Synapse‐dependent and independent mechanisms of thalamocortical axon branching are regulated by neuronal activity

Axon branching and synapse formation are critical processes for establishing precise circuit connectivity. These processes are tightly regulated by neural activity, but the relationship between them remains largely unclear. We use organotypic coculture preparations to examine the role of synapse formation in the activity‐dependent axon branching of thalamocortical (TC) projections. To visualize TC axons and their presynaptic sites, two plasmids encoding DsRed and EGFP‐tagged synaptophysin (SYP‐EGFP) were cotransfected into a small number of thalamic neurons. Time‐lapse imaging of individual TC axons showed that most branches emerged from SYP‐EGFP puncta, indicating that synapse formation precedes emergences of axonal branches. We also investigated the effects of neuronal activity on axon branching and synapse formation by manipulating spontaneous firing activity of thalamic cells. An inward rectifying potassium channel, Kir2.1, and a bacterial voltage‐gated sodium channel, NaChBac, were used to suppress and promote firing activity, respectively. We found suppressing neural activity reduced both axon branching and synapse formation. In contrast, increasing neural activity promoted only axonal branch formation. Time‐lapse imaging of NaChBac‐expressing cells further revealed that new branches frequently appeared from the locations other than SYP‐EGFP puncta, indicating that enhancing activity promotes axonal branch formation due to an increase of branch emergence at nonsynaptic sites. These results suggest that presynaptic locations are hotspots for branch emergence, and that frequent firing activity can shift branch emergence to a synapse‐independent process. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 323–336, 2016     

Vesicular release mechanisms in astrocytic signalling

Astrocytes play an important role in chemical signalling, acting as receptive as well as secretory elements. They can express receptors for essentially all classical neurotransmitter substances and for a large variety of peptides. Recent evidence indicates that astrocytes are involved in the information processing within the nervous system. Astrocytes respond to various neurotransmitters with elevations in intracellular calcium which can either be long-duration Ca(2+) spikes or oscillations in Ca(2+) levels. Astrocytic excitation can be propagated to adjacent astrocytes in the form of Ca(2+) waves. Due to their intimate spatial relationship with synaptic contacts, astrocytes can directly respond to synaptically released messengers and communicate, via signalling substances, with neurons in a reciprocal manner. Cultured astrocytes and astroglioma cells express synaptic vesicle proteins and members of the synaptic SNARE complex. Astrocytes can release a variety of messenger substances via receptor-mediated mechanisms implicating their potential for regulated exocytosis and the participation of proteins of the SNARE complex.     

Modeling absence seizure dynamics: implications for basic mechanisms and measurement of thalamocortical and corticothalamic latencies

A successful physiologically based continuum model of the corticothalamic system is applied to determine the relative contributions of axonal and intrinsic cellular delays to the waveforms of absence seizures. The predicted period of the absence seizure depends linearly on model parameters describing thalamocortical, corticothalamic, intracortical, and synaptodendritic delays, and these dependences are linked to the seizure mechanism by showing how time intervals between peaks in the waveforms depend on the parameters. Counterintuitively, it is found that a peak in the local field potential recorded in the thalamic relay nuclei can precede the peak in the cortical field that drove it, without violating causality, but rendering naive interpretation of time intervals between peaks invalid. We argue that a thalamocortical loop mechanism for absence seizures is consistent with intrathalamic cellular properties being the leading determinant of the frequency of spike-wave discharges in rat genetic models, with the combination of network and cellular properties providing a natural explanation for the lower frequency of human absence seizures. Finally, our results imply that the seizure frequency is not determined by the fastest thalamocortical and corticothalamic fibers, but rather depends on an effective weighted conduction velocity of all pathways present.     

Efficient DNA transfection in neuronal and astrocytic cell lines

We have studied different parameters for efficient DNA transfection in various cell types and with different size of the promoter. Here we report that the optimum condition for DNA transfection by electroporation is 350 V/960 microF for PC12, 450V/960 microF C6 cells, and 250 V/500 microF for COS-1 cells. For the human neuroblastoma (SK-N-SH) cells the optimum condition for DNA transfection is by the calcium phosphate method. In promoter mapping studies, a serial deletion approach is commonly used. To optimize transfection we have selected three DNA constructs that varied in size from 4.5 to 12.4 kilobases (kb). We measured the promoter activity of these constructs under conditions of 'equal amount', 'equimolar', and 'equimolar plus carrier DNA to make it equal amount'. We recommend that for comparative purpose, transfection should be carried out under 'equimolar condition' without a need to adjust the total amount of DNA by carrier DNA. Taken together, our results suggest that efficient methods for DNA transfection are important to study gene regulation by devising better ways to deliver DNA into the mammalian cells.     

NT2 derived neuronal and astrocytic network signalling

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.     

How astrocytic ATP shapes neuronal activity and brain circuits

Astrocytes play a key role in processing information at synapses, by controlling synapse formation, modulating synapse strength and terminating neurotransmitter action. They release ATP to shape brain activity but it is unclear how, as astrocyte processes contact many targets and ATP-mediated effects are diverse and numerous. Here, I review recent studies showing how astrocytic ATP modulates cellular mechanisms in nearby neurons and glia in the grey and white matter, how it affects signal transmission in these areas, and how it modulates behavioural outputs. I attempt to provide a flowchart of astrocytic ATP signalling, showing that it tends to inhibit neural circuits to match energy demands.     

Calcium dynamics of cortical astrocytic networks in vivo

Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca²⁺]_i events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca²⁺]_i events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca²⁺]_i activity in individual cells and a robust coordination of [Ca²⁺]_i signals in neighboring astrocytes. These findings indicate potential neuron–glia communication in the intact brain.     

Immunohistochemical demonstration of neuronal and astrocytic markers and oncofoetal antigens in retinoblastomas.

General opinion is that retinoblastomas, though not everyone agrees with that view. Some authors suggest that retinoblastomas are derived from a primitive retinal cell able to differentiate into both neuronal and glial cell lines. The aim of the present work was to study immunohistochemically the expression of neuronal and astrocytic markers in retinoblastomas and at the same time the presence of the oncofoetal antigens carcinoembryonic antigen (CEA) and alpha Foeto Protein (AFP), since patients with retinoblastomas often show high oncofoetal antigen in serum levels. For this purpose we employed the streptavidin-biotin immunoperoxidase technique in 13 cases of retinoblastoma to evaluate the presence and distribution of neuron-specific enolase (NSE), neurofilament protein (NF), glial fibrillary acidic protein (GFAP), S-100 protein, CEA and AFP. All 13 tumours studied stained for NSE. Seven of them showed GFAP- and S-100 positive perivascular glial cells as well as cells distributed randomly in the tumour that were interpreted as non tumour cells. All 13 retinoblastomas lacked detectable NF, CEA, and AFP. These results support the idea that retinoblastomas are neuronal tumours, although retinal glial cells may become incorporated in the tumour and proliferate in response to the tumour.     

Behavior-related neuronal reactions and dynamics of neuronal activity

Functionally, behavior-related discharges of associative neurons are an efferent flow of pulses continuously generated over the course of each behavioral act of an animal. However, predominant research approaches are based on the "stimulus - reaction" principle. Analysis of the dynamics of unit activity in monkeys during performance of a multi-step behavioral complex showed that differences related to different behavioral acts consist in composition changes in the active neurons (or their recombination) rather than in a number of responsive cells or involvement of action-specific neurons. Each combination of active neurons ensures the distribution of efferent signals characteristic of the given combination. These findings suggest the addressing coding of the efferent neuronal signals.     

CNF1 improves astrocytic ability to support neuronal growth and differentiation in vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.     

P2Y receptors activate neuroprotective mechanisms in astrocytic cells

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.     

Microtubule dynamics in neuronal morphogenesis

Microtubules (MTs) are essential for neuronal morphogenesis in the developing brain. The MT cytoskeleton provides physical support to shape the fine structure of neuronal processes. MT-based motors play important roles in nucleokinesis, process formation and retraction. Regulation of MT stability downstream of extracellular cues is proposed to be critical for axonogenesis. Axons and dendrites exhibit different patterns of MT organization, underlying the divergent functions of these processes. Centrosomal positioning has drawn the attention of researchers because it is a major clue to understanding neuronal MT organization. In this review, we focus on how recent advances in live imaging have revealed the dynamics of MT organization and centrosome positioning during neural development.     

Mean field model of acetylcholine mediated dynamics in the thalamocortical system

A recent continuum model of the large scale electrical activity of the thalamocortical system is generalized to include cholinergic modulation. The model is examined analytically and numerically to determine the effect of acetylcholine (ACh) on its steady states, linear stability, spectrum, and temporal responses. Changing the ACh concentration moves the system between zones of one, three, and five steady states, showing that neuromodulation of synaptic strength is a possible mechanism by which multiple steady states emerge in the brain. The lowest firing rate steady state is always stable, and subsequent fixed points alternate between stable and unstable. Increasing ACh concentration changes the form of the spectrum. Increasing the tonic level of ACh concentration increases the magnitudes of the N100 and P200 in the evoked response potential (ERP), without changing the timing of these peaks. Driving the system with a pulse of cholinergic activity results in a transient increase in the firing rate of cortical neurons that lasts over . Step-like increases in cortical ACh concentration cause increases in the firing rate of cortical neurons, with rapid responses due to fast acting nicotinic receptors and slower responses due to muscarinic receptor suppression of intracortical connections.     

Organized inclusions in astrocytic and amorphous inclusions in neuronal mitochondria of human frontal brain tissue

Brain tissue specimens were obtained early post mortem from the frontal lobe of seven unselected elderly subjects known to have been free of neurologic disease. Electron microscopically, two types of intramitochondrial inclusions were seen in four of seven cases. In two cases a few astrocyte mitochondria of the gyral white matter showed dense, elongated inclusions with an ordered linear substructure. These inclusions were, as a rule, accompanied by a row of prismatic cristae. In three cases some nerve cell mitochondria contained amorphous material of medium density and compact appearance. The globular masses often occupied the whole width of the mitochondrion. A relationship between the observed finding and a particular disease or morbid condition was not apparent. The inclusions are regarded as the morphologic substrate of a nonspecific metabolic change or degenerative process of the mitochondrion.     

Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury

Glutamate excitotoxicity plays an important role in the development of secondary injuries that occur following traumatic brain injury (TBI), and contributes significantly to expansion of the total volume of injury. Acute increases in extracellular glutamate levels have been detected in both experimental brain trauma models and in human patients, and can lead to over-stimulation of glutamate receptors, resulting in a cascade of excitotoxic-related mechanisms culminating in neuronal damage. These elevated levels of glutamate can be effectively controlled by the astrocytic glutamate transporters GLAST (EAAT1) and GLT-1 (EAAT2). However, evidence indicate these transporters and splice variant are downregulated shortly following the insult, which then precipitates glutamate-mediated excitotoxic conditions. Lack of success with glutamate receptor antagonists as a potential source of clinical intervention treatment following TBI has resulted in the necessity for a better understanding of the mechanisms that underlie the process of excitotoxicity, including the function and regulation of glutamate transporters. Such new insight should improve the likelihood of development of novel avenues for therapeutic intervention following TBI.