Early synaptogenesis in vitro: Role of axon target distance

Pol, Anthony N. van den; Obrietan, Karl; Belousov, Andrei B.; Yang, Yang; Heller, H. Craig

doi:10.1002/(sici)1096-9861(19981005)399:4<541::aid-cne7>3.0.co;2-1

Cited by 25 publications

(17 citation statements)

References 44 publications

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“…Moreover, the effects of astrocytic presence on synaptogenesis may be evaluated within 3-D matrices (Irons et al, 2008). In addition to influencing inter-neuronal distances and the number of synaptic partners in close contact, neuronal density also influences trophic factor concentrations and availability (van den Pol et al, 1998). Neurotrophic factors are involved in regulation of survival, neurite outgrowth, and synapse formation/plasticity in developing as well as mature neuronal networks (Oppenheim, 1991; Sayer et al, 2002; Waimey and Cheng, 2006).…”

Section: Discussionmentioning

confidence: 99%

“…In immature developing neuronal cultures (3-4 DIV), neurite outgrowth was enhanced (based on neurite length and growth rate) in higher density compared to low-density cultures, specifically implicating neuron-neuron contact (i.e., increased availability of membrane surface area for axonal guidance) in this enhanced neurite outgrowth (van den Pol et al, 1998). These neuronal cultures exhibited GABA and glutamate release, indicative of synaptogenesis, after 3-4 DIV in high-density cultures but not in low-density cultures at this time point (van den Pol et al, 1998). Enhanced neurite outgrowth in higher density cultures was thought to increase the probability of synapse formation, and thus explained the finding that the total synapses in these cultures increased with neuronal density at this early time point.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, plating density may influence the rate of neuronal maturation, as population-based bursting was first recorded at an earlier day in vitro (DIV) in cultures plated at higher density versus lower density (Wagenaar et al, 2006). In another culture system, high-density rat hippocampal neuronal cultures exhibited greater neurite extension than lower density cultures, suggesting that contact between cells influences neurite outgrowth (van den Pol et al, 1998). Additionally, at very early culture time points (3-4 DIV), higher density neuronal cultures exhibited a three-fold increase in synapse formation than low-density cultures, implicating the distance that axons/dendrites must traverse to reach a target in the rate of synaptogenesis in vitro (van den Pol et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

“…In another culture system, high-density rat hippocampal neuronal cultures exhibited greater neurite extension than lower density cultures, suggesting that contact between cells influences neurite outgrowth (van den Pol et al, 1998). Additionally, at very early culture time points (3-4 DIV), higher density neuronal cultures exhibited a three-fold increase in synapse formation than low-density cultures, implicating the distance that axons/dendrites must traverse to reach a target in the rate of synaptogenesis in vitro (van den Pol et al, 1998). While these studies provide evidence that neuronal density influences network electrophysiological behavior and initial synaptogenesis, the influence of neuronal density on the number of synapses per neuron in immature versus mature cultures has not been studied.…”

Section: Introductionmentioning

confidence: 99%

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Synapse-to-neuron ratio is inversely related to neuronal density in mature neuronal cultures

Cullen¹,

Gilroy²,

Irons³

et al. 2010

Brain Research

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Synapse formation is a fundamental process in neurons that occurs throughout development, maturity, and aging. Although these stages contain disparate and fluctuating numbers of mature neurons, tactics employed by neuronal networks to modulate synapse number as a function of neuronal density are not well understood. The goal of this study was to utilize an in vitro model to assess the influence of cell density and neuronal maturity on synapse number and distribution. Specifically, cerebral cortical neurons were plated in planar culture at densities ranging from 10 – 5000 neurons/mm2, and synapse number and distribution were evaluated via immunocytochemistry over 21 days in vitro (DIV). High-resolution confocal microscopy revealed an elaborate three-dimensional distribution of neurites and synapses across the heights of high-density neuronal networks by 21 DIV, which were up to 18 μm thick, demonstrating the complex degree of spatial interactions even in planar high-density cultures. At 7 DIV, the mean number of synapses per neuron was less than five, and this did not vary as a function of neuronal density. However, by 21 DIV, the number of synapses per neuron had jumped 30- to 80-fold, and the synapse-to-neuron ratio was greatest at lower neuronal densities (<500 neurons/mm2; mean approximately 400 synapses/neuron) compared to mid and higher neuronal densities (500 – 4500 neurons/mm2; mean approximately 150 synapses/neuron) (p < 0.05). These results suggest a relationship between neuronal density and synapse number that may have implications in the neurobiology of developing neuronal networks as well as processes of cell death and regeneration.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Synapse-to-neuron ratio is inversely related to neuronal density in mature neuronal cultures

Cullen¹,

Gilroy²,

Irons³

et al. 2010

Brain Research

View full text Add to dashboard Cite

show abstract

“…This could be due to the fact that the synaptic contacts are already established in mature cultures and are still developing in the immature cultures (van den Pol et al, 1998). Moreover, because the GABA A R-mediated activity is largely excitatory during early cell culture development (Ganguly et al, 2001; Obrietan and van den Pol, 1995), the network-driven glutamatergic activity may not be revealed: the application of bicuculline will result in neuronal inhibition, not disinhibition until about DIV10.…”

Section: Discussionmentioning

confidence: 99%

Recovery of network-driven glutamatergic activity in rat hippocampal neurons during chronic glutamate receptor blockade

Leininger

Belousov

2009

Brain Research

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Previous studies indicated that a long-term decrease in the activity of ionotropic glutamate receptors induces cholinergic activity in rat and mouse hypothalamic neuronal cultures. Here we studied whether a prolonged inactivation of ionotropic glutamate receptors also induces cholinergic activity in hippocampal neurons. Receptor activity was chronically suppressed in rat hippocampal primary neuronal cultures with two proportionally increasing sets of concentrations of NMDA plus non-NMDA receptor antagonists: 100 μM/10 μM AP5/CNQX (1X cultures) and 200 μM/20 μM AP5/CNQX (2X cultures). Using calcium imaging we demonstrate that cholinergic activity does not develop in these cultures. Instead, network-driven glutamate-dependent activity, that normally is detected in hyper-excitable conditions, reappears in each culture group in the presence of these antagonists and can be reversibly suppressed by higher concentrations of AP5/CNQX. This activity is mediated by non-NMDA receptors and is modulated by NMDA receptors. Further, non-NMDA receptors, the general level of glutamate receptor activity and CaMK-dependent signaling are critical for development of this network-driven glutamatergic activity in the presence of receptor antagonists. Using electrophysiology, western blotting and calcium imaging we show that some neuronal parameters are either reduced or not affected by chronic glutamate receptor blockade. However, other parameters (including neuronal excitability, mEPSC frequency, and expression of GluR1, NR1 and βCaMKII) become up-regulated and, in some cases, proportionally between the non-treated, 1X and 2X cultures. Our data suggest recovery of the network-driven glutamatergic activity after chronic glutamate receptor blockade. This recovery may represent a form of neuronal plasticity that compensates for the prolonged suppression of the activity of glutamate receptors.

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Surface-accessible GABA supports tonic and quantal synaptic transmission

Vautrin

Maric

Sukhareva

et al. 2000

Synapse

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Exocytosis is commonly viewed as the only secretory process able to account for quantal forms of fast synaptic transmission. However, the demonstrated variability and composite properties of miniature postsynaptic signals are not easily explained by all-or-none exocytotic discharge of transmitter in solution from inside vesicles. Recent studies of endocrine secretion have shown that hormone release does not coincide with exocytosis due to its trapping in the core matrix of the granule. Thus, we tested whether the synaptic transmitter GABA could also be held in a matrix before being released. Using confocal microscopy and flow cytometry of embryonic rat hippocampal neurons, we found a GABA immunoreaction at the surface of live cell bodies and growth cones that coincided spatially and quantitatively with the binding of tetanus toxin fragment C (TTFC). TTFC binds predominantly at membrane sites containing the trisialoglycosphingolipid GT1b. Using flow cytometry, GT1b-containing liposomes preincubated in 100 nM GABA exhibited the same relationship between GABA and TTFC surface binding as found on neurons and growth cones. Embryonic neurons differentiated in culture expressed initially a tonic, and after 3-5 days, transient, postsynaptic signals mediated by GABA acting at GABA(A) receptor/Cl(-) channels. A stream of saline applied to the neuronal surface rapidly and reversibly suppressed both tonic and transient signals. A brief application of the GABAmimetic isoguvacine immediately transformed both tonic and transient GABAergic signals into tonic and transient isoguvacinergic signals. These results and those in the literature are consistent with an immediately releasable compartment of transmitter accessible from the presynaptic surface.

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Early synaptogenesis in vitro: Role of axon target distance

Cited by 25 publications

References 44 publications

Synapse-to-neuron ratio is inversely related to neuronal density in mature neuronal cultures

Synapse-to-neuron ratio is inversely related to neuronal density in mature neuronal cultures

Recovery of network-driven glutamatergic activity in rat hippocampal neurons during chronic glutamate receptor blockade

Surface-accessible GABA supports tonic and quantal synaptic transmission

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