Action Potentials Issue #9 - ALA Scientific Instruments

Proudly Celebrating Our
16 th Year Anniversary
Issue #9 Spring / Summer 2002 A L A Scientific Instruments
Furthering Science through Innovative Instrumentation
Issue Contents
Product Forum Update:
+ VA-10 - Voltammeter Amplifier
+ CFE-1 - Carbon Fiber Electrodes
+ EPC-10 - Automated Patch Clamp
+ BAM-10 - Biological Audio Amplifier
Articles:
+ Renger Article (Iontophoresis Products)
Miscellaneous:
+ Upcoming Sponsored Symposium
+ ALA...Technical Tips
+ ALA...News
+ ALA...Around the World
+ ALA...Scientific User Web Forum
Product Forum Update
VA-10 - Voltammeter Amplifier
The VA-10 is a sensitive (picoampere range) current
amplifier that can be used to detect quantal
transmitter release (amperometry) from single cells in
tissue culture. It was designed at the Max-Planck
Institute for Experimental Medicine in G ttingen as an
economically priced alternative to do-it-yourself systems
and expensive commercial systems.
The amplifier is available in two versions: 19" cabinet
with built-in power supply, or plug-in units for the
EPMS modular system. Both versions are equipped
with a small headstage with a BNC connector.
VA-10 -Rack Mount (19 )
VA-10 - Modular
Product Forum Update...
EPC-10 - Automated Patch Clamp Amplifier
Revolutionary Advantages:
+ Full Computer Control
+ Automatic Self-Test and Calibration
+ Automatic Capacitance Neutralization
+ Capacitance Tracking
+ Automatic Leak Subtraction
New Features from it s predecessor EPC-9:
+ New Interface with Fiber Optic Connection
+ Low Frequency Voltage Clamp
+ True Current Clamp
+ Ultra-Slim Headstage
New!!
BAM-10 - Biological Audio Amplifier
Audio monitors have a long history as partofthe
electrophysiology
setup. An audio monitor provides signals audible to the
human ear while eyes and hands are fully occupied during experiments.
Audio signals are intuitive, economic, and in some cases,
essential.
CFE-1 - Carbon Fiber Electrodes
The CFE-1 Carbon Fiber Electrodes are superior in
design to all other types on the market. An insulation
method process called EDP (electrodeposition of paint)is
applied in the manufacturing process. This improves the
noise performance over polypropylene types, improves
electrode stability, and allows the electrode to be cut back
after each use, thereby increasing the useful life- span of
each electrode by a factor of at least 10 over other types.
.118 (3.0 mm)
2.250 (57.15 mm) +/- .25
*
High frequency signals, in the sensitive range of human ears, can be
amplified directly and then sent to our ears through speakers or earphones
(DIRECT mode). Both amplitude and frequency can be discriminated
by the human ear. DC or very slow changing signals (i.e.
resting membrane potential) can not be heard by human ears directly.
However, using the frequency
modular technique (MODULA-
BAM-10
Price Reduction!!!
From: $ 895.00
To: $ 650.00
TION), such slow changing biological signals
can be monitored by coding the signal
into frequency changes of an audible
signal with fixed amplitude. That is, the
tone of an audible sound follows the
amplitude changes of the biological
signal under recording.
(*Fitsstandard BNC female - Exact size may vary.)

Feature Article: Iontophoresis Products - Non-equilibrium activation of ligand-gated receptors
through ultrafast micro-iontophoresis
John J. Renger, Ph. D.
Synapses are the fundamental functional unit within the nervous system
for communication between neurons and essential in processes
such as learning and memory, yet these dynamic structures remain relatively
poorly understood. Synapses continue to conceal their
secrets largely because techniques are unavailable to directly measure
the process of synaptic transmission in real time and at individual
identifiable synapses. Further, the study of synaptic transmission
is easily confounded by ongoing changes in the state of the presynaptic
terminal (e.g. synaptic vesicle depletion, residual calcium,
changes in the number of vesicle fusion events, etc.), the post synaptic
membrane (density and distribution of receptors, receptor
conductance, etc.), or other, as yet unknown, alterations. Additionally,
much of what has been inferred about synaptic function is based on
the population behavior of synapses (ie. field recording, recording from
various neuromuscular junction preparations, whole-cell recording
from neurons with many tens to hundreds of synaptic connections), not
on the properties of individually identifiable release sites. To more precisely
examine synaptic transmission at identifiable synapses, we can
eliminate many assumptions regarding presynaptic neurotransmitter
release by using ultrafast micro-iontophoresis both in
conjunction
with, or in place of, endogenous neurotransmitter release.
Ultrafast micro-iontophoresis is a powerful research tool for examining
functional receptor distribution on neuronal arbors. In addition, this
technique permits the direct determination of receptor activation, inactivation,
and desensitization with non-equilibrium applications of
ligand. This approach has further revealed a new mechanism for the
relative activation of receptors within synapses that contain mixed
receptor types.
Often research focuses on the properties of
neurotransmitter-gated
receptors under equilibrium conditions of ligand.
This may largely reflect the ease with which bulk application of
ligand can be carried out. However, bath application of a neurotransmitter
is non-physiological condition and akin to pathological
conditions. At equilibrium, ligand-gated receptors fail to recapitulate
the normal physiological
characteristics seen when neurotransmitter
is applied very briefly (over the range of microseconds to
milliseconds) as occurs during synaptic transmission. Thus, it
should be understood that during normal synaptic transmission ligandgated
receptors are activated under non-equilibrium conditions.
Furthermore, complications from receptor internalization, desensitization,
and inactivation are most likely to occur over long periods (seconds
or greater) of bath applied transmitters/ ligands which may lead
to inaccurate conclusions regarding receptor function. Therefore, to
best understand the physiological properties of neurotransmitter
receptors, the application of neurotransmitter must closely mimic the
temporal and spatial characteristics of normal
neurotransmission.
It is possible to apply neurotransmitter in a near-physiological manner
using a state-of-the-art iontophoresis amplifier (MVCS-02C, npi
electronic; c.f. Figures 4A and 4C in Liu et al., 1999). Comparisons of
iontophoresis-evoked A M PA
(a-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid) receptor responses to brief applications
(1 ms duration, 100 nA; 100 m (Rseries iontophoresis electrode)
of neurotransmitter (150 mM Na-glutamate, pH 7.0) with
endogenous miniature synaptic transmission events, showed that this
iontophoresis system was capable of faithfully and stably reproducing
synaptic-like responses with less variability than endogenous
neurotransmission (c.f. Figure 5 Liu et al., 1999).
The ultrafast micro-iontophoresis technique can also be applied to
answering questions as to how, when, what types, and under what
conditions, functional ligand-gated receptors enter synapses during
development. Cottrell et al., (2000) used micro-iontophoresis to
establish the early appearance of functional NMDA (n-methyl-Daspartate)
receptors and A M PA receptors and their clustering at
synapses during development. Using repetitive iontophoresis applications
in combination with computer-controlled robotic micromanipulation,
this group was able to "map" functional glutamate receptors along
dendritic arbors.
A recent application of ultrafast micro-iontophoresis demonstrated
that varying the amplitude-duration parameters of iontophoresis
ejection pulse leads to dramatic alterations in the ratio of A M PA and
NMDA receptor activation (Figure 1; and see Renger et al., 2001).
This approach directly demonstrated that the concentration-duration
profile of neurotransmitter is a critical feature for determining how the
physiological function of the different receptor types can be selectively
controlled within a single synapse. In these experiments, it was
shown brief pulses of glutamate (ejection pulse = 1 ms duration, 100
nA amplitude) were effective at activating both A M PA and NMDA
receptors, while applying the same total amount of transmitter over a
longer time period (ejection pulse = 10 ms duration, 10 nA amplitude)
was efficacious for the preferential activation of NMDA receptors. This
selective activation of receptor types during non-equilibrium application
of neurotransmitter demonstrates the necessity of using nearphysiological
activation of ligand-gated receptors to understand their
biological function.
To test hypotheses regarding receptor activation, inactivation, and
desensitization during endogenous transmitter release, ligandgated
receptors need to be examined with stable ultrafast applications
of neurotransmitter that reproduce endogenous release parameters as
closely as possible. To carry out this approach in our experiments,
several important technical issues needed to be addressed. As reported
in Liu et al. (1999), for mimicking endogenous release it was
important that the time constant of the iontophoresis electrode be minimized.
In order to accomplish this, we used quartz capillary glass and
a Flaming-Brown laser puller to design electrodes that have a tip
inner diameter

Feature Article: Iontophoresis Products - Non-equilibrium activation of ligand-gated receptors
through ultrafast micro-iontophoresis...
To guide the electrode among synapses, a confocal microscope was
used in conjunction with robotic manipulators so that the tip of the electrode
could be placed within 1 micron of the putative synaptic terminals.
The distance between the electrode tip and the synapseiscritical
for determining the efficacy of the response, as shown by the dramatic
drop in the response in mapping experiments from Liu’s group
(Cottrell et al., 2000, Renger et al., 2001). Thus, careful consideration
must be made in designing the optics of the system with which this
technique is used. It is also important to note that this system can
theoretically be used for the application of many charged molecules.
For some neurotransmitters, it is necessary to consider the
pKa of the molecule and adjust the pH accordingly in order achieve a
net charge on the molecule for its use in iontophoresis. We have
been successful in using this device for GABA iontophoretic application
by acidifying the solution in which GABA is dissolved (pH 4.0).
Figure 1: Concentration-duration profile of neurotransmitter determines
A M PAR/ NMDAR activation at an individual synapse.
Iontophoresis - MVCS-02C Series
The ONLY iontophoresis
system that simulates
synaptic events!
Allows receptor mapping. Spatial resolution: 1 M
Using an advanced capacitance compensation circuit the MVCS
system allows very fast drug applications. Time
resolution:500 s
Automated balancing of iontophoretic current
Currents from tens of pA to hundreds of A
High-voltage, high-speed current source
Automated electrode resistance test
Select References:
1) G. Liu et al. (1999), Neuron, Vol. 22, 395-409. 1999
2) J.J. Renger et al. (2001), Neuron, Vol. 29, 469-484. 2001
3) Cottrell et al., J. Neurophysiol., Vol 84, 1573-1587. 2000
A. Schematic of the experimental design for iontophoretic examination
of postsynaptic receptors. The iontophoresis electrode is
brought to within 1 m of an isolated synapse. Filled vertical and horizontal
bars represent the "Fast" (1 ms, 100 nA) and "Slow" (10 ms, 10
nA) iontophoretic application parameters.
B. "Slow" iontophoretic pulses preferentially elicited NMDAR-only
responses from synapses on a voltage clamped hippocampal neuron,
while "Fast" pulses elicited A M PAR and NMDAR responses from the
same site. A M PA receptors failed to activate in the presence of a
"Slow" flux of glutamate. This data demonstrates that "AMPA-quiet"
responses can be generated at synapses with functional A M PAR’s
(modified from Renger et al., 2001).
As our understanding of the complexities of the synaptic transmission
process advance, we will design better and more accurate tests
of the function of these intriguing structures called synapses. A significant
requirement for this advancement is our ability to identify and utilize
the technologies that allow us to precisely reproduce physiological
events, as we understand them. Currently, ultrafast micro-iontophoresis
is a technology that most faithfully reproduces endogenous
synaptic
transmission and as such, enables experiments to
uncover the next trove of hidden secrets of the synapse.
A Merck-MIT Fellowship and RIKEN-MIT Fellowship supported JJR.
W ork described here was also funded by NIH and RIKEN grants to G.
Liu at MIT.
John J. Renger, Ph. D.
RIKEN Neuroscience Center, Center for Learning and Memory
Massachusetts Institute of Technology, 77 Massachusetts Avenue
Cambridge, MA 02139
Current Address: WP 26-265, Molecular Pharmacology
Merck & Co. Inc., 770 Sumneytown Pike, West Point, PA 19486
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ALA...News
Action Potentials Fall/Winter 2002 Preview
ALA s new 8-Trode , for in vivo extracellular recoding with chronic
or active implantation
ALA...Around the World
Society for Research on Biological Rhythms Meeting (SRBR) 2002
-Amelia Island, FL - May 22-26, 2002
Forum of European Neuroscience (FENS) 2002 - Paris, France,
July 13-17, 2002 - Symposium From Networks to Molecules
Society for Neuroscience 2002 - Orlando, FL - November 2-7, 2002
-Sponsored Symposium Planned - TBA
Biophysical Society 2003 - San Antonio, TX - March 2-4 2003
M E A Recording Discussion & Feedback on MEA system from
MultiChannel Systems.
Perfusion Forum Setup, Troubleshooting, General Questions.
Amplifiers General Questions & Comments

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