Using Electrophysiology to Study Synaptic and Extrasynaptic Ionotropic Receptors in Hippocampal Neurons

Abstract

Electrophysiology is an exceptionally useful tool for neuroscience research due to the intrinsic electrical excitability of neurons and the significant array of ion channels present in both neurons and glia. Diverse electrophysiological techniques may be applied to neurobiology ranging from measurements of cell populations in broad brain regions to measurements of single channels in patches of plasma membrane. One of the strengths of electrophysiology as a tool is the ability to measure the properties of known ion channels in heterologous systems, then dissect the diverse pharmacology and biophysics of neuronal responses to finally better understand which component channels and their features determine the biologically critical outputs of neurons and circuits.

Patch-clamp electrophysiology allows recording of neuronal receptors at both the synaptic and extrasynaptic level. The specific techniques described here permit the study of both populations independently by measuring miniature excitatory synaptic currents and currents derived from somatic receptors. The resolution and accuracy of the techniques described in the chapter are high (in the sub-picoampere and sub-millisecond ranges). Further, these methodologies provide valuable information about the behavior of the receptors in their native environment where they coexist with auxiliary, modulatory, and anchoring proteins.

In this chapter, we firstly describe the methodology for preparing hippocampal neuronal cultures. Secondly, we describe the process of recording and analyzing miniature postsynaptic currents. Finally, we describe in detail the technique of fast agonist application onto outside-out patches obtained from the soma of neurons. We discuss common problems found with these approaches and present tips to assist researchers new to the field so they may rapidly master the techniques.

Appendices

Appendix 1: Igor Macro for Peak-Scaled Non-stationary Fluctuation Analysis

#pragma rtGlobals=1 // Use modern global access method.

Macro PeakscaledNSNA()

execute "PSNSNA()"

end

Function PSNSNA()

//start by initializing strings (the names of important bits) and waves (the contents)

string NSNAAvg,Identifier,BackgroundVarName

string /g Variance,Average,BinnedVariance,BinnedAverage,STDEV, Binnedgraph, CurrentAvg,FittedParabola

string /g layoutName="DisplayFormat"

variable bslnstart=0 // begin time to calculate back ground variance

variable bslnend=4

variable smoothing_in=0

variable numbins_in=30

variable bin=1 //0 if no, 1 if yes

variable /g smoothing=0

variable /g BackgroundVar //calculated background variance

variable /g xLim

variable /g yLim

variable /g ScalePt

variable /g ScaleMag

variable /g ScaleFact

variable /g numbins=0

variable /g binstart=0 //pA

variable /g binend=0

variable DrivingForce=-55

variable /g Conduct=0

variable /g Popen

//Make a prompt window for user input of important values

Prompt bslnstart, "Baseline start point (ms)"

Prompt bslnend, "Baseline end point (ms)"

Prompt NSNAAvg,"Average Wave",popup,WaveList("avg_*",";","")

Prompt smoothing_in, "smooth factor (binomial)"

Prompt Identifier,"enter file suffix for identification"

Prompt numbins_in, "number of bins"

Prompt DrivingForce, "enter voltage (mV)"

DoPrompt "enter wave",NSNAAvg,bslnstart,bslnend,smoothing_in,Identifier,numbins_in,DrivingForce

if(V_flag==1) //If there is no selected wave then it cancels

return 0 //cancel do prompt

endif

smoothing=smoothing_in //rename input values for use

numbins=numbins_in

Duplicate /o $NSNAAvg NonStatAvg

Duplicate /o NonStatAvg NonStatVar

Duplicate /o NonStatAvg ScaleAvg

Average="Avg"+Identifier //This is why you need to enter suffix, to give fresh names to these values

Variance="Var"+Identifier

BinnedAverage="Avg_Bin_"+Identifier

BinnedVariance="Var_Bin_"+Identifier

CurrentAvg="Current_"+Identifier

BackgroundVarName="BackVar"+Identifier

FittedParabola="FittedNoise"+Identifier

STDEV="Var_SD"+Identifier

variable chncnt,wvcnt,numSweeps ///initialize more variables to name each sweep

string wvlist,wvName,wvName2,NonStatAvgName

wave NonStatVar

string tempwave

wave chanselect

wave waveselect

nvar smoothing

NonStatVar=0

numSweeps=0

wavestats /q/R=(xcsr(A),xcsr(B)) NonStatAvg ////Gets stats of the average current wave in analysis window

xLim=V_min+(0.05*V_min) ////sets a limit of 5% under the minimum

ScalePt=xcsr(A)

ScaleMag=V_min

for (chncnt = 0; chncnt < numpnts(ChanSelect); chncnt += 1) // loops through each time point in the input waves, normally in ChanA (ChanSelect)

if (ChanSelect[chncnt] != 1) ///If only one sweep, end the macro as noise analysis cannot be performed.

continue

endif

wvlist = GetChanWaveList(chncnt) ///this is a neuromatic function that returns the list of selected waves for a particular channel

for (wvcnt = 0; wvcnt < ItemsInList(wvList); wvcnt += 1) ///Loops through each sweep (for the timepoint in question from chncnt)

wvName = StringFromList(wvcnt, wvList) ///gets the current wave

wvName2 = StringFromList(wvcnt+1, wvList) ///gets the next wave for pairwise comparison

if (exists(wvName) == 0) ///keep going until wvName is 0 i.e. it has gone through every wave

continue

endif

duplicate /o $wvName, Sweep ///Get values for each pair of waves

ScaleFact = Sweep(ScalePt)/ScaleMag ///These two lines scale the average peak to the correspnding datapoint in the sweep

ScaleAvg = NonStatAvg*ScaleFact

if (smoothing>1) ///Smooth out the records

smooth smoothing, Sweep

endif

NonStatVar+=(ScaleAvg-Sweep)^2 ///Square the difference to get the variance, add variances

numSweeps+=1

endfor

NonStatVar/=(numSweeps-1) ///final calculation of variance by dividing by total number,-1

endfor

////calculate and print background variance

wavestats /q /R=(bslnstart,bslnend) NonStatVar ////NonStatVar is the wave with the variance at each timepoint, this is just in the baseline window

BackgroundVar=V_avg //This gives the average variance in the background range

print "background variance (pA^2) is:", BackgroundVar

wavestats /q/R=(xcsr(A),xcsr(B)) NonStatAvg ////Gets stats of the average current wave in analysis window

xLim=V_min+(0.05*V_min) ////sets a limit of 5% under the minimum

wavestats /q/R=(xcsr(A),xcsr(B)) NonStatVar ////Gets stats of average Variance wave

yLim=V_max+(0.05*V_max) ////sets a limit of 5% over the maximum

duplicate /o /R=(xcsr(A),xcsr(B)) NonStatVar $Variance ////Duplicates section of variance between cursors

duplicate /o /R=(xcsr(A),xcsr(B)) $NSNAAvg $Average ////Renames section of current between cursors

duplicate /o /R=(xcsr(A),xcsr(B)) $NSNAAvg BckVr ////Renames section as other type of string/wave, never got that distinction

if (bin==1)

duplicate /o $Average AvgForBin ////more renaming to put things in the right form

duplicate /o $Variance VarForBin

make /o/N=(numbins) NonStatAvg_Bin ////Gets new waves to contain data from binned records

make /o/N=(numbins) NonStatVar_Bin

make /o/N=(numbins) NonStatVarSD

wave VarForBin, AvgForBin,NonStatAvg_Bin,NonStatVar_Bin,NonStatVarSD

variable incwave

variable incAvg //increment through binning

nvar numbins,binstart,binend

variable /g binwidth=0

variable count_if=0

variable count_if_sd=0

variable /g waveEndForSD=0

NonStatAvg_Bin=0 /////initializing loops

NonStatVar_Bin=0

NonStatVarSD=0

incavg=0

wavestats /Q AvgForBin

waveEndForSD=V_npnts-2 /////The number of comparisons is less because the end points have only one neighbour

Binwidth=(V_min-V_max)/numbins /////Binwidth is in current and is the full range sliced up

print "binwidth is", binwidth

variable countbins=0

for (incAvg=1;incAvg<=numbins;incAvg+=1) /////Binning routine, loops for each bin

binstart=V_min-(incAvg*Binwidth) /////calculates upper and lower limits of the bin

binend=binstart+binwidth

count_if=0 /////reinnitializes counts

count_if_sd=0

for (incwave=0;incwave<(V_npnts-1);incwave+=1) /////Runs through each datapoint in cursor range

if((AvgForBin[incwave]<binstart) && (AvgForBin[incwave]>=binend)) /////If in bin range then include in bin

NonStatAvg_Bin[incavg-1]+=AvgForBin[incwave] /////Add current and variance to binpoint

NonStatVar_Bin[incavg-1]+=VarForBin[incwave]

if(incwave<waveEndForSD) ///// This calculates errors for error bars on each variance point

NonStatVarSD[incavg-1]+=(VarForBin[incwave+1]-VarForBin[incwave])^2

count_if_sd+=1

endif

count_if+=1

endif

endfor

NonStatAvg_Bin[incavg-1]/=count_if /////Total value divided by number of contributors

NonStatVar_Bin[incavg-1]/=count_if

NonStatVarSD[incavg-1]/=2*(count_if_sd-1) /////Divide error bars by number of contributors*2

NonStatVarSD[incavg-1]=sqrt(NonStatVarSD[incavg-1]) /////Take sqrt of it. to give SD

endfor

print "number of executed bins",countbins

duplicate /o NonStatAvg_Bin $BinnedAverage //////Back again, duplicate binned stuff

duplicate /o NonStatVar_Bin $BinnedVariance

duplicate /o NonStatVar_Bin $BinnedVariance

duplicate /o NonStatAvg_Bin $FittedParabola

duplicate /o NonStatVarSD $STDEV //for standard error bars

endif

BckVr=BackgroundVar //////Duplicting background var

duplicate /o BckVr $BackgroundVarName //////More renaming

Binnedgraph="MeanVarBin"+Identifier

if(wintype(Binnedgraph)==0)

display /k=1 $BinnedVariance vs $BinnedAverage //make graphs for binned data

DoWindow /C $Binnedgraph

ModifyGraph mode=4,marker=19,msize=4

ModifyGraph rgb=(0,0,0)

AppendToGraph $BackgroundVarName vs $Average //////Add background variance

ModifyGraph mode=0

ShowInfo

ErrorBars $BinnedVariance Y,wave=($STDEV,$STDEV) //////Stick error bars on

SetAxis/A

SetAxis bottom 2, xLim

ModifyGraph mode( $BinnedVariance)=4

endif

Make/D/N=3/O W_coef //////Do a fit

W_coef[0] = {BackgroundVar,-0.5,100} //////Make first guesses for variance fit

FuncFit/H="100" SigworthNSNA W_coef $BinnedVariance /X=$BinnedAverage /D=$FittedParabola /W=$STDEV /I=1 /////Hold backvar,;fit N,i

Conduct = w_coef[1]/DrivingForce*1000

Popen = V_min/(w_coef[1]*w_coef[2])

TextBox /w=$Binnedgraph/N=test/C/F=0/E=1/A=MT "G(pS)="+num2str(Conduct)+"\r"+"N="+num2str(w_coef[2])+ "\r"+"PoPeak ="+num2str(Popen) //////Display it

AppendToGraph $FittedParabola vs $BinnedAverage

ModifyGraph lstyle($FittedParabola)=3,rgb($FittedParabola)=(0,0,0),mode($BinnedVariance)=3,rgb($BackgroundVarName)=(34816,34816,34816)

Label left "Variance (pA\\S2\\M)";DelayUpdate

Label bottom "Current (pA)"

display NonStatAvg

DoWindow /C $CurrentAvg

ModifyGraph rgb=(0,0,0)

ModifyGraph tick=3,noLabel=2,axThick=0

NewLayout /N=Layoutname

AppendLayoutObject graph $Binnedgraph

AppendLayoutObject graph $CurrentAvg

ModifyLayout frame=0,trans=1;DelayUpdate

ModifyLayout left($Binnedgraph)=100,top($Binnedgraph)=370,width($Binnedgraph)=300,height($Binnedgraph)=350;DelayUpdate

ModifyLayout left($CurrentAvg)=60,top($CurrentAvg)=100,width($CurrentAvg)=400,height($CurrentAvg)=300

print "Conductance = ",Conduct

print "PoPeak = ",Popen

end

////////////////////////////////////////////////////////////////////////////

Appendix 2: Sigworth NSNA Function

#pragma rtGlobals=1 // Use modern global access method.

Function SigworthNSNA(w,x) : FitFunc //sigworth non-stationary noise analysis fit with background variance

Wave w

Variable x

//CurveFitDialog/ These comments were created by the Curve Fitting dialog. Altering them will

//CurveFitDialog/ make the function less convenient to work with in the Curve Fitting dialog.

//CurveFitDialog/ Equation:

//CurveFitDialog/ f(x) = var_back + i*I -((I*I)/N)

//CurveFitDialog/ End of Equation

//CurveFitDialog/ Independent Variables 1

//CurveFitDialog/ x

//CurveFitDialog/ Coefficients 3

//CurveFitDialog/ w[0] = var_back

//CurveFitDialog/ w[1] = i

//CurveFitDialog/ w[2] = N

return w[0] + w[1]*x-((x*x)/w[2])

End

Appendix 3: Igor Macro for Non-stationary Fluctuation Analysis

#pragma rtGlobals=1 // Use modern global access method.

Macro PairwiseNSNA()

execute "NSNA()"

end

Function NSNA()

//start by initializing strings (the names of important bits) and waves (the contents)

string NSNAAvg,Identifier,BackgroundVarName

string /g Variance,Average,BinnedVariance,BinnedAverage,STDEV, Binnedgraph, CurrentAvg,FittedParabola

string /g layoutName="DisplayFormat"

variable bslnstart=0 // begin time to calculate back ground variance

variable bslnend=20

variable smoothing_in=0

variable numbins_in=10

variable bin=1 //0 if no, 1 if yes

variable /g smoothing=0

variable /g BackgroundVar //calculated background variance

variable /g xLim

variable /g yLim

variable /g numbins=0

variable /g binstart=0 //pA

variable /g binend=0

variable DrivingForce=-55

variable /g Conduct=0

variable /g Popen

//Make a prompt window for user input of important values

Prompt bslnstart, "Baseline start point (ms)"

Prompt bslnend, "Baseline end point (ms)"

Prompt NSNAAvg,"Average Wave",popup,WaveList("avg_*",";","")

Prompt smoothing_in, "smooth factor (binomial)"

Prompt Identifier,"enter file suffix for identification"

Prompt numbins_in, "number of bins"

Prompt DrivingForce, "enter voltage (mV)"

DoPrompt "enter wave",NSNAAvg,bslnstart,bslnend,smoothing_in,Identifier,numbins_in,DrivingForce

if(V_flag==1) //If there is no selected wave then it cancels

return 0 //cancel do prompt

endif

smoothing=smoothing_in //rename input values for use

numbins=numbins_in

Duplicate /o $NSNAAvg NonStatAvg

Duplicate /o NonStatAvg NonStatVar

Average="Avg"+Identifier //This is why you need to enter suffix, to give fresh names to these values

Variance="Var"+Identifier

BinnedAverage="Avg_Bin_"+Identifier

BinnedVariance="Var_Bin_"+Identifier

CurrentAvg="Current_"+Identifier

BackgroundVarName="BackVar"+Identifier

FittedParabola="FittedNoise"+Identifier

STDEV="Var_SD"+Identifier

variable chncnt,wvcnt,numSweeps ///initialize more variables to name each sweep

string wvlist,wvName,wvName2,NonStatAvgName

wave NonStatVar

string tempwave

wave chanselect

wave waveselect

nvar smoothing

NonStatVar=0

numSweeps=0

for (chncnt = 0; chncnt < numpnts(ChanSelect); chncnt += 1) // loops through each time point in the input waves, normally in ChanA (ChanSelect)

if (ChanSelect[chncnt] != 1) ///If only one sweep, end the macro as noise analysis cannot be performed.

continue

endif

wvlist = GetChanWaveList(chncnt) ///this is a neuromatic function that returns the list of selected waves for a particular channel

for (wvcnt = 0; wvcnt < ItemsInList(wvList)-1; wvcnt += 1) ///Loops through each sweep (for the timepoint in question from chncnt)

wvName = StringFromList(wvcnt, wvList) ///gets the current wave

wvName2 = StringFromList(wvcnt+1, wvList) ///gets the next wave for pairwise comparison

if (exists(wvName) == 0) ///keep going until wvName is 0 i.e. it has gone through every wave

continue

endif

duplicate /o $wvName, Sweep ///Get values for each pair of waves

duplicate /o $wvName2, Sweep2

if (smoothing>1) ///Smooth out the records

smooth smoothing, Sweep

smooth smoothing, Sweep2

endif

NonStatVar+=(Sweep2-Sweep)^2 ///Square the difference to get the variance, add variances

numSweeps+=1

endfor

NonStatVar/=2*(numSweeps) ///final calculation of variance by dividing by total number, and 2 since each wave was used twice

endfor

////calculate and print background variance

wavestats /q /R=(bslnstart,bslnend) NonStatVar ////NonStatVar is the wave with the variance at each timepoint, this is just in the baseline window

BackgroundVar=V_avg //This gives the average variance in the background range

print "background variance (pA^2) is:", BackgroundVar

wavestats /q/R=(xcsr(A),xcsr(B)) NonStatAvg ////Gets stats of the average current wave in analysis window

xLim=V_min+(0.05*V_min) ////sets a limit of 5% under the minimum

wavestats /q/R=(xcsr(A),xcsr(B)) NonStatVar ////Gets stats of average Variance wave

yLim=V_max+(0.05*V_max) ////sets a limit of 5% over the maximum

duplicate /o /R=(xcsr(A),xcsr(B)) NonStatVar $Variance ////Duplicates section of variance between cursors

duplicate /o /R=(xcsr(A),xcsr(B)) $NSNAAvg $Average ////Renames section of current between cursors

duplicate /o /R=(xcsr(A),xcsr(B)) $NSNAAvg BckVr ////Renames section as other type of string/wave, never got that distinction

if (bin==1)

duplicate /o $Average AvgForBin ////more renaming to put things in the right form

duplicate /o $Variance VarForBin

make /o/N=(numbins) NonStatAvg_Bin ////Gets new waves to contain data from binned records

make /o/N=(numbins) NonStatVar_Bin

make /o/N=(numbins) NonStatVarSD

wave VarForBin, AvgForBin,NonStatAvg_Bin,NonStatVar_Bin,NonStatVarSD

variable /g binsize //calculated bin cutoff /////seems to not be used at all

variable incwave

variable incAvg //increment through binning

nvar numbins,binstart,binend

variable binsize_prev

variable /g binwidth=0

variable count_if=0

variable count_if_sd=0

variable /g waveEndForSD=0

NonStatAvg_Bin=0 /////initializing loops

NonStatVar_Bin=0

NonStatVarSD=0

incavg=0

wavestats /Q AvgForBin

waveEndForSD=V_npnts-2 /////The number of comparisons is less because the end points have only one neighbour

Binwidth=(V_min-V_max)/numbins /////Binwidth is in current and is the full range sliced up

print "binwidth is", binwidth

variable countbins=0

for (incAvg=1;incAvg<=numbins;incAvg+=1) /////Binning routine, loops for each bin

binstart=V_min-(incAvg*Binwidth) /////calculates upper and lower limits of the bin

binend=binstart+binwidth

count_if=0 /////reinnitializes counts

count_if_sd=0

for (incwave=0;incwave<(V_npnts-1);incwave+=1) /////Runs through each datapoint in cursor range

if((AvgForBin[incwave]<binstart) && (AvgForBin[incwave]>=binend)) /////If in bin range then include in bin

NonStatAvg_Bin[incavg-1]+=AvgForBin[incwave] /////Add current and variance to binpoint

NonStatVar_Bin[incavg-1]+=VarForBin[incwave]

if(incwave<waveEndForSD) ///// This calculates errors for error bars on each variance point

NonStatVarSD[incavg-1]+=(VarForBin[incwave+1]-VarForBin[incwave])^2

count_if_sd+=1

endif

count_if+=1

endif

endfor

NonStatAvg_Bin[incavg-1]/=count_if /////Total value divided by number of contributors

NonStatVar_Bin[incavg-1]/=count_if

NonStatVarSD[incavg-1]/=2*(count_if_sd-1) /////Divide error bars by number of contributors*2

NonStatVarSD[incavg-1]=sqrt(NonStatVarSD[incavg-1]) /////Take sqrt of it. to give SD

endfor

print "number of executed bins",countbins

duplicate /o NonStatAvg_Bin $BinnedAverage //////Back again, duplicate binned stuff

duplicate /o NonStatVar_Bin $BinnedVariance

duplicate /o NonStatVar_Bin $BinnedVariance

duplicate /o NonStatAvg_Bin $FittedParabola

duplicate /o NonStatVarSD $STDEV //for standard error bars

endif

BckVr=BackgroundVar //////Duplicting background var

duplicate /o BckVr $BackgroundVarName //////More renaming

Binnedgraph="MeanVarBin"+Identifier

if(wintype(Binnedgraph)==0)

display /k=1 $BinnedVariance vs $BinnedAverage //make graphs for binned data

DoWindow /C $Binnedgraph

ModifyGraph mode=4,marker=19,msize=4

ModifyGraph rgb=(0,0,0)

AppendToGraph $BackgroundVarName vs $Average //////Add background variance

ModifyGraph mode=0

ShowInfo

ErrorBars $BinnedVariance Y,wave=($STDEV,$STDEV) //////Stick error bars on

SetAxis/A

SetAxis bottom 2, xLim

ModifyGraph mode( $BinnedVariance)=4

endif

Make/D/N=3/O W_coef //////Do a fit

W_coef[0] = {BackgroundVar,-0.5,100} //////Make first guesses for variance fit

FuncFit/H="100" SigworthNSNA W_coef $BinnedVariance /X=$BinnedAverage /D=$FittedParabola /W=$STDEV /I=1 /////Hold backvar,;fit N,i

Conduct = w_coef[1]/DrivingForce*1000

Popen = V_min/(w_coef[1]*w_coef[2])

TextBox /w=$Binnedgraph/N=test/C/F=0/E=1/A=MT "G(pS)="+num2str(Conduct)+"\r"+"N="+num2str(w_coef[2])+ "\r"+"PoPeak ="+num2str(Popen) //////Display it

AppendToGraph $FittedParabola vs $BinnedAverage

ModifyGraph lstyle($FittedParabola)=3,rgb($FittedParabola)=(0,0,0),mode($BinnedVariance)=3,rgb($BackgroundVarName)=(34816,34816,34816)

Label left "Variance (pA\\S2\\M)";DelayUpdate

Label bottom "Current (pA)"

display NonStatAvg

DoWindow /C $CurrentAvg

ModifyGraph rgb=(0,0,0)

ModifyGraph tick=3,noLabel=2,axThick=0

NewLayout /N=Layoutname

AppendLayoutObject graph $Binnedgraph

AppendLayoutObject graph $CurrentAvg

ModifyLayout frame=0,trans=1;DelayUpdate

ModifyLayout left($Binnedgraph)=100,top($Binnedgraph)=370,width($Binnedgraph)=300,height($Binnedgraph)=350;DelayUpdate

ModifyLayout left($CurrentAvg)=60,top($CurrentAvg)=100,width($CurrentAvg)=400,height($CurrentAvg)=300

print "Conductance = ",Conduct

print "PoPeak = ",Popen

end

////////////////////////////////////////////////////////////////////////////