Synaptogenesis in rat cerebral cortex cultures is affected during chronic blockade of spontaneous bioelectric activity by tetrodotoxin

Huizen, F. van; Romijn, H.J.; Habets, A.M.M.C.

doi:10.1016/0165-3806(85)90232-9

Cited by 103 publications

(58 citation statements)

References 39 publications

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“…Occasionally, low-threshold, slow calcium spikes were observed, but these did not evoke synaptic potentials. In agreement with previous reports (35)(36)(37), replacement of the medium with fresh, TTX-free medium resulted in recovery of spontaneous activity and action potentials within a few minutes. We observed no obvious effects of chronic exposure to TTX on overall levels of immunoreactivity or on postsynaptic clustering for either GluRl or GABAAR,32/3 (Fig.…”

Section: Resultssupporting

confidence: 80%

Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters.

Craig

Blackstone

Huganir

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

225

170

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Several immunocytochemical and physiological studies have demonstrated a concentration of neurotransmitter receptors at postsynaptic sites on neurons, but an overall picture of receptor distribution has not emerged. In particular, it has not been clear whether receptor clusters are selectively localized opposite terminals that release the corresponding neurotransmitter. By using antibodies against the excitatory glutamate receptor subunit GluRl and the inhibitory type A yaminobutyric acid (GABA) receptor P2/3 subunits, we show that these different receptor types cluster at distinct postsynaptic sites on cultured rat hippocampal neurons. The GABAA receptor .32/3 subunits clustered on cell bodies and dendritic shafts opposite GABAergic terminals, whereas GluRl clustered maiuly on dendritic spines and was associated with glutamatergic synapses. Chronic blockade of evoked transmitter release did not block receptor clustering at postsynaptic sites. These results suggest that complex mechanisms involving nerve terminal-specific signals are requlired to allow different postsynaptic receptor types to cluster opposite only appropriate presynaptic terminals.At the neuromuscular junction, postsynaptic receptors are precisely localized opposite presynaptic terminals (1). There is now considerable evidence that neurotransmitter receptors are also clustered at postsynaptic sites on neurons, albeit in many cases less tightly than at the neuromuscular junction. By immunoelectron microscopy, Triller et al. (2) (21, 22). We found that glutamate receptor subunits GluRl and GluR2/3 cluster at a subset of postsynaptic sites on cultured hippocampal neurons, which is suggestive of an element of specificity in receptor clustering (13). These results are consistent with the possibility that receptors selectively cluster opposite appropriate presynaptic terminals, but direct evidence has been lacking.To resolve this question, we have performed a series of double-label immunolocalization experiments to compare the distributions of different types of postsynaptic receptors in individual cells and to relate the localization of postsynaptic receptors with that of different types of presynaptic terminals. We present here direct evidence that the glutamate receptor subunit GluRl and the GABAA receptor 82/3 subunits (GABAARP82/3) selectively cluster opposite terminals releasing the corresponding neurotransmitters and, furthermore, that postsynaptic receptor clustering occurs in the absence of evoked transmitter release. MATERIALS AND METHODSHippocampal cells were prepared from 18-day rat embryos by dissociation with trypsin, plated on poly(L-lysine)-coated glass coverslips at 2400 cells per cm2, and maintained in serum-free medium above an astroglial monolayer (23, 24).Cytosine arabinoside (5 ,AM) was added 3 days after plating to inhibit the proliferation ofnonneuronal cells. For inhibition of synaptic activity, tetrodotoxin (TTX; 1 ,uM) was added to the culture medium on day 0 when the coverslips were placed over glial monolayers and ...

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Section: Resultssupporting

confidence: 80%

Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters.

Craig

Blackstone

Huganir

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

225

170

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“…For instance, K max may have values up to several thousands of synaptic contacts. In order to set realistic connectivity ranges we used approximations based on the mean number of synapses and neuron seeding density per volume unit, as observed in our own experiments or by others (Van Huizen et al 1985;Habets et al 1987). According to the experimental protocol in Habets et al (1987), the neuronal seeding density was about 2000 cells/ mm 2 , which is similar to our experiments, and number of synapses per 10 3 µm 3 ranged from about 30 to 160.…”

Section: Network Parameterssupporting

confidence: 50%

“…Neurons in cortical networks receive several thousands of synaptic inputs in vivo and about one thousand in vitro (Van Huizen et al 1985;Ichikawa et al 1993), delays may exceed the 40-ms range in vivo (Swadlow and Waxman 1975) or 20-ms in vitro (Muller et al 1997). Moreover, several experimental studies on synaptogenesis in cortical neuronal cultures show that network activity is correlated with the average number of synapses per neuron (further referred to as connectivity; Habets et al 1987;Muramoto et al 1993).…”

Section: Introductionmentioning

confidence: 99%

Network bursts in cortical cultures are best simulated using pacemaker neurons and adaptive synapses

et al. 2010

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One of the most specific and exhibited features in the electrical activity of dissociated cultured neural networks (NNs) is the phenomenon of synchronized bursts, whose profiles vary widely in shape, width and firing rate. On the way to understanding the organization and behavior of biological NNs, we reproduced those features with random connectivity network models with 5,000 neurons. While the common approach to induce bursting behavior in neuronal network models is noise injection, there is experimental evidence suggesting the existence of pacemaker-like neurons. In our simulations noise did evoke bursts, but with an unrealistically gentle rising slope. We show that a small subset of 'pacemaker' neurons can trigger bursts with a more realistic profile. We found that adding pacemaker-like neurons as well as adaptive synapses yield burst features (shape, width, and height of the main phase) in the same ranges as obtained experimentally. Finally, we demonstrate how changes in network connectivity, transmission delays, and excitatory fraction influence network burst features quantitatively.

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“…Using morphological indicators of neuronal maturation, we have established an inverse correlation between the frequency of junctional communication connecting neurons and astrocytes and the extent of neuronal maturation in these cultures. It is well established that neuronal morphological differentiation is accompanied by maturation of chemical synapses, as observed in both dissociated and organotypic primary CNS cultures (30)(31)(32)(33)(34)(35). In addition, traditionally, it has been suggested that neuronal differentiation and, more specifically, final maturation of chemical synapses, follow the silencing of electrical ones (36,37).…”

Section: Discussionmentioning

confidence: 99%

Gap-junctional coupling between neurons and astrocytes in primary central nervous system cultures

Fróes

Correia

Garcia-Abreu

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

162

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Gap-junctional communication between neurons and astrocytes dissociated from rat brain was identified in culture by using dye-transfer assays and electrophysiological measurements. Cell types were identified by using antibodies against ␤-tubulin III, glial fibrillary acidic protein, and 2,3-cyclic-nucleotide phosphohydrolase, which are antigenic determinants of neurons, astroglia, and oligodendrocytes, respectively. Dye coupling was examined as a function of time after dissociated embryonic brain cells were plated onto confluent monolayers of postnatal astrocytes by intracellularly injecting the fluorochrome Lucifer yellow. Coupling of neurons to the astrocytic monolayer was most frequent between 48 h and 72 h in culture and declined over the next 4 days. This gradual uncoupling was accompanied by progressive neuronal maturation, as indicated by morphological measurements in camera lucida drawings. Dye spread was abolished reversibly by octanol, an agent that blocks gap junction channels in other systems. Double whole-cell voltage-clamp measurements confirmed the presence of heterocellular electrical coupling in these cocultures. Coupling was also seen between neurons and astrocytes in cocultures of cells dissociated from embryonic cerebral hemispheres but was rarely detectable in cocultures of postnatal brain cells. These data strongly suggest that junctional communication may provide metabolic and electrotonic interconnections between neuronal and astrocytic networks at early stages of neural development and that such interactions are weakened as differentiation progresses.The nervous system has great complexity in terms of the diversity of cellular contacts and the broad range of information processing performed by neural cells. Thoroughly debated and overwhelmingly accepted nearly a century ago, the neuron doctrine (1) not only established cellular individuality in the brain parenchyma but also designated neurons as the functional elements in signaling in the central nervous system (CNS). As a consequence, research has focused on elucidating the cellular and molecular details of neuronal pathways, whereas glial cells have been regarded as elements of structural and trophic support, with no direct influence in information processing. However, in the past few years, evidence has accumulated indicating that glial networks may provide functional support to neuronal activity and may constitute dynamic pathways for electrical and chemical signaling in the CNS. Studies showing synchronous metabolic and electrical responses in astrocytes mediated by neuronal-glial interactions demonstrate that, in culture, both slow and rapid calcium waves propagate through the functional syncytium provided by the astrocytic network in response to iontophoretic application of glutamate to astrocytes (2) and to firing of glutamatergic neurons (3, 4). Conversely, the propagation of astrocytic calcium waves to neurons has been more recently reported (5-7). Calcium signaling from astrocytes to neurons in culture was proposed to ...

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Synaptogenesis in rat cerebral cortex cultures is affected during chronic blockade of spontaneous bioelectric activity by tetrodotoxin

Cited by 103 publications

References 39 publications

Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters.

Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters.

Network bursts in cortical cultures are best simulated using pacemaker neurons and adaptive synapses

Gap-junctional coupling between neurons and astrocytes in primary central nervous system cultures

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