AXOBITSIntegrated Solutions for Cellular Neuroscience, Cell Based Screening and Functional Genomics Vol. 36 October 2002

IN THIS ISSUE:

2 A MESSAGE FROMThe Product Line Manager, High-Throughput Electrophysiology,Patch-Clamp Systems

3 NEW PRODUCT NEWSImageXpress 5000AOpusXpress 1.0 SoftwarepCLAMP 9.0

6 FOCUS ON METHODSThe Use of Net-Charge Analysis for the Study of Ion Channel Pharmacology

9 PROFILEYuri Opsichuk, Engineer Extraordinaire

10 TOOL TIPSANNOUNCING IMAGINGWORKBENCH 5.0 FROMINDEC BIOSYSTEMS

11 AXON REPLIES

Axon Instruments proudly introduces the release of the PatchXpress 7000A, Automated Parallel Patch-Clamp System. PatchXpress isa logical product to come from Axon, a company known for nearly two decades as the highest quality instrumentation and softwareprovider for electrophysiology. Not surprisingly, drug discovery companies have looked to Axon to provide an automated patch-clampsystem that increases ion channel screening throughput with the highest data quality. The PatchXpress 7000A records whole-cell currents from 16 channels simultaneously using high-performance planar patch-clamp electrodes provided exclusively by AvivaBiosciences.

Independent pressure control on all 16 channels results in high quality recordings with tight gigaseals and low access resistance values.The same team that brought you the versatile and powerful pCLAMP software suite developed the all new screening-orientedPatchXpress software. With it any user can control the automated procedures in PatchXpress like a veteran patch-clamper.

If you would like to see the PatchXpress 7000A, please visit the Axon booth at the upcoming Society for Neuroscience conference inOrlando, FL (www.sfn.org). Alternatively, contact our sales department for a demo (sales@axon.com). If you have further questionsabout the revolutionary PatchXpress 7000A, please check out our website (www.patchxpress.com) or contact me directly(chrism@axon.com).

Sincerely,

2 w w w . a x o n . c o m

A MESSAGE FROM...

Editors:Al Walter, Ph.D.Simone Elletson

Marketing:Debbie QuinnSimone Elletson

Cover:The cover of this issue of AxoBitsis a montage of images capturedusing ImageXpress.

Enclosed:Take a look at the enclosed CDwhich contains an animated shortfeature starring the OpusXpress6000A workstation.

Contributors:Al Walter, Ph.D.

Andrew Olson, Ph.D.

Damian Verdnik, Ph.D.

Burt Maertz

Cathy Smith-Maxwell, Ph.D.

Chang Wang, Ph.D.

Chris Mathes, Ph.D.

James Rountree, Ph.D.

Sean Carriedo, Ph.D.

Steven Smith, Ph.D.

Tony Figl, Ph.D.

Ward Yuhas, Ph.D.

...the Product Line Manager, High-Throughput Electrophyisiology, Patch-Clamp Systems

Chris Mathes, Ph.D.

Learn more about Axon Instruments at theseupcoming conferences:

TIGR Genome Sequencing and Analysis Conference, Boston, MA

October 2-5, 2002 Booth # 1402

American Society of Human Genetics, Baltimore, MD

October 15-19, 2002 Booth # 1213

Chips to Hits, Philadelphia, PA

October 27-31, 2002 Booth # 216

Society for Neuroscience, Orlando, FL

November 2-7, 2002 Booth # 457-462

Axon Instruments will present two symposia at the

Society for Neuroscience MeetingNovember 2, 2002

Rosen Centre Hotel, Salon 2

2:00 pm - 4:00 pm Recording and Analyzing Evoked and Spontaneous Events:LTP/LTD, Action Potentials, Miniatures, Single ChannelsAn overview of a wide variety of electrophysiological phenome-na will be presented using pCLAMP 9.0. Any and all interestedparties are welcome to attend.

4:00 pm - 5:00 pm An Introduction to Using DNA MicroarraysTopics to be touched upon include microarray construction,microarray scanners, issues involved when analyzing a scannedmicroarray image, and the bioinformatic approaches employedfor analyzing multiple microarray experiments.

Microarray Analysis Training

Get the most out of Acuity and GenePix Pro by choosing from oneof several programs of instruction: seminars, on-site training, andinternet-based training. For more detail and to schedule training,visit the Axon Instruments home page. Next conference seminar:

Chips to HitsPhiladelphia, PA, October 27-31 2002 GenePix Pro: Tuesday, October 29Acuity: Wednesday, October 30

Axon Instruments is proud to announce the release of theImageXpress 5000A automated cellular imaging and anal-ysis system. The ImageXpress system is designed for

rapid, high-resolution fluorescence imaging and analysis.

The ImageXpress optical system increases screening throughputwithout compromising image quality. It uses a full-spectrumhigh-power xenon arc lamp, custom light guide and Abbé illumi-nation optics to deliver efficient, uniform illumination to the sam-ple, and keep exposure times to a minimum. The motorized exci-tation and emission filter wheels (10 positions each) and patent-ed, motorized dichroic wheel (4 positions) accept off-the-shelffilter sets for maximum flexibility. The motorized turret holds upto six objectives. Thus, the system supports a full range of bio-logical microscope objectives and fluorescent dyes.

The precision motorized stages (X-Y stage for plate motion andZ stage for focusing) provide rapid movement and trouble-freecontinuous operation. Digital images are acquired by a cooled,high-resolution (1280 × 1024 pixels) CCD camera designed byAxon specifically for ImageXpress. High-speed laser autofocus-ing ensures rapid capture of crisp images without the risk of pho-tobleaching. The host computer communicates with theImageXpress hardware through Axon's own high-speed frame-grabber board.

ImageXpress 5000A control and analysis software is as versatileand flexible as the hardware, combining powerful features withease-of-use. The software consists of three separate modules: aconsole (user interface), an instrument server (hardware control),and a database server (storage and retrieval of annotated images).The modular design also allows you to monitor one or moreImageXpress systems over a network, and to share a centraldatabase among multiple ImageXpress systems.

The console's graphical user interface provides intuitive controlof the instrument in interactive mode, scripting of commands forautomated acquisition and analysis, plus visualization tools formonitoring automated acquisition and on-screen display of analy-sis results. All commands are fully scriptable, allowing unattend-ed operation and unparalleled flexibility. Script Wizards makewriting custom scripts a breeze.

The integrated client-server database makes managing the largevolume of image data easy. The ImageXpress database automati-cally stores images, annotated with complete acquisition informa-tion (e.g., time and date of acquisition, exposure time, excitationand emission filters and dichroics, objective lens, X-, Y- and Zpositions, etc.) and allows unlimited user annotations (e.g., drugcompounds and concentrations, cell type, incubation time, or anyother experimental conditions). Two query tools—a fully config-urable tree view and a flexible query dialog—make it simple toretrieve images based on the annotated information. The

database is fully network-enabled and scalable. The database canalso archive images to and retrieve images from mass storagemedia.

Automated image analysis tools for a wide range of cellularimaging tasks include: illumination and background correction,real-time cell counting, cell nucleus detection, cytoplasmic detec-tion or sampling, and neurite detection. A host of measurements(both fluorescence intensity measurements and object shape) areavailable for any or all of these regions. You can choose fromobject pixel statistics (average intensity, median, variance, etc.),fluorescence texture characterization, area, perimeter, best-fitellipse, angle of major axis, bounding box, center of mass, con-vex hull. The Analysis Script Wizard helps you to quickly createyour own custom script using these powerful algorithms alongwith the flexibility to create any number of derived measure-ments, including ratiometric analyses between separate fluores-cence channels. All image annotations (both system and user-supplied) are available for further characterizing the analysisresults.

Analysis results are displayed on-screen in spreadsheet formatand can be visualized in scatterplot and histogram windows. Anydata column can be instantly graphed. All of the results viewsare linked; for example, double-clicking on a data point in agraph automatically selects the object in the image view and theappropriate line in the spreadsheet. Analysis results can also beeasily exported to an external database or spreadsheet program.

Stop by our booth, # 457-462, at the Society for NeuroscienceAnnual Meeting for a demonstration.

NEW PRODUCT NEWS

AxoBits Vol. 36 October 2002 3

The ImageXpress Interface

OpusXpress software controls the OpusXpress 6000Aworkstation, an automated eight-channel voltage clampfor oocytes. This system increases the efficiency of drug

discovery for ion channels and transporters through automationand parallel recording. Data acquisition and on-line analysis arecarried out for up to eight oocytes in parallel using acquisitionprotocols designed in a protocol editor derived from Axon'sindustry-standard pCLAMP data acquisition software.OpusXpress software offers a high degree of flexibility, allowingusers control over all aspects of experimental design. As aresult, users can readily acquire data from ligand-gated and volt-age-gated ion channels, as well as from ion transporters. Forvoltage-gated ion channels, OpusXpress software supports com-plex voltage stimulus profiles with voltage steps and ramps aswell as p/n leak subtraction, permitting detailed studies of thevoltage-dependence of channel function and drug interaction.

All eight oocytes can be impaled automatically at the click of abutton. Amplifier controls, which reside in software, allow usersto go from setup mode to voltage clamp mode in all eight chan-nels simultaneously. However, the system is flexible enough toallow replacement of a subset of oocytes, leaving the remainingoocytes voltage clamped.

The Edit Experiment Procedure window (Figure 1) features astraightforward and intuitive user interface for quick and easyexperimental design. Each line represents a single fluid condi-

tion for which the fluid source, step duration, flow rate, and dataacquisition protocol and start time (delay) can be individuallyselected. Selection of multiple wells at once, as shown in step 2,is an easy shorthand for rapidly setting up procedures to testdrugs from any number of wells. The shorthand is translatedinto individual fluid steps in the Procedure window (Figure 2,bottom). Besides two choices of buffer (A and B), as many as24 drugs can be applied to a single oocyte (wells A1 through H3are used for oocyte 1). Up to six different one- or two-step flow

rate profiles can be specified for any one experiment, allowingtremendous flexibility in fluid delivery. Steps are identified asrepresenting Control or Test measurements (Acquisition Type) sothat users can normalize data from on-line analysis to easilycompare replicate drug responses between oocytes with differentcurrent magnitudes. Cell viability testing prevents addition ofdrugs to oocytes when recovery from previous drug addition isinadequate or when holding (leak) currents become too large.Therefore, drugs are not wasted on poor quality oocytes and areeasily saved for testing on viable oocytes. Cut-off thresholds forviability testing are entirely user-selectable.

Users can monitor all important aspects of an ongoing experi-ment with the two large 17" flat screen monitors that come aspart of each OpusXpress system. Recorded currents and volt-ages can be observed readily in the oscilloscope window duringan experiment (Figure 2, top middle). Extracted data measure-ments can be observed simultaneously in the on-line analysiswindow (Figure 2, right of the oscilloscope window). Displaysof normalized values of the data measurements are also readilyavailable for easy comparison of replicate measurements.Experiment progress can be followed simultaneously from astep-by-step outline of the experiment procedure (Figure 2, bot-tom) and from a window containing 96-well plate displays thatindicate the testing progress for each well (Figure 2, left). Whentesting from compound plates identified by bar code, pointingthe mouse at wells of interest identifies well content, both com-pound and concentration, and double-clicking on a well that hasalready been tested displays the data file.

OpusXpress software provides users of the OpusXpress 6000Aworkstation with a powerful tool for recording from eightoocytes in parallel and automating data acquisition and on-lineanalysis. The seamlessly integrated software makes it easy forusers to design and run experimental procedures, to follow theprogress of experiments during data acquisition, and to analyzedata during testing.

FOCUS ON SOFTWARE

4 w w w . a x o n . c o m

Figure 1

Figure 2

OPUSXPRESS 1.0 SoftwareParallel Oocyte Voltage Clamp

pCLAMP 9.0Comprehensive Electrophysiology

Software

Axon Instruments is proud to announce the release ofpCLAMP 9.0. This major upgrade to our widely usedelectrophysiology data acquisition and analysis software

suite offers expanded flexibility for performing a myriad of exper-iments.

The investigation of memory looks at synaptic plasticity throughLong-Term Potentiation/Depression (LTP/LTD) experiments.Due to the nature of the preparations, online monitoring andinteractive stimulation of the data is extremely important.Clampex 9.0 is now enhanced in several key areas, such asmulti-region statistics, analog/digital train stimulations, andinterruptible sequencing keys, to allow you to conduct this classof experiments.

Long-term monitoring is even easier with sweep lengths of onemillion samples. If that were not enough, our new MiniDigi 1Atwo-channel digitizer (included in pCLAMP 9.0) provides con-tinuous background chart recording, so you'll have a completerecord of all activity even in the absence of a stimulus. Monitorseal quality (resistance) during episodic acquisition, output trig-gers for sweep/event-starts, or view a running counter of detect-ed events. You're bound to discover new ways of usingClampex 9.0 that you'll soon find indispensable.

Multi-spike analysis covers a wide range of event types, such asevoked action potentials and spontaneous "miniature" events (i.e.EPSPs and EPSCs). Traditional amplitude-level thresholds andwindow discriminators are used to capture multiple events basedupon peak amplitudes. However, with "minis", a single class ofevents can widely vary in peak amplitude, so it is desirable toseparate events based upon other criteria. A scalable shape-matching detection algorithm is provided just for this purpose.Furthermore, all categories of detected events can be automati-

cally marked, captured and extracted in a single pass of the data.An extra bonus is the dynamic linking of all views of the data;from raw data to extracted events to spreadsheet measurementsto scatter plots—you'll like what you see!

Biophysicists rely upon statistical methods to analyze single-channel data, and need to process many thousands of single-channel events. Clampfit 9.0's Windows implementation ana-lyzes up to one hundred thousand events and supports both con-tinuous and episodic data. Typical results include amplitude his-tograms, dwell-time histograms, fitting, probability of opentimes Po and burst analyses.

You'll find many more analysis gems in Clampfit 9.0, such ascursor-pair delta values, variable rise and decay slope percentagesettings, data normalization and saved graphs, as well as moreesoteric analyses such as the Kolmogorov-Smirnoff statisticaltest, nonstationary fluctuation analysis for single-channel con-ductance, and variance-mean analysis for post-synaptic currents.Last but not least, for experimental flexibility, concatenate eithercontinuous or episodic files together.

With all these program enhancements (and more), you're sure tofind the right tools for success in pCLAMP 9.0!

NEW PRODUCT NEWS

AxoBits Vol. 35 October 2002 5

Sharing Data using AxoScope

Sharing data is easy using AxoScope! When you need to shareoriginal pCLAMP data with a colleague who does not usepCLAMP, have the colleague download AxoScope to use as aviewer. A fully functional version of AxoScope can be down-loaded for free from the Axon website (www.axon.com). UsingAxoScope, your colleague will be able to make cursor measure-ments, print the data, save the results sheet and save an editedversion of the data file. If need be, the data can also be savedas ASCII text for further analysis by almost any other program.AxoScope, like other Axon software, has extensive, context-sensitive online help that is accessed using the F1 key.

Train Generation

Template Event Detection

IntroductionConcentration-response studies are the keystone of ion channelpharmacology. For example, to characterize an experimentalagonist, responses to the drug are compared to responses evokedby a reference compound considered to be a full agonist. Suchconcentration-response comparisons provide information aboutboth the potency and efficacy of the experimental drugs relativeto the reference compound. Typically, these comparisons arebased on the measurement of the peak currents recorded inresponse to the agonist applications. However, by definition,peak currents are not equilibrium responses but rather representthe inflection point of a complex function determined by ligandbinding rates as well as channel activation, inactivation, anddesensitization rates. The kinetic features of the solutionexchange (i.e. drug delivery) also impact the amplitude and tim-ing of peak responses. Channels with rapid desensitization rates,such as the nicotinic α7 receptor, may produce peak currents thatoccur well before the dynamics of solution exchange can be com-pleted [1]. In such a case, how can a given concentration be saidto produce a certain response, when the peak current occurs at atime when the concentration of drug reaching the receptors isonly a small fraction of the full concentration applied? Oneapproach to this problem is to consider an alternative measure ofresponse such as the net charge associated with the agonist-evoked response (Figure 1).

In a voltage-clamp experiment the net charge equates to the totalamount of channel activation produced by a drug application.Since net charge is a measure of the total number of ions movingacross the membrane, it may be more predictive of some crucialin vivo effects such as changes in intracellular calcium, than ameasurement of peak current.

We have recently re-evaluated the pharmacology of the nicotinicα7 receptor, based on the use of net-charge analysis [2].Previous studies of α7 receptors suggest that this receptorresponds to acetylcholine (ACh) with relatively low affinity, hav-ing an EC50 (half-activation concentration) ranging from 100 µMto 300 µM. However, these estimates were all based on analysisof peak currents, and as mentioned above, with high agonist con-centrations, peak currents occur well before complete solutionexchange is achieved. Most of the published data on α7 recep-tors have come from study of the receptors expressed in Xenopusoocytes, however similar findings have been reported for nativereceptors on tissue-cultured [3] or acutely dissociated neurons[4, 5].

Here we show how net-charge analysis can be applied to thestudy of α7-type nicotinic acetylcholine receptors (nAChR) andwe address the question of whether this form of analysis might beappropriate for the characterization of other nAChR subtypes.

The α7 nAChR subunit functions as a homo-oligomeric channel,and channels containing this subunit represent one of the twomajor nAChR subtypes in the brain. These receptors do not bindnicotine with high affinity but can be labeled with α-bungarotox-in. Most of the nAChR that do bind nicotine with high affinityare composed of α4 and β2 subunits. The activation and desensi-tization properties of those receptors are very different from thoseof α7-type receptors. We compare and contrast the ACh respons-es of these two types of nicotine receptors found in the centralnervous system.

MethodsExperimental protocols and data analysisOocyte recordings were made using the OpusXpressTM 6000Aworkstation (Axon Instruments, Union City, California), unlessotherwise noted. The OpusXpress system is an integrated hard-ware and software package that provides automated impalement,fluidics, voltage clamp, data acquisition, and on-line analysis per-mitting the study of multiple oocytes in parallel. Cells were auto-matically perfused with bath and agonist solutions. The latterwere delivered from 96-well compound plates. Both the voltageand current electrodes were filled with 3 M KCl. The agonistsolutions were applied via disposable tips, which eliminated anypossibility of cross-contamination. Drug applications alternatedbetween ACh controls and experimental applications of ACh atconcentrations increasing from 0.1 µM to 3 mM. Flow rateswere set at 2 ml/min for α7 (except where noted) and 1 ml/minfor the other receptor subtypes. Cells were voltage-clamped at aholding potential of -60 mV. Data were collected at 50 Hz andfiltered at 20 Hz. ACh applications were 12 s in duration for α7receptors and 10 s for the other receptor subtypes. Drug applica-tions were followed by 400 s washout periods.

A standard manual recording method was used for one data set,using a recording chamber from Warner Instruments that wasmodified to reduce turbulence. Drug was supplied through a per-fusion line by manually filling a 1.8 ml drug loop [2]. The bathflow rate was 6 ml/min.

FOCUS ON METHODS

The Use of Net-Charge Analysis for the Study of Ion Channel PharmacologyRoger L. Papke1 and Julia K. Porter PapkeDepartment of Pharmacology and TherapeuticsBox 100267 JHMHSCUniversity of FloridaGainesville, Florida 32610-02671Corresponding author

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Acquire eight days worth of quality data in under 2 hours.

Calculations of net charge can be made by selecting the "area"statistic, either during data acquisition or during subsequent off-line analysis in Clampfit. However, net-charge (i.e. area) calcula-tion can be very sensitive to even small amounts of baseline drift.Therefore our area calculations were made in Clampfit so thatmanual baseline adjustment could be made when appropriate.The baseline was defined as the mean current for the 20 s periodbefore drug application. The analysis region for peak and net-charge analysis went from 5 s before the initiation of drug appli-cation through 135 s following. Mean and standard error of themean (SEM) were calculated from the normalized responses of atleast three oocytes for each experimental concentration.

Responses to experimental drug applications, both peak currentand net charge, were calculated relative to the preceding AChcontrol responses in order to normalize the data, compensatingfor the varying levels of channel expression among the oocytes.Data were initially normalized relative to a standard ACh concen-tration that activated easily measured responses but did not pro-duce significant accumulated desensitization with repeated appli-cation. The concentrations of the ACh controls were 30 µM forα4β2 and 300 µM for α7. For α4β2 receptors these values weresubsequently recalculated relative to the empirically determinedmaximum response. For concentration-response relations, datawere fit with the Hill equation

where Imax denotes the maximal response for a particular ago-nist/subunit combination, and n represents the Hill coefficient.Imax, n, and the EC50 were all unconstrained for the fitting proce-dures.

The solution exchange profiles shown in Figure 2 were generatedas voltage measurements of junction potential when 115 mMCsCl was introduced into the recording chamber using theOpusXpress drug delivery system. These solution exchange pro-files were compared with the evoked responses of different AChRsubtypes.

ResultsFigure 1 shows responses of an oocyte expressing human α7nAChR to the application of 30 µM, 300 µM, and 3 mM ACh.There is a 20-fold increase in the peak amplitude of the currentover this concentration range. However, this appears to be morea reflection of the synchronization of channel opening than thetotal amount of channel opening, as the net charge flow thatoccurred during these responses increased by less than a factor of 2.

In Figure 2A, the responses of an α7-expressing oocyte to theapplication of relatively low and high concentrations of ACh arecompared to an estimated profile of solution flow (green line)into the experimental chamber. The responses of oocytesexpressing α7 receptors show a dramatic change in waveformwhen the ACh concentration is increased. The response to

30 µM ACh continues to mount through much of the rising edgeof the drug application. However, when 300 µM ACh is applied,the response essentially terminates long before the concentrationof drug in the chamber approaches 300 µM. While in this exam-ple the peak of the 300 µM ACh response is about seven timesthat of the 30 µM ACh response, the net charge stimulated by the300 µM application is only about twice that stimulated by the 30 µM ACh application. Note that at the time of the peak currentresponse to the application of 300 µM ACh, the actual ACh con-centration in the chamber is likely to be less than 60 µM. In con-trast to the α7 responses, the responses of oocytes expressingα4β2 receptors show relatively little change in waveformbetween the 10 µM and 100 µM ACh-evoked currents (Figure2B). The rising phase of both responses follows the estimatedonset of solution exchange closely, so that the peak amplitude andnet charge measurements increase proportionately.

As shown in Figure 2C, for α7 receptors the net-charge concen-tration-response curve is shifted to the left by about a factor often relative to the curve for peak currents, while for α4β2 recep-tors the peak current and net-charge concentration-responsecurves are nearly superimposable (Figure 2D). Results are sum-marized in Table 1.

Peak responses Net-charge analysisReceptor Imax Hill Coef. EC50 (µM) Imax Hill Coef. EC50 (µM)α7 0.99 ± 0.02 1.4 ± 0.12 159 ± 11 0.94 ±.03 1.6 ± 0.2 37.3 ± 3.8α4β2 0.97 ± 0.07 0.7 ± 0.1 11 ± 3.7 0.92 ± 0.05 1.2 ± 0.3 17 ± 4

The net-charge analysis would seem to be a better representationof α7 receptor activation, considering that with the application ofhigh agonist concentrations, the peak currents themselves areoccurring when the concentration is only about a tenth of the fullconcentration applied.

FOCUS ON METHODS

[ ][ ] ( )

nmax

n50

I agonistResponse

agonist EC=

+

Figure 1. Responses of an oocyte expressing human α7 nicotinic AChR to a 100-fold rangeof ACh concentrations. Such responses are normally characterized in reference to the peakcurrent amplitudes which in this experiment increase by more than twenty times over this con-centration range (top). However, when net charge is calculated (illustrated in red below), thereis less than a two-fold change in response.

Table 1

AxoBits Vol. 36 October 2002 7

Typically, oocyte recording systems vary from one lab to anotherin the details of chamber design, drug application, flow rates, etc.In order to determine whether or not net-charge analysis is sensi-tive to such technical differences, we compared the data obtainedfrom multiple oocytes expressing human α7 receptors recorded inparallel with the OpusXpress system at two different flow rates (1 ml/min and 2 ml/min) with previously published data obtainedwith a standard manual recording method and a flow rate of 6 ml/min (see Methods). It is important to note that the dataobtained with the manual recording method took 8 days to collect.In a typical OpusXpress concentration-response study, the sameamount of data can be acquired and analyzed over the span of lessthan 2 hours.

As shown in Figure 3, the net charge analyses from theOpusXpress system give dose-response curves that are equivalentto those obtained with conventional recording methods. Results ofthese three net-charge analyses have an average EC50 value of 29.0µM ± 8 µM (SD). Five previously published studies, based on thepeak current responses of human α7 receptors expressed inXenopus oocytes, have reported EC50 values ranging from 107 µMto 334 µM, with an average of 195 µM and standard deviation 83µM (summarized in Papke and Papke, 2002 [2]).

DiscussionA concentration-response analysis based on the peak amplitudes ofα7 receptor mediated currents leads to the false conclusion thatthis receptor subtype responds best to relatively high concentra-tions of agonist, which is clearly not the case. In fact, thereappears to be a strong concentration dependence for the fast inacti-vation/desensitization of α7 receptors such that, during the appli-cation of a steep concentration ramp, receptors open only in a nar-row band of concentration and close when the agonist concentra-tion goes above that range. Therefore, most of the charge throughα7 receptors produced by an ACh application occurs when theconcentration is in the range of 30 µM to 100 µM. Applying con-centrations of ACh higher than 100 µM has the effect of synchro-nizing receptor activation during the time period when the concen-tration ramp goes through that range. This produces progressivelylarger peak currents when higher concentrations of agonist areapplied because the band of effective concentration is tighter in atemporal sense. The large peak currents evoked by the applicationof high agonist concentrations are due to increased synchronizationand not due to increased activation.

The α7 nAChR appears to function as a homomeric receptor withfive subunits per receptor and, therefore, potentially five agonistbinding sites. The concentration dependence of both channelopening and channel closing is consistent with models that havethe highest probability of opening associated with submaximallevel of agonist occupancy. That is, the channel may be more like-ly to open if only two or three of the five agonist binding sites areoccupied than if four or five sites are occupied. The high level ofreceptor occupancy that occurs with high agonist concentrationsseems to actually decrease receptor activation rather than promote

FOCUS ON METHODS

Figure 3. Comparison of concentration-response studies based on measurements of net chargeaccumulation with a standard manual recording system (black) and a perfusion flow rate of 6ml/min with an 18 s drug application (data from Papke and Papke [1]) and the OpusXpress auto-mated recording system run at either 1 ml/min with a 20 s drug application (in blue) or 2 ml/minwith a 12 s drug application (red).

8 w w w . a x o n . c o m

Figure 2. The effects of ACh concentration on the amplitude and kinetics of neuronal nico-tinic AChR subtypes α7 and α4β2 in panels A and B, respectively. In each panel, the top tracesshow representative responses of oocytes to the application of a relatively low ACh concen-tration in blue and a higher concentration in black, as indicated. In the lower sets of traces,both responses have been scaled to the same amplitude and are compared to an estimate of thedrug application time course obtained with open tip recordings of junction potential duringsolution exchange in the bath (see Methods), indicated in green. The traces represent 200 s ofdata for α7 responses and 400 s for the α4β2 receptor subtypes. The concentration-responsecurves of the nicotinic AChR subtypes α7 and α4β2 are shown in panels C & D, respective-ly. For each receptor, the response was calculated both in terms of peak current amplitude andnet charge during the entire drug response and normalized to the empirically determined max-imum response.

it. Supporting the hypothesis that the "rapid" desensitization of α7receptors is more concentration-dependent than time-dependent,we have reported similar findings in studies with acutely dissociat-ed neurons, where the concentration ramps are only a few millisec-onds long [5]. Interestingly, one difference between the net-chargeanalysis of oocyte responses and net-charge analysis of responsesfrom acutely dissociated neurons is that the neuronal responseswith high peak currents contain less net charge than responses toten-fold lower concentrations of agonist, while the oocyte respons-es reach a plateau maximum net charge. This apparently isbecause even the steepest concentration ramps in an oocyte experi-ment present the optimal range of agonist (e.g., 30 µM to 100 µMACh) for a sufficiently long period of time (hundreds of millisec-onds) for the net amount of channel opening to be limited by slowdesensitization or another form of inactivation. In experimentswith acutely dissociated neurons, when a high agonist concentra-tion is applied, the receptors are exposed to the optimal band ofconcentration for no more than a couple of milliseconds, which isnot long enough to promote the maximal amount of channel open-ing.

While net-charge analysis is well suited for the study of α7-typenAChR, does it have an advantage over peak current analysis forthe study of other nAChR subtypes? As shown in Figure 2, netcharge and peak current analysis give essentially identical resultsfor α4β2 receptors expressed in oocytes (further discussion of thispoint is included in an expanded version of this report, availableon the Axon Instruments web site).

In conclusion, we propose that net-charge analysis offers specialinsights into the properties of the α7-type nAChR and gives amore reasonable assessment of this receptor's pharmacologicalproperties. Net-charge analysis is easily done and the results arerelatively insensitive to technical aspects of the data acquisition.Additionally, net-charge analysis provides an important tool forunderstanding use-dependent processes such as open channelblock.

AcknowledgementsThis work was supported by NIH grants NS32888-02 and GM57481-01A2. We thank Bernadette Schoneburg and Clare Stokes for technicalassistance. We are very grateful to Axon Instruments for the use of thebeta release OpusXpress system and pCLAMP 9.

References1. Papke, R.L. and J.S. Thinschmidt, The Correction of Alpha7 NicotinicAcetylcholine Receptor Concentration-Response Relationships in XenopusOocytes. Neurosci. Let., 1998. 256: p. 163-166.2. Papke, R.L. and J.K.P. Papke, Comparative pharmacology of rat andhuman alpha7 nAChR conducted with net charge analysis. Br. J. ofPharmacol., 2002. in Press.3. Mike, A., N.G. Castro, and E.X. Albuquerque, Choline and acetyl-choline have similar kinetic properties of activation and desensitization onthe alpha7 nicotinic receptors in rat hippocampal neurons. Brain Res.,2000. 882(1-2): p. 155-68.4. Papke, R.L., et al., Alpha7-selective agonists and modes of alpha7receptor activation. Eur. J. Pharmacol., 2000. 393(1-3): p. 179-95.5.Uteshev, V.V., E.M. Meyer , and R.L. Papke, Activation and inhibitionof native neuronal alpha-bungarotoxin-sensitive nicotinic ACh receptors.Brain Res., 2002. in Press.

The resume of Dr. Yuri Opsichuk, Advanced ResearchGroup engineer at Axon Instruments, reads like that oftwo individuals. His outstanding accomplishments in

engineering and biology parallel one another in time. Afterearning a B.S. in physics and a M.S. in electrical engineering atthe Moscow Physical-Technical Institute, he succumbed to thelure of physiology and took his Ph.D. in Biophysics at theBogomoletz Institute of Physiology. It is here that Yuri beganhis study of ion channel function. When Yuri was building thefirst patch clamp rig in the USSR, the flow of technology fromthe West to the former Soviet Union was restricted. Undauntedby the lack of personal computers or the components to buildthem, Yuri and his colleagues constructed the first IBM-com-patible PC in the USSR, overcoming missing hardware compo-nents through a hardware/software emulation strategy. As oneof the founders of cooperative Electronika, Yuri helped designand build many of the system components that today are com-mercially available, such as: data acquisition systems, patchclamp amplifiers, patch electrode pullers, perfusion chambers,temperature controllers and electrode holders. Through hisefforts a rapid solution exchanger was realized and later com-mercialized. Yuri demonstrated that his engineering talentextended to optics as well, by designing and constructing anoptical setup for measuring the rapid pH changes accompany-ing synaptic transmission in hippocampal slices. Issues thatwould have been serious impediments to others representedexciting opportunities for Yuri to test his ingenuity, intellect andindomitable nature.

To continue his study of ion channels, Yuri conducted his post-doctoral studies in the laboratories of Dr. Akaike in Japan, Dr. Peterson in England and Dr. Cahalan in the USA. Soonafter joining the laboratory of Dr. Cahalan at the University ofCalifornia in Irvine, Yuri became an associate researcher. Atthis point Yuri had already established a distinguished career asa bench scientist, publishing his research in several prestigiousjournals and contributing to two book chapters. Although hisresearch was in full swing, Yuri still found time to consult withETM Systems in Irvine, CA. He designed a real-time videomemory board that became part of a microfluorescence ion

PROFILE

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TOOL TIPS

imaging system. Yuri realized that through his engineering skillsand understanding of bench science, he could make significantcontributions to the development of equipment and toolsrequired in biological research.

Shortly thereafter, Yuri seized the opportunity to join AxonInstruments. He quickly gained the well-deserved reputation ofsomeone who can rapidly master a wide range of technologies.Even as the roles of the engineers have become more defined,Yuri continues to participate in all phases of a project. He ledthe design of Axon's GenePix 4000A microarray scanner, wherehe was personally responsible for a substantial portion of electri-cal, firmware, mechanical and optical design of the instrument.Through this project he perfected the first microarray scannercapable of simultaneous two-wavelength scanning. This is animpressive engineering feat with a truly practical outcome, as ithalves the time required to obtain a full scan ratio. Althoughthere are several companies that produce a microarray scanner,only one other corporation to date has produced a scanner withthe same capability. The digital CCD camera and data acquisi-tion system that are the heart of Axon's new ImageXpress highthroughput cell-based screening system also received majordesign input by Yuri.

Though Yuri's efforts are never consigned to just one project, hehas of late devoted a considerable amount of the time to therefinement of Axon's first high-throughput automated patch-clamp system. In a sense, he has come full circle and hasreturned to his roots. As he puts the final touches on the first-generation instrument, he is already working on the design of thesecond-generation instrument. We look forward to an excitingfuture for our high-throughput patch clamp systems with Yuri asthe clever catalyst. In this endeavor as well as many others, Yuriis Axon's engineer extraordinaire.

MultiClamp TelegraphsIn Clampex versions 8.2 and above, protocols can be automaticallylaunched when the mode of the MultiClamp is changed from volt-age clamp to current clamp and vice versa. By clicking the Modebutton on the MultiClamp Commander, Clampex will automaticallystart the appropriate protocols.

To use this feature select the Configure / Sequencing Keys menuitem in Clampex and press Add. A list box appears with a ‘tradi-tional’ sequencing key toolbutton or key combination (e.g., Alt + 0,Ctrl + Shift + 5, etc.). Scroll down to see the sequencing keys thatare MultiClamp specific. There is a set of 16 entries beginning with“I-Clamp,” corresponding to the 16 input channels of the Digidata.Following these entries are 16 more, beginning with “V-Clamp.”Choose a Sequencing Key that correctly represents the Digidatachannel to which you've connected the MultiClamp Scaled Output.Telegraphs (Configure / Telegraphed Instrument) for this channelmust be enabled.

A typical example of operation would be the following:1. Design two protocols, one for V-Clamp, one for I-Clamp, using

Clampex.2. In Clampex, select Configure / Sequencing Keys or press the but-ton on the Sequencing Keys toolbar:

3. Press the New Set button, and name and save the set.4. Click the Add button and select the I-Clamp IN 0 entry (if theScaled Output from the MultiClamp 700A is connected to theAnalog IN 0) from the pull-down menu. Click OK.5. In the Sequencing Keys / Properties dialog box, click on theOperations tab.6. Select to launch a protocol when the Mode on the MultiClamp isswitched to I-Clamp. Click on the Protocol radio button.7. Under the Action pull-down menu, you can select to load a proto-col, actually record data or simply view data on the screen withoutsaving it to disk. Select “Record.”8. Click the Browse button to open a Windows Explorer interface,and select and Open the I-Clamp protocol that you designed in step 1. 9. Click OK to add the entry to the Sequencing Keys list.10. Repeat this process with the V-Clamp protocol, starting again atstep 4, but this time loading the V-Clamp protocol. The SequencingKeys list will look like this:

11. These protocols will now be run automatically when you switchrecording modes on the MultiClamp Commander. In the exampleshown, clicking on the MultiClamp IC Mode button will launchSequencing Key “I-Clamp IN 0,” which will record data obtainedusing protocol “InterHigh.pro.” Similarly, clicking on theMultiClamp VC Mode button will launch Sequencing Key “V-Clamp IN 0,” which will record data obtained using protocol “es 16sweeps.pro.”

The Sequencing dialogs are very rich. For an exhaustive explana-tion of all the entries, please refer to the Online Help.

A New Life for Imaging Workbench!

Looking for a dynamic fluorescence & ion imaging sys-tem compatible with Axon Instruments' pCLAMP?INDEC Biosystem's Imaging Workbench 5.0 with itsimproved functionality is the answer. Based on Axon’sImaging Workbench 4, Imaging Workbench 5.0 fromINDEC is an interactive, easy-to-use software tool for

acquiring, visualizing, and analyzing processes occurring in millisecondtimeframes. INDEC wants to share it’s vision for this product with youand discuss upgrades, future features, and its commitment to ongoingdevelopment and support. Come see the improvements they've madeand give them your thoughts at Neuroscience—booth #451. Also besure to visit their web site at www.ImagingWorkbench.com

Q: Can I use my GenePix Personal 4100A scanner to evaluatespot morphology on a microarray?

A: Irregular or missing spots can be determined prior to stainingand hybridization by reflectance imaging with the GenePixPersonal 4100A microarray scanner. This method allows the non-destructive evaluation of your microarrays because no treatmentor staining is required for reflectance imaging. The principle ofreflectance imaging is that laser light is scattered from sampledeposited onto the glass slide (e.g., microarray spots). A portionof the scattered light is reflected back into the optical path andcollected by the detector. The optical path of the GenePixPersonal 4100A scanner allows one to collect reflected light fromthe specimen when the emission filter is removed (thus permit-ting the 635 nm and 532 nm reflected light to be collected).Reflectance imaging is not possible with the GenePix 4000 scan-ners because the optical path contains rejection filters that don'tpermit reflected excitation light to reach the detector.

Configuring the GenePix Personal 4100A scanner for reflectanceimaging is fairly simple.

Select an unused filter position, indicated as "Empty" in the dropdown list under Filter in the Hardware Settings dialog box:

Next, load the slide and scan. This will generate an image of thelight reflected from the microarray chip:

The image shown above was obtained from a pre-hybridizationslide of DNA probes in 3x SSC buffer spotted onto a glass slide(courtesy of Dr. Joe DeRisi, Department of Biochemistry,University of California, San Francisco).

The scanner was set as follows: 800 V PMT, 100% laser power,and no emission filters for both red and green channels. Note thatthere is some autofluorescence signal from the DNA or SSCbuffer in the green channel, but autofluorescence was not seen inthe red channel (data not shown).

The Feature Alignment tool identifies the missing spots and auto-matically flags them as “Not Found." If desired, users can notethe locations and record them as "Empty" in the GAL file ID col-umn.

Q: I forgot to save my settings file (gps file) and exited the pro-gram, can I recover the changes I made to my blocks and fea-tures?

A: The state of the settings at the time of exiting GenePix Pro isautomatically saved in a gps file located in the application direc-tory (e.g., c:/Axon/GenePix Pro 4.1) The file is named accordingto the user name of the individual that is presently logged-in:(e.g., j_doe.gps)

AxoBits Vol. 36 October 2002 11

AXON REPLIES

GeneClamp and Axoclamp Feature Enhancement

If you perform two-electrode voltage-clamp (TEVC) experimentswhere the currents normally exceed several µA, then you may beinterested in a feature enhancement to your GeneClamp 500B orAxoclamp 2B amplifier. The expression of channel proteins inoocytes is a good example. Whole-cell oocyte currents can exceedtens of µA, during which time the membrane resistance may dropto values of a few kΩ. This circumstance can lead to voltageerrors in the clamp command if the recording conditions are notoptimal. Typical steps to optimize clamp performance include theuse of a virtual ground to zero the bath potential, minimizing theelectrode resistance and maximizing the amplifier feedback gain.For a review on the subject of voltage-clamp fidelity under condi-tions of large currents/low Rm, see Axobits 31 (Feb. 2001), yourAxoclamp or GeneClamp user manual, or the Axon Guide (freelyavailable on our website, www.axon.com).

If large currents and low membrane resistance are unavoidable inyour preparation, and you are concerned about the fidelity of yourTEVC, we can help you to enhance the performance of yourGeneClamp 500B or Axoclamp 2B amplifier. Steady-state holdingpotentials will be improved, without affecting AC components ofthe signal. A component modification on the main circuit board isrequired, and can be performed by you in the field at no cost!Alternatively, if you wish for Axon to perform the modification, afixed charge of US$250 plus shipping expenses will apply. ContactAxon Instruments' Technical Support Department at+1-510-675-6200 or send an email to tech@axon.com.

Australia, New ZealandGenomics ProductsGeneWorks Pty. Ltd.P.O. Box 11Rundle MallAdelaide, SA 5000, AustraliaFreecall: 1800-882-555Ph: +61 8-8234-2644Fax: +61 8-8234-2699E-mail: service@geneworks.com.auWeb: www.geneworks.com.au

CanadaCarsen Group Inc.151 Telson RoadMarkham, Ontario L3R 1E7CANADAPh: +1 800-387-0437Fax: +1 905-479-2595e-mail: roxanned@carsengroup.comWeb: www.carsengroup.com

China, TaiwanGenomics ProductsCold Spring Biotechnology (Three offices)Beijing OfficeMr. Leo TaiCold Spring BiotechRoom 1603 (A) Vantone New World PlazaNo. 2 Fu Cheng Men Wai StreetXi Cheng District, Beijing 100037,P.R. ChinaPh: +86 10-6858-8166Fax: +86 10-6858-8166 or -7980E-mail: fengjih@ms3.hinet.net

Shanghai OfficeCold Spring Biotech158 Hanzhong Road, Room 1310Hanzhong PlazaShanghai 200070, P.R. ChinaPh: +86 21-6353-5972Fax: +86 21-6353-5973E-mail: fengjih@ms3.hinet.net

Taiwan OfficeMr. Pasteur Tai2F-1, No. 31, Lane 169Kang Ning St.Da Hu Science ParkHsichih City, Taipei Hsien, Taiwan,R.O.ChinaPh: +886 2-2695-9990Fax: +886 2-2692-3410E-mail: fengjih@ms3.hinet.net

France, BelgiumGenomics ProductsCellular NeuroscienceProductsDIPSI IndustrieImmeuble Vecteur-Sud70-86, avenue de la RépubliqueF-92325 Chatillon Cedex, FrancePh: +33 1-4965-6720Fax: +33 1-4965-6729E-mail: info@dipsi.comWeb: www.dipsi.com

Germany, AustriaGenomics ProductsBiozym Diagnostik GmbHPostfach31833 Hess. Oldendorf, GermanyPh: +49 5152-9020Fax: +49 5152-2070E-mail: array@biozym.comWeb: www.biozym.com

IndiaGenomics ProductsMicro Devices MetrohmLtd.New No. 13, Old No. 4/1First Avenue, Indira NagarAdyar, Chennai-600 020, IndiaPh: +91 44 441-0444Fax: +91 44 443-0386E-mail: chennai@mdml.comWeb: www.mdml.com

ItalyGenomics ProductsM-Medical GenencoVia Pier Capponi 5750132 Firenze, ItalyPh: +39 55-5001871Fax: +39 55-5001875E-mail: m.petilli@mmedical.itWeb: www.mmedical.it

JapanGenomics ProductsCellular NeuroscienceProductsInter Medical Co. Ltd.Inter Bldg.40-4 Imaike 3-chome, Chikusa-kuNagoya 464-0850, JapanPh: +81 52-731-8000Fax: +81 52-731-5050E-mail: intermed@po.sphere.ne.jpWeb: www1.sphere.ne.jp/intermed

KoreaGenomics ProductsDae Myung Scientific Co.,Ltd.252-77 Kuui-DongKwang Jin-KuHyang-Won B/D, 3FSeoul, KoreaPh: +82 2-458-5835Fax: +82 2-452-1221E-mail: daemyung2000@hotmail.comWeb: www.dm4you.com

Singapore, MalaysiaGenomics ProductsResearch Biolabs Pte Ltd.211 Henderson Road # 14-01Henderson Industrial ParkSingapore 159552, SingaporePh: +65 273-1066Fax: +65 273-4914 E-mail: biolabs@singnet.com.sgWeb: www.researchbiolabs.com

Spain, PortugalGenomics ProductsDurviz, s.l.Parque Tecnológico de ValenciaLeonardo da Vinci, 1046980 Paterna (Valencia), SpainPh: +34 96-136-6107Fax: +34 96-136-6168E-mail: durviz@durviz.comWeb: www.durviz.com

SwitzerlandGenomics ProductsBucher Biotec AGSchützengraben 7CH-4051 Basel, SwitzerlandPh: +41 61-269-1111Fax: +41 61-269-1112E-mail: info@bucher.chWeb: www.bucher.ch

The Netherlands,Luxembourg, Denmark,Norway, Sweden, Finland,IcelandGenomics ProductsWestburg B.V.Arnhemseweg 87P.O. Box 2143830 AE Leusden, The NetherlandsPh: +31 33-495-0094Fax: + 31 33-495-1222E-mail: info@westburg.nlWeb: www.westburg.nl

United Kingdom, IrelandGenomics Products GRI Ltd.Gene HouseQueenborough Lane, Rayne, BraintreeEssex CM7 8TFUnited KingdomPh: +44 1376 332900Fax: +44 1376 344724E-mail: gri@gri.co.ukWeb: www.gri.co.uk

United StatesAxon Instruments, Inc.In the U.S., all sales are directly fromAxon Instruments. All prices are statedand must be paid in U.S. dollars(US$). FOB Factory, California,U.S.A. All prices are subject tochange without notice.

www.axon.com

3280 Whipple Road, Union City, CA 94587 USAPhone: +1 (510) 675-6200 Fax: +1 (510) 675-6300

e-mail: sales@axon.com

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