|A Proposal to Examine the Role of N-
and P- type Voltage-Dependent Calcium Channels in Spontaneous Bursting
Activity of Cultured Neuronal Networks.
The University of North Texas
requests $[to be determined] from the National Science Foundation (NSF)
to explore the role of N- and P-type voltage-dependent calcium channels
(VDCCs) in bursting of cultured neuronal networks. Spontaneous bursts
are a ubiquitous feature of the activity of cultured neuronal networks
as well as in vivo systems.
Bursts may be a more fundamental
method of interneuronal signaling than single action potentials.
Endogenous bursting is frequently
associated with and believed to play a significant role in central pattern
generators (CPGs), the circuitry that produces stereotypic movements such
as those used in walking or swimming (Wenner, 2001).
Presently N- and P-type VDCCs
are unique among the six known classes of VDCCs blocked by specific agents.
Many other VDCC antagonists exist, but act over a broader spectrum of channels.
Specifically, omega-Conotoxin GVIA is a peptide neurotoxin which selectively
and reversibly blocks N-type VDCCs. Omega-Agatoxin IVA selectively and
reversibly blocks P-type VDCCs.
Rhoades and Gross (1994) established a
dependence on extracellular calcium in bursting using more broad spectrum
VDCC antagonists such as veratridine and ditiazem. The greater specificity
of the compounds employed in the proposed study will greatly resolve the
roles of two calcium channels.
Both channels possess different gating
characteristics and tissue specificity. For example, N-type VDCCs
are activated by high voltage, while P-type VDCCs are activated by low
voltage. Further, N-type VDCCs are opened transiently while T-type
VDCCs remain open for a greater duration. The differences between
these and other types of VDCCs almost certainly to convey neuronal networks
the ability of modulate various parameters of their bursts.
Activity of the networks will
be recorded using micro-electrode arrays, glass plates onto which electrodes
are photoetched (Rhoades and Gross, 1994). Frontal cortex and spinal
cord tissue will be cultured from embryonic murine tissue on these arrays.
The data files are then processed by our
in-house software, IBurst, which derives a number of parameters of activity
such as spike and burst production, burst duration, interburst interval,
frequency of spikes in a burst, and so on. Bursts lend themselves
to a number of more complex analyses than spike production alone.
Whereas spike production is limited to relatively few measures (primarily
frequencies counts), a number of parameters may be analyzed from bursting
neurons. These parameters may be influenced directly by mechanisms
tied to specific subtypes of VDCCs.
Further, relationships may exist between
apparently independent measures of activity which can be teased out by
correlation analyses such as factor or principal components analysis.
Attempts at these analyses will lay the groundwork for additional applications
of statistical inference on other data sets collected in this laboratory
outside the scope of this study.
The tremendous advances in computing power
over the past decade have allowed the shift from primarily qualitative
research (comparisons of strip chart recordings) to considerably more quantitative
work. For example, the number of parameters our software may derive
from the activity of a neuron has increased from roughly half a dozen to
more than 40 variables. Additional, biologically relevant ratios
between these variables may be revealed in this study.
While these innovations increase the quantity
of data, they simultaneously create a greater workload for the researcher
in the analysis stage of the research. It is imperative that data
analysis be refined and automated as much as possible. A programmer
will be required to develop macros for sorting and analyzing this vast
quantity of values. Since data will be collected for the same series
of activity parameters for all experiments, regardless of their aim, these
macros will be utilized for additional studies, including previously collected
for Network Neuroscience
This research will be conducted
in the laboratories of the Center for Network Neuroscience (CNNS), which
includes a reliable infrastructure of equipment and software and its own
staffed cell culture facility.
Since its inception in 1987 the CNNS has
worked to develop a library of activity profiles of neuroactive compounds.
Such "fingerprints" have assisted in the prediction of the clinical effects
of compounds through the comparison of blind samples with the effects of
previously tested compounds (Keefer, 2001). However, the existing
library contains few examples of calcium channel antagonists and no examples
with the specificity of the agents described above.
Another goal of this work is the development
of new approaches for summarizing data into readily identifiable profiles
of influences on network activity. Because preliminary results indicate
that VDCCs preferentially influence burst production, this study represents
a unique opportunity to refine techniques in data analysis and data.
As such, this work may provide guidance in optimizing the existing compound
Programmer: SPSS macros
Cell Culture: technician and materials
(medium, mice, pipettes, etc.)
removed for individuals' privacy.
Keefer EW, Gramowski
A, Stenger DA, Pancrazio JJ, Gross GW. (2001) Characterization of acute
neurotoxic effects of trimethylolpropane phosphate via neuronal network
Biosens Bioelectron 16:513-25.
Rhoades, BK and
Gross, GW. (1994) Potassium and calcium channel dependence of bursting
in cultured neuronal networks.
Brain Research 643: 310-318.
Wenner P, O'Donovan
MJ. (2001) Mechanisms that initiate spontaneous network activity in the
developing chick spinal cord. J Neurophysiol 86:1481-98.