhinal cortex, which projects to distal dendrites of CA1 but not area CA3, is critical for the establishment and maintenance of distal dendritic enrichment of HCN1. Moreover blockade of excitatory neurotransmission using tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, or 2-aminophosphonovalerate redistributed HCN1 evenly throughout the dendrite without significant changes in protein expression levels. Inhibition of calcium/calmodulin-dependent protein kinase II activity, but not p38 MAPK, also redistributed HCN1 in CA1 pyramidal neurons. We conclude that activation of ionotropic glutamate receptors by excitatory temporoammonic pathway projections from the entorhinal cortex establishes and maintains the distribution pattern of HCN1 in CA1 pyramidal neuron dendrites by activating calcium/calmodulin-dependent protein kinase II-mediated downstream signals.

The numerous γ-aminobutyric acid type A receptor (GABAAR) subtypes are differentially expressed and mediate distinct functions at neuronal level. In this study we have investigated GABAAR-mediated modulation of the spontaneous activity patterns of primary neuronal networks from murine frontal cortex by characterizing the effects induced by a wide selection of pharmacological tools at a plethora of activity parameters in microelectrode array (MEA) recordings. The basic characteristics of the primary cortical neurons used in the recordings were studied in some detail, and the expression levels of various GABAAR subunits were investigated by western blotting and RT-qPCR. In the MEA recordings, the pan-GABAAR agonist muscimol and the GABABR agonist baclofen were observed to mediate phenotypically distinct changes in cortical network activity. Selective augmentation of αβγ GABAAR signaling by diazepam and of δ-containing GABAAR (δ-GABAAR) signaling by DS1 produced pronounced changes in the majority of the activity parameters, both drugs mediating similar patterns of activity changes as muscimol. The apparent importance of δ-GABAAR signaling for network activity was largely corroborated by the effects induced by the functionally selective δ-GABAAR agonists THIP and Thio-THIP, whereas the δ-GABAAR selective potentiator DS2 only mediated modest effects on network activity, even when co-applied with low THIP concentrations. Interestingly, diazepam exhibited dramatically right-shifted concentration-response relationships at many of the activity parameters when co-applied with a trace concentration of DS1 compared to when applied alone. In contrast, the potencies and efficacies displayed by DS1 at the networks were not substantially altered by the concomitant presence of diazepam. In conclusion, the holistic nature of the information extractable from the MEA recordings offers interesting insights into the contributions of various GABAAR subtypes/subgroups to cortical network activity and the putative functional interplay between these receptors in these neurons.

We analyze the effect of noise on spontaneous activity of a excitatory-inhibitory neural network model. Analytically different regimes can be distinguished depending on the network parameters. In one of the regimes noise induces synchronous aperiodic oscillatory activity in the isolated network (regime B). The Coherent Stochastic Resonance phenomena occur. Activity is highly spatially correlated (synchrony), it’s oscillatory on short time scales and it decorrelates in time on long time scale (aperiodic). At zero noise the oscillatory activity vanishes in this regime. Changing parameters (for example increasing the excitatory-to-excitatory connection strength) we get spontaneous synchronous and periodic activity, even without noise (regime C). The model is in agreement with the measurements of spontaneous activity of two-dimensional cortical cell neural networks placed on multi-electrode arrays performed by Segev et.al [2].

During the early development of neocortical networks, many glutamatergic synapses lack AMPA receptors and are physiologically silent. We show in neocortical cultures that spontaneous synchronous network activity is able to convert silent synapses to active synapses by the incorporation of AMPA receptors into synaptic complexes throughout the network within a few minutes. To test the effect of synaptic activation on the connectivity of neuronal populations, we created separated neuronal networks that could innervate each other. We allowed outgrowing axons to invade the neighboring network either before or after the onset of synchronous network activity. In the first case, both subnetworks connected to each other and synchronized their activity, whereas in the second case, axonal connections failed to form and network activity did not synchronize between compartments. We conclude that early spontaneous synchronous network activity triggers a global AMPAfication of immature synapses, which in turn prevents later-arriving axons from forming afferent connections. This activity-dependent process may set the range of corticocortical connections during early network development before experience-dependent mechanisms begin elaborating the mature layout of the neocortical connections and modules.

Event Abstract Back to Event Characterization of 4-Aminopyridine-induced network plasticity in human iPSC-derived neuronal cultures Gaye Tanriöver1* and Katja Nieweg1 1 Phillips University, Institute of Pharmacology and Clinical Pharmacy, Germany Motivation Homeostatic network plasticity is a key regulatory mechanism for fine-tuning network activity. 4-aminopyridine (4-AP), which is commonly used to model epilepsy in vitro and in vivo has recently been described to trigger homeostatic plasticity in cultures of mouse hippocampal neurons [1]. The inhibition of several types of K+ channels (D-type and A-type K+ currents and a fraction of delayed rectifier currents) by 4-AP leads to prolonged action potentials and increased neurotransmitter release [2]. This induces hyperactivity, which is counteracted by mechanisms leading to decreased neuronal excitability. Human induced pluripotent stem cell (hiPSC)-derived neurons have been proposed to be a highly valuable in vitro model to be used in neurotoxicology, drug screening and regenerative medicine. Since spontaneous synchronous network activity develops in hiPSC-derived neuronal networks, our aim was to study the capacity of hiPSC-derived neuronal networks to exhibit homeostatic plasticity in response to 4-AP. Material and Methods hiPSCs were differentiated to cortical neurons as described previously [3]. Fifty thousand cells were plated after enrichment of neurons by immunopurification. After long-term (2-3 months) culture of the neurons for maturation, networks were transferred on the PO and laminin-coated 60-electrode multi-electrode arrays (Multichannel Systems, 200 µm electrode distance, 30µm electrode diameter) (MEAs) for recording. Calcium imaging was performed using Fura-2-AM ratiometric dye to identify single cell activity. Cells were loaded with 3µM Fura-2 AM for 20 min at 37°C in HEPES buffer. Fluorescence intensities at the two wavelengths (F340 and F380) were recorded separately and combined (fluorescence ratio: r=F340/F380) after background subtraction. Cells were imaged before and during 4-AP treatment as well as after removal of 4-AP. Neuronal networks with spontaneous network activity were incubated with 4-AP (100 µM) in order to trigger hyperactivity and measured during 4 days of 4-AP treatment as well as post-washout to monitor homeostatic plasticity. Results We modified our previously published [3] protocol for immunopanning of human iPSC derived neurons in order to permit differentiation of astrocytes from persisting neural precursor cells. Thus, we were able to generate mature, spontaneously active cortical like astrocyte-neuronal networks, shown by the presence of synchronous calcium transients around two months after immunoisolation. Analysis of network maturation was performed by culturing on 60-electrode MEAs. Single spikes and spike trains could be detected long before the occurrence of synchronous network bursts, which appeared two to three months after immunoisolation. To test, whether 4-AP triggered hyperactivity can induce homeostatic plasticity in human iPSC derived neuronal networks as it has been described for mouse hippocampal neurons [1], we treated human neurons of different maturational stages with 100µM 4-AP for 2-4 days and analyzed spontaneous network activity by MEA and calcium imaging. Mature, bursting human networks responded to 4-AP treatment with increased spiking and bursting rates (Fig. 1). Within the first 24h of treatment, mechanisms of homeostatic plasticity lead to downregulation of the activity close to baseline levels. One day after 4-AP removal, we observed that the overall network activity was strongly reduced even below baseline levels. The activity slowly recovered during the following days. Calcium imaging, which permits analyzing the activity of single cells within a network revealed similar results in mature human networks. However, immature human cultures without synchronous bursting activity did not respond to 4-AP and thus homeostatic plasticity could not be triggered in these cultures. Discussion 4-AP treatment of dissociated mouse hippocampal neurons cultured on MEAs was described, as a model to study homeostatic network plasticity by Pozzi et al. [1]. They showed that 4-AP induced hyperactivity lead to downregulation of neuronal excitability in mouse cultures. In our study, we applied this model to hiPSC derived neuronal networks and could show, that the maturational status of the network is a crucial parameter. Only spontaneously bursting cultures were able to respond in the expected manner. Whether downregulation of overall network activity occurs at the level of neuronal excitability or by synaptic downscaling will be analyzed by patch clamp recording. Conclusion In our study, we could show that mature hiPSC-derived neuronal networks cultured on MEAs are a valuable model to investigate mechanisms of homeostatic network plasticity in vitro. Since homeostatic plasticity is impaired in many psychiatric disorders and also at early stages of neurodegenerative diseases, a combination of patient derived iPSCs and the 4-AP plasticity model could serve as an innovative platform for drug screening.

Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone

Modeling of spontaneous synchronized periodic activity observed in in vitro networks

Fast changes of NMDA and AMPA receptor activity under acute hyperammonemia in vitro

Many immunocytochemical studies have identified different types of neurotransmitters localized in the corpus callosum (CC) axons in the adult mammal. Few studies have looked at the development of different neurochemically identified CC systems. Previous studies on the development of cat CC axons have indicated that a large number of transitory CC axons project to the cortex during early postnatal development. The present study focuses on the development of one neurochemically identified group of CC axons in the cat, labeled with an antibody against neuropeptide Y (NPY), to determine if this group participates in transitory CC axonal growth. Cats at specified ages from birth to adulthood were studied with a routine method of immunocytochemistry for antiserum to NPY. NPY-immunoreactive (ir) CC axons were detected at all stages examined, from newborn to adult; the peak density occurred during postnatal weeks (PNW) 3-4. During PNW 1-2, the density of NPY-ir CC axons increased gradually; some NPY-ir axons at this age had growth cones located within the CC bundle between the cerebral hemispheres. The density of the NPY-ir CC axons decreased gradually during PNW 5-7, and from PNW 8 to maturity only a few NPY-ir CC axons were observed. These results indicate that at least two types of NPY-ir CC axons (i.e., transitory and permanent) exist during development, and that most of these axons are eliminated or only express NPY-ir for a short period during development. The results also indicate that neurochemical subsets of CC axons participate in the extensive transitory growth observed by means of the membrane tracer DiI but they may follow unique developmental timetables.

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Cytokeratins are intermediate filament proteins found in most epithelial cells including the mammary epithelium. Specific cytokeratin expression has been found to mark different epithelial cell lineages and also to associate with putative mammary stem/progenitor cells. However, a comparative analysis of the expression of cytokaratins during embryonic and postnatal mammary development is currently lacking. Moreover, it is not clear whether the different classes of putative mammary stem/progenitor cells exist during embryonic development. Here, we use double/triple-label immunofluorescence and immunohistochemistry to systematically compare the expression of cytokeratin 5 (K5), cytokeratin 6 (K6), cytokeratin 8 (K8), cytokeratin 14 (K14) and cytokeratin 19 (K19) in embryonic and early postnatal mouse mammary glands. We show that K6+ and K8+/K14+ putative mammary progenitor cells arise during embryogenesis with distinct temporal and spatial distributions. Moreover, we describe a transient disconnection of the expression of K5 and K14, two cytokeratins that are often co-expressed, during the first postnatal weeks of mammary development. Finally, we report that cytokeratin expression in cultured primary mammary epithelial cells mimics that during the early stages of postnatal mammary development. These studies demonstrate an embryonic origin of putative mammary stem/progenitor cells. Moreover, they provide additional insights into the use of specific cytokeratins as markers of mammary epithelial differentiation, or the use of their promoters to direct gene overexpression or ablation in genetic studies of mouse mammary development.

We investigated the morphological features of long range intrinsically projecting neurons and their pattern of axonal arborization in cat area 17 at different stages of postnatal development. In one set of experiments intracortically projecting cells were retrogradely labeled in vivo with rhodamine latex beads and then visualized by in vitro filling with Lucifer yellow. In another approach, intracortical fibers including the cells of origin were labeled postmortem in fixed brains with the lipophilic carbocyanine dye Dil. The results of this study indicate that the long-range intrinsic circuitry of the primary visual cortex develops in three major steps. The first step consists of the development of unclustered long-range axons in the two outer compartments of the cerebral cortex, layer I and the subplate. These early connections could serve as a scaffold for the organization of the tangential architecture of the neocortex as they originate from cells that are the first to receive synaptic input from extrinsic afferents. The second step consists of the outgrowth of horizontal axon collaterals originating from cells located in layers II-VI. During the first 2-3 weeks these connections still differ from those in the adult because they span shorter distances, originate more often from neurons with morphological features of nonpyramidal cells, and lack the precise clustering of the mature connections. The third step consists of a selection process that leads to the elimination of axon terminals from locations between the clusters of tangentially projecting neurons. This selection starts at the end of the second postnatal week and, hence, overlaps in time with the still proceeding elongation of axons and continues beyond the end of the fourth postnatal week when axon length has reached its maximal extent. This refinement process enhances the specificity of long-range connections and is probably influenced by visual experience.

During wakefulness, neocortical neurons are subjected to an intense synaptic bombardment. To assess the consequences of this background activity for the integrative properties of pyramidal neurons, we constrained biophysical models with in vivo intracellular data obtained in anesthetized cats during periods of intense network activity similar to that observed in the waking state. In pyramidal cells of the parietal cortex (area 5-7), synaptic activity was responsible for an approximately fivefold decrease in input resistance (Rin), a more depolarized membrane potential (Vm), and a marked increase in the amplitude of Vm fluctuations, as determined by comparing the same cells before and after microperfusion of tetrodotoxin (TTX). The model was constrained by measurements of Rin, by the average value and standard deviation of the Vm measured from epochs of intense synaptic activity recorded with KAc or KCl-filled pipettes as well as the values measured in the same cells after TTX. To reproduce all experimental results, the simulated synaptic activity had to be of relatively high frequency (1-5 Hz) at excitatory and inhibitory synapses. In addition, synaptic inputs had to be significantly correlated (correlation coefficient approximately 0.1) to reproduce the amplitude of Vm fluctuations recorded experimentally. The presence of voltage-dependent K+ currents, estimated from current-voltage relations after TTX, affected these parameters by <10%. The model predicts that the conductance due to synaptic activity is 7-30 times larger than the somatic leak conductance to be consistent with the approximately fivefold change in Rin. The impact of this massive increase in conductance on dendritic attenuation was investigated for passive neurons and neurons with voltage-dependent Na+/K+ currents in soma and dendrites. In passive neurons, correlated synaptic bombardment had a major influence on dendritic attenuation. The electrotonic attenuation of simulated synaptic inputs was enhanced greatly in the presence of synaptic bombardment, with distal synapses having minimal effects at the soma. Similarly, in the presence of dendritic voltage-dependent currents, the convergence of hundreds of synaptic inputs was required to evoke action potentials reliably. In this case, however, dendritic voltage-dependent currents minimized the variability due to input location, with distal apical synapses being as effective as synapses on basal dendrites. In conclusion, this combination of intracellular and computational data suggests that, during low-amplitude fast electroencephalographic activity, neocortical neurons are bombarded continuously by correlated synaptic inputs at high frequency, which significantly affect their integrative properties. A series of predictions are suggested to test this model.

During early development neocortical neurons develop synchronous oscillatory network activity with the beginning of the second week in culture. This spontaneous synchronous activity is considered to be a requisite for the maturation of the synaptic networks. To simulate this activity it is common to use networks of neurons and analyze different connection strategies. On the other hand global population models (complex oscillators) are able to simulate the average activity of a large number of neurons. In the present work we combine both approaches to extend a population model to a set of distributed oscillators. In this way we get a spatial distribution of parameters and activity and are able to study effects of local external stimulation and propagation of excitation waves in large network areas. Small world connection strategy allows to investigate different behaviors by adjusting only one parameter.

Previous studies in rat, showing a transient pattern of expression of the alpha 7 nicotinic acetylcholine receptor in the ventrobasal thalamus and barrel cortex during the first 2 postnatal weeks, suggest that these receptors may play a role in development of the thalamocortical system. In the present study, in situ hybridization and radiolabeled ligand binding were employed to examine the spatiotemporal distribution of alpha 7 mRNA and alpha-bungarotoxin binding sites in the thalamocortical pathway of mouse during early postnatal development. As in the rat, high levels of alpha 7 mRNA and alpha-bungarotoxin binding sites are present in the barrel cortex of mouse during the first postnatal week. Both alpha 7 mRNA and its receptor protein are observed in all cortical laminae, with the highest levels seen in the compact cortical plate, layer IV, and layer VI. When viewed in a tangential plane, alpha 7 mRNA and alpha-bungarotoxin binding sites delineate a whisker-related barrel pattern in layer IV by P3-5. Quantitative analysis reveals a dramatic decrease in the levels of expression of alpha 7 mRNA and alpha-bungarotoxin binding sites in the cortex by the end of the second postnatal week. Unlike in the rat, only low levels of alpha 7 mRNA or alpha-bungarotoxin binding sites are present in the ventrobasal complex of the mouse thalamus. The broad similarities between the thalamocortical development of rat and mouse taken together with the present results suggest that alpha 7 receptors located on cortical neurons, rather than on thalamic neurons, play a role in mediating aspects of thalamocortical development.