Action Potential Issue #15 - ALA Scientific Instruments

A Practical Guide to Pressure Polishing
Miriam B. Goodman, Assistant Professor
Dept. of Molecular & Cellular Physiology
Stanford University School of Medicine
Stanford, CA 94305 USA
Seminars, symposia, and technical workshops
sponsored by ALA Scientific, npi electronic,
Multi Channel Systems, and e.IP
Typical patch pipettes have tip openings of ~2 µm in
diameter and are sharply tapered. For a variety of applications,
smaller tip openings and a blunt shape are desirable.
For example, my colleagues and I routinely record
from C. elegans neurons (see Goodman et al., 1998;
Pierce-Shimomura et al., 2001; O'Hagan et al., 2005)
whose tiny cell bodies (1 to 3 µm in diameter) demand
pipettes with smaller tip openings. A blunt shape offers
several advantages even for pipettes with more typical tip
openings. First, a blunt pipette has a lower resistance
than a sharply tapered pipette with a similar tip opening
(13±2 MΩ vs. 5±1 MΩ , see Table 1 of Goodman and
Lockery, 2000). Second, a blunt pipette results in a larger
membrane bleb, presenting a larger surface area for
perforated patch recordings.
About five years ago, Shawn Lockery and I described a
method for shaping patch pipettes during pressure polishing
(Goodman and Lockery, 2000). We were motivated
to develop this method by the need to reliably fabricate
pipettes with
Fig. 1
sub-micron tip
openings and
resistances
less than 10
Closeup of CPM-2 stage MΩ. We found
that glasses
that naturally
formed blunt
tips (e.g. R-6)
were amenable
to making such
pipettes.
CPM-2 controller Unfortunately,
such glasses
are notoriously
noisy (Rae and
Levis, 1992), while glasses with adequate electrical properties
do not readily form blunt shapes (Table 1).
Pressure polishing offers a solution to this puzzle: a reliable
way to make blunt pipettes with sub-micron tip openings
from low-noise borosilicate glasses.
This is a practical guide for using pressure polishing in
your laboratory. Our pressure polishing rig consists of an
inverted microscope designed for routine tissue culture
work equipped with a CPM-2 kit from ALA Scientific
Instruments including a high-pressure pipette holder, an
ultra-long working distance 100X dry objective, a manual
valve for directing pressurized air through the pipette
lumen, and a sure regulator connected to the building's
pressurized air supply (Fig. 1). (My lab uses a Leica PL
FL L 100x/0.75, 4.7 mm working distance; comparable
lenses are also available from the metallurgical lines for
other microscope.) This set-up offers superior optics
compared to dedicated microforges that lack a condenser
and cannot accommodate a long working-distance highpower
objective.
In practice, pressure polishing involves four steps, is as
quick as standard fire-polishing, and, once the technique
has been mastered, it is quite reliable:
1) Mount the pipette in the high-pressure holder and position
it so that the pipette tip is level with the polishing filament
at high magnification. It is wise to position the polishing
filament just outside the field of view of the 100X
objective, as it will expand during heating.
2) Direct pressurized air (~40 PSI) down the lumen of the
pipette and activate the polishing coil. During this step
the pipette tip should expand slightly. If this step is prolonged,
the tip will expand like a balloon and burst!
3) Turn off the pressured air and polish the tip to the
desired tip opening size.
4) Double-check to ensure that the pressurized air is off,
then remove the pipette from its holder.
References:
Goodman MB, Lockery SR (2000) Pressure polishing: a method for re-shaping
patch pipettes during fire polishing. J Neurosci Methods 100:13-15.
Goodman MB, Hall DH, Avery L, Lockery SR (1998) Active currents regulate
sensitivity and dynamic range in C. elegans neurons. Neuron 20:763-772.
O'Hagan R, Chalfie M, Goodman MB (2005) The MEC-4 DEG/ENaC channel
of Caenorhabditis elegans touch receptor neurons transduces mechanical signals.
Nat Neurosci 8:43-50.
Pierce-Shimomura JT, Faumont S, Gaston MR, Pearson BJ, Lockery SR (2001)
The homeobox gene lim-6 is required for distinct chemosensory representations
in C. elegans. Nature 410:694-698.
Rae JL, Levis RA (1992) Glass technology for patch clamp electrodes. Methods
Enzymol 207:66-92.
Future issues of Action Potentials:
Multielectrode array analysis of circadian synchrony within
the suprachiasmatic nucleus
Sara J. Aton and Erik D. Herzog
Department of Biology, Washington University in St. Louis; St. Louis,
MO 63130
Gap Junction Recordings with two SEC amplifiers
Jose F. Ek-Vitorin and Janis M. Burt
Department of Physiology, University of Arizona, Tucson, AZ 85724
Please stay tuned for news about the sixth annual
Drug Discovery for Ion Channels satellite meeting at
the Biophysical Society meeting in Salt Lake City,
which ALA Scientific will be cosponsoring with
Molecular Devices.
SFN 2005
Symposium: Microelectrode &
Multielectrode Recording Techniques
When: Tuesday, November 15, 2005
Time: 5:30pm - 8:00pm
Where: Washington Convention Center -
Room: 209A
Introduction and Overview
Alan Kriegstein, President, ALA Scientific Instruments
Reiner Polder, Vice President, e.IP
“Pharmacology of two Nicotinic Acetylcholine Receptor
Subtypes on Insect CNS Neurons Studied with the Single-
Electrode Voltage Clamp Technique”
Dr. Vincent L. Salgado, BASF Corporation
“Neuronal avalanches and information storage: A few strong
connections”
Dr. John Beggs, Indiana University
"Leading and Dominance Asymmetry In Coupled Neural
Networks"
Itay Baruchi, Tel Aviv University
“The synapse is not everything: Modulation of excitability and
electrical coupling and their contribution to learning”
Dr. Brian D. Burrell, University of South Dakota
“Perforated Microelectrode Arrays Optimize Oxygen
Availability and Signal-to-Noise Ratio in Brain Slice
Recordings”
Dr. Uli Egert, University Freiburg
O'Hagan R., Chalfie M. and Goodman M.B. (2005) The MEC-
4 DEG/ENaC channel of Caenorhabditis elegans touch receptor
neurons transduces mechanical signals. Nat Neurosci. 8
pp. 43-50.
This study features the use of the "pressure-polishing technique" to form lowimpedance
small-tip patch pipettes, an essential capability for in vivo patch
clamp recordings of C. elegans neurons (see applications note this issue).
Fukuda, K., Nakajima, T., Viswanathan, P.C. and Balser, J.R.
(2005) Compound-specific Na+ channel pore conformational
changes induced by local anesthetics. J Physiol. 564 pp 21-
31.
This article is a mechanistic study of sodium channel pharmacology that features
the use of the ALA HSSE system for high-speed solution exchange.
Akitake, B., Anishkin, A. and Sukharev, S. (2005) The
"Dashpot" mechanism of stretch-dependent gating in MscS. J.
Gen. Physiol. 125 pp. 143-154
The ALA HSPC, high-speed pressure clamp, is used to induce rapid pressure
changes to mechanosensitive ion channels in the whole-cell patch
Interesting Current Papers:
FENS 2006
Technical Workshop: Noninvasive, highresolution
recording
When: July 8-12, 2006
Where: Vienna, Austria
Organized by: Andreas Draguhn (Univ.
Heidelberg) and Hans Reiner Polder (npi
electronic)
The recent merge of cellular and network physiology makes it
necessary to analyze the behavior of individual cells within
multi-cellular ensembles. Therefore, various approaches have
been developed to combine single-cell techniques with largescale
approaches. Many of these techniques allow recording
neuronal activity without penetration of the cell by an electrode.
Such recordings can be combined with staining and
structural analysis of the cells. Moreover, elegant stimulation
techniques by highly focal photo release of caged agonists
allow for a mapping of cortical circuits. Together, optical, electrophysiological
and histological approaches make it possible
to combine high-resolution cell physiology with large-scale
network analysis. Our workshop shall present several new
methodological approaches from leading laboratories in the
field of cellular and network physiology.
Invited speakers:
H.U. Dodt (Munich, Germany), D. Pinault (Strasbourg,
France), D. Schubert (Düsseldorf, Germany), Y. Kovalchuk
(Munich, Germany), R. Mason (Cambridge, United Kingdom),
R. Bruno (Heidelberg, Germany)
http://fens2006.neurosciences.asso.fr/pages/sympo.html#52
ALA Scientific Instruments is dedicated to providing the scientific community with instruments that can address the most fundamental
questions in neuroscience and biophysics. We are proud to present the following examples of seminal studies employing
our instruments:
configuration.
The ability to record from multiple electrodes in a defined
spatial arrangement is a significant advance in extracellular
recording techniques. Below are three very diverse applications
of this important technology:
Aton SJ, Colwell C.S, Harmar A.J, Waschek J and Herzog
E.D. (2005) Vasoactive intestinal polypeptide mediates circadian
rhythmicity and synchrony in mammalian clock neurons.
Nat Neurosci. 8 pp. 476-83.
E. Kolossov , Z. Lu , I. Drobinskaya, N. Gassanov , Y. Duan,
H. Sauer, O. Manzke, W. Bloch, H. Bohlen, J. Hescheler and
Fleischmann, B.K. (2005) Identification and characterization
of embryonic stem cell-derived pacemaker and atrial cardiomyocytes.
FASEB J. 19 pp. 577-9
M. J. Berry, J. L. Puchalla, E. Schneidman and Harris, R.A.
(2005) Redundancy in the population code of the retina.
Neuron 46 pp. 493-50