Journal of Neuroscience Methods 188 (2010) 1–6

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Journal of Neuroscience Methods

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Simultaneous recording of the field-EPSP as well as the population spike in theCA1 region in freely moving rats by using a fixed “double”-recording electrode

Thomas Scherf, Julietta U. Frey, Sabine Frey ∗

Leibniz-Institute for Neurobiology, Department of Neurophysiology, Brenneckestrasse 6, D-39118 Magdeburg, Germany

a r t i c l e i n f o

Article history:Received 24 November 2009Received in revised form 15 January 2010Accepted 18 January 2010

Keywords:“Double”-recordingFreely moving ratsHippocampal CA1Contralateral CA3Population spikeField-EPSPElectrophysiological recordings

a b s t r a c t

The recording of field potentials in freely moving rats is a very appropriate and commonly used methodto describe changes in cellular mechanisms underlying synaptic plasticity. Recently, we introduced amethod for the simultaneous recording of both the field-EPSP as well as the population spike in thedentate gyrus of freely moving rats. We used self-made “double”-recording electrodes, consisting of twowires straighten together with a constant distance between both tips. This method was now furtherdeveloped to obtain stable long-term recordings of CA1 field potentials. Rats were chronically implantedwith a bipolar recording electrode; one tip of which reached the stratum radiatum to record the field-EPSP, the other tip was lowered into the stratum pyramidale of the same neuron population to record thepopulation spike by stimulation of the contralateral CA3 (cCA3). In such prepared rats, simultaneouslyrecorded field-EPSP as well as the population spike where thus obtained from their places of generation ina very reliable manner. This kind of preparation allowed a better standardization of stimulation intensitiesbetween different animals and stable electrophysiological recordings of both CA1-potentials over a time

period of at least 24 h in freely behaving animals. Furthermore, primed burst stimulation of the cCA3(a single biphasic priming pulse was followed by a burst of 10 pulses (frequency of 100 Hz) 190 mslater; pulse duration per half-wave: 0.1 ms) resulted in an early-LTP of both measured parameters, the

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. Introduction

Processes of long-lasting changes in synaptic efficacy such asong-term potentiation (LTP) and long-term depression (LTD) in thentact animals are commonly investigated by recording of extra-ellular field potentials. In 1973, LTP was described by Bliss andomo (1973) first in the dentate gyrus of anaesthetised rabbits. Arief high-frequency stimulation of the perforant path can induce a

ong-lasting enhancement in synaptic efficacy, which can last in thentact animal several days or even weeks (Abraham, 2003; Abrahamt al., 1995, 2006; Abraham and Williams, 2008; Bekenstein andothman, 1991; Lynch et al., 1990; Manahan-Vaughan et al., 2003).or investigation of different forms of synaptic plasticity in theA1 region of the hippocampus in the intact animal usually a

ecording electrode is implanted in the ipsilateral stratum radia-um for recording the field-EPSP, while stimulation electrodes aremplanted in the ipsilateral CA3 (e.g. Manahan-Vaughan, 1997) totimulate the associational input, or in the contralateral CA1 or

∗ Corresponding author. Tel.: +49 391 6263 426; fax: +49 391 6263 421.E-mail address: sfrey@ifn-magdeburg.de (S. Frey).URL: http://www.ifn-magdeburg.de/departments/dep3/dep3 home.jsp

S. Frey).

165-0270/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jneumeth.2010.01.020

spike in the CA1 region of freely moving rats.© 2010 Elsevier B.V. All rights reserved.

CA3 region to stimulate the commissural input to the ipsilateralCA1 (Bliss et al., 1983; Diamond et al., 1988; Leung et al., 1992).The recorded field potential in the stratum radiatum of the CA1region, i.e. the field-EPSP, after the stimulation of the afferentsare characterized by a typical waveform depending on the placeof the stimulation electrode, e.g. ipsi- or contralaterally, stimulat-ing the associational or commissural pathways, respectively. Thiskind of preparation was used for many studies to investigate synap-tic plasticity such as LTP and LTD (Bliss et al., 1983; Diamondet al., 1988; Doralp and Leung, 2008; Kaibara and Leung, 1993;Kemp and Manahan-Vaughan, 2004, 2008; Leung et al., 1992, 2003;Manahan-Vaughan, 1997; Staubli and Lynch, 1987). However, itis well known that for induction of long-lasting LTP, postsynap-tic depolarisation as well as spike generation is required (Lismanand Spruston, 2005; Pike et al., 1999; Raymond, 2008). Recordingof just the field-EPSP does not allow to characterize postsynap-tic spike generation per stimulus. For a better characterization ofthe used stimulus parameters and the inter-comparison (standard-ization) of used stimulation intensities between different animals,

the recording of both, the EPSP as well as the spike is useful (seealso Frey and Frey, 2009). As mentioned above, the stimulationstrength for induction of different forms of long-lasting changesin synaptic plasticity is not only dependent on the applied pat-tern, its frequency and number of stimuli but also on the stimulus

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ntensity (Frey and Frey, 2008; Navakkode et al., 2007; Reymannnd Frey, 2007; Sajikumar et al., 2008). Leung and Au (1994) couldhow, that LTP is a function of pulse intensity. They induced inA1 of hippocampal slices LTP by theta-frequency primed burstst a fixed stimulus intensity. The time course of both, the field-PSP and the population spike was investigated at different testulse intensities defined in an input–output (I/O) relation takenefore experiments. The stimulus intensity was chosen accordingo the normalized slope of the field-EPSP and the amplitude ofhe population spike, respectively. In both signals the amount ofotentiation 10 s after primed bursts were dependent on stimu-

us intensity of test pulses. The degree of LTP was larger for themaller signals and decreased with higher test stimulation, whichan be explained by the input–output characteristics, a given maxi-al outcome of a given neuronal population is limited by factors as

he number of neurons. Recently, we had shown, that for inductionf LTP in the CA1 region in freely moving rats the applied stim-lus intensity per LTP-protocol is important for the time coursef the recorded field-EPSP (Hassan et al., 2006). From the valuesbtained from I/O curves – taken before the experiments – stim-lation intensities were determined to induce 30% and 60% of theaximal slope of the field-EPSP. With these intensities test record-

ngs and different LTP-inducing protocols were applied showinghat the higher stimulus intensity often resulted in a transientepression followed by long-lasting potentiation. We now triedo normalise parameters for electrophysiological investigations inreely moving animals. It is known, that the EPSP does not reachn asymptotic level, so that it is impossible to determine exactlyhe stimulus intensity for a distinct amount of a maximal EPSP. Tovercome this limit it seems to be necessary to record the pop-lation spike too, because this parameter reaches an asymptotic,aximal level at higher stimulus intensities, thus guaranteeing a

etter standardization/normalization of stimulus intensities.Recently, we established a method for simultaneously recording

f both, the field-EPSP as well as the population spike using sep-rate recording electrodes in the dentate gyrus in freely movingats (Frey and Frey, 2009). This methods allowed stable record-ngs of both parameters over the entire experimental session andhe characterization of both potentials during different forms ofynaptic plasticity.

. Materials and methods

.1. Animals and electrode implantation

All experiments have been performed with permission of theocal legislatives authorities of the Land Sachsen-Anhalt. All efforts

ere made to reduce the number of animals and their suffering. Allxperiments were performed on male Wistar rats (7 weeks old;eight 270–320 g at the time of preparation). After surgery for

lectrode implantation animals were housed individually in plasticranslucent cages with free access to water and food.

Surgery was performed under deep pentobarbital narcosis40 mg/kg, i.p.) supplemented with additional doses if required. Thenimals were mounted in a stereotaxic frame (with bregma andambda in the same horizontal lane). For the placement of stimula-ion electrodes, recording electrodes and miniscrews with attachedilver wire, served as indifferent and ground electrode, small accessoles were drilled in the skull and the dura was pierced for theccess to the brain.

A special “double”-recording electrode for simultaneous record-

ng of the field-EPSP and the population spike was designed. Twotainless steel wires (lacquer-coated, with a diameter of 125 �mer wire) were straightened/tightened close together using a spe-ial device made by the workshop of our institute. The wires wereightened between two metallic bars for several days, the dis-

nce Methods 188 (2010) 1–6

tance between the bars could be adjusted by screws. Because ofthe straightening the wires got more stable and lost their softness.Afterwards, a small piece of cardboard was fixed in the middle of thewires using clue. After removal from the bar-device the loose endswere twisted and connected with the sockets and the recordingapparatus, the electrode wires were cut carefully under microscopeat the given distance with very sharp surgical scissors. The card-boards served as “holder” to connect to the stereotaxical apparatus.The two tips of this “double”-recording electrode were cut with adistance of 260–280 �m, for the adjustment of the shorter tip intothe stratum pyramidale for the recording of the population spikeand the longer tip into the stratum radiatum for the recording of thefield-EPSP. This “double”-recording electrode was implanted intothe CA1 region of the right hippocampus (CA1; coordinates: antero-posterior (AP) −4.0 mm from bregma, mediolateral (ML) 2.3 mm,and dorsoventral (DV) approximately 2.4 mm from dural surface).A bipolar stimulation electrode (lacquer-insulated stainless steelwires, 125 �m in diameter) was implanted into the contralateralCA3 region (cCA3; coordinates: AP: −2.1 mm, ML: −1.4 mm, DV2.6–3.3 mm from dural surface) (see Fig. 1). The correct place-ment of the electrodes was adjusted by monitoring of both evokedpotentials, the field-EPSP and population spike. The electrodes wereattached to a miniature plastic socket and fixed with acrylic dentalcement. The wounds were treated with a chlorhexidine containingmedical powder.

In such prepared animals it is possible to record both elec-trophysiological parameters the field-EPSP as well the populationspike for several weeks, in average for 3–6 weeks after preparation.

2.2. Evoked potentials and experimental design

The animals were given at least 10 days of recovery after prepa-ration. During electrophysiological recordings, changes in the slopeof the field-EPSP and the population spike amplitude were regis-tered. The slope of the field-EPSP was measured at the steepest riseof the first negative deflection of the potential (Fig. 1D). The ampli-tude of the population spike was measured from the peak of thefirst positive deflection of the evoked potential to the peak of thefollowing negative deflection (Fig. 1E).

The day before the electrophysiological experiment, rats werehabituated in special recording chambers for at least 4 h with freeaccess to food and water during the entire experiment. Biphasicconstant current pulses (0.1 ms per cycle) were applied to the cCA3in order to evoke CA1 field potentials of about 60% of the slope ofthe field-EPSP and 25% of the maximal amplitude of the populationspike. To determine these stimulus intensities an I/O curve wasestimated. Biphasic, constant current pulses (0.1 ms per half-wave,50–600 �A) were applied to the cCA3 (Fig. 2). From this I/O theaccording E–S curve was calculated (−24 h). Due to the prepared“double”-recording electrode a simultaneous recording of both, thefield-EPSP and the population spike was possible.

For test recordings, biphasic constant current pulses (0.1 ms percycle) were applied to the cCA3 in order to evoke CA1 field poten-tials of about 60% of the maximal slope function of the field-EPSPand 25% of the maximum population spike amplitude. After record-ing a stable baseline for 1 h, (every 5 min 4 test pulses at a frequencyof 0.5 HZ were averaged and stored, alternately every 10 min forthe field-EPSP and the population spike) test stimuli were given foranother 8 h and at the next day.

In another experimental group, after recording a baseline for1 h, rats were tetanized by a primed burst stimulation protocol

(consisting of a single priming pulse followed 190 ms later by aburst of 10 pulses delivered at a frequency of 100 Hz, with 0.1 mspulse duration per half-wave) with the stimulus intensity of 25% ofthe maximal population spike amplitude. Initially, after 5 min andthen every 10 min, four test stimuli were applied, and the averaged,

T. Scherf et al. / Journal of Neuroscience Methods 188 (2010) 1–6 3

Fig. 1. Schematic illustration of rat frontal brain sections (Paxinos and Watson, 1998) (A) and histological proof of electrode localization: (B) stimulation electrode in the cCA3and (C) localization of the fixed “double”-recording electrode in CA1 (arrow (b) represents the localization of the longer tip in stratum radiatum for recording the field-EPSP,a aractem plitudo is fiels

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rrow (a) the shorter tip for recording the population spike). (D) and (E) show cheasured between the represent markers; and from the stratum pyramidale (E) am

f the contralateral CA3. Calibration 10 ms/2 mV. (Abbreviations: CA3, cornu ammon.r., stratum radiatum.)

ean value of the slope of field-EPSP and the population spikemplitude were recorded for another 8 h and after 24 h. Two hoursfter the 24 h recording an I/O curve was estimated to comparehe input–output relations before and after the experiment. Fromhis I/O the according E–S curve was calculated (+26 h).

Statistical significance was estimated by using the Wilcoxon-igned rank test when compared within one group or 2-way ANOVAhen compared between groups. The statistical significance was

et at p < 0.05.After the end of experiments the correct placement of the elec-

rodes was histologically verified and only those rats with correctocalization of the electrodes were included in the final analysis.

. Results

.1. Preparation procedure

Here, we introduce a method for the simultaneous recording ofhe field-EPSP and the population spike in the CA1 region in freely

oving rats by using a chronically implanted “double”-recording

ristic waveforms recorded from the stratum radiatum (D) slope of the field-EPSPe of the population spike measured between the represent markers, by stimulationd 3; CA1, cornu ammonis field 1; hf, hippocampal fissure; s.p., stratum pyramidale;

electrode. In Fig. 1A schematic illustration of the localization ofthe bipolar stimulation electrode in the contralateral CA3 and the“double”-recording electrode in the CA1 is shown. Further, his-tological proofs for localization of the implanted electrodes areprovided (Fig. 1B—stimulation electrode; C—recording electrode,respectively). During preparation, electrodes were implantedunder visual electrophysiological control, i.e. electrodes were low-ered into their final place verified by the optimal possible recordedsignal. The distance between the two tips of the recording elec-trode was around 260–280 �m. The longer tip of the recordingelectrode crossed the stratum oriens and the stratum pyramidaleduring preparation, finding its final position in the stratum radia-tum for recording the field-EPSP (Fig. 1C arrow b and D). The shortertip reached its final position in the stratum pyramidale for record-ing the population spike (Fig. 1C arrow a and E). The given distancebetween the two tips ensured, that by searching for an optimal pop-

ulation spike the longer tip of the recording electrode automaticallyreached the stratum radiatum for recording the field-EPSP.

Fig. 2A shows an example of a depth-profile of evoked poten-tials in the CA1 region by stimulating the contralateral CA3 region.The left panel shows a schematic pyramidal cell of the CA1, on

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Fig. 2. Recording of field potentials during the implantation of chronical electrodesand the input–output relation between the field-EPSP and population spike in freelymoving animal are provided. (A) Schematic illustration of a pyramidal CA1 neuronwith related field potentials. Representative recorded field potentials in relationto the depth beyond dural surface are given. (A(a)) shows the population spikerecorded in the pyramidal layer of the CA1, lowering the recording electrode further,the form of the recorded potential changes and in the layer of the apical dendrites,it turns into the characteristic field-EPSP (A(b)). Calibration 10 ms/2 mV. (Abbrevi-ations: s.o., stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum; s.lm.,stratum lacunosum moleculare.) (B) Representative analogous potentials for thefield-EPSP and the population spike in the CA1 region obtained by determining theinput–output relation. Analogous traces for CA1 field-EPSP recordings in the stra-tum radiatum (left panel) and analogous traces for CA1 population spike recordingsin the stratum pyramidale (right panel) and by stimulation of the cCA3 with ris-ing stimulation intensities of 50, 100, 150, 200, 300, 400 and 600 �A (from top tobottom) are given. Calibration 10 ms/2 mV.

Fig. 3. Effects of test stimulation and weak tetanization of the contralateral CA3 on both, thsamples of recorded signals—upper part field-EPSP to test stimulation of the cCA3 (left: bthe experiment), lower part field-EPSP with the weak tetanization protocol (left: baseli(calibration 10 ms/2 mV). (B) Represents the time course of the slope of field-EPSP measthe field-EPSP after weak tetanization symbolized by the arrow at time point “0” (filled cibaseline) are shown. (C) Analogous trace samples of recorded signals - upper part: populafter beginning the experiment; right: 9 h after beginning the experiment), lower part: pomiddle: 5 min after weak tetanus; right: 8 h after weak tetanus) (calibration 10 ms/2 mV).at its place of generation after test stimulations for test stimulation (open circles, n = 10)by the arrow (filled circles, n = 9). Mean values (±SEM) of the amplitude of the populatiorecorded signals before (open circles) and after 24 h (filled circles) of the beginning of thesignals before (open circles) and after 24 h (filled circles) of tetanization.

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the right panel representative evoked potentials in dependency onthe place of recordings are presented. Fig. 2A(a) represents a typ-ical population spike recorded in the stratum pyramidale of theCA1 on the shorter tip of the recording electrode; Fig. 2A(b) repre-sents the field-EPSP obtained from the longer tip of the electrodereaching the stratum radiatum of the CA1. The final position of therecording electrode for the optimal evoked population spike wasat around 2.20 mm from the dural surface and for the field-EPSParound 2.50 mm from the dural surface determined by the fixed dis-tance of both tips of the recording electrode of about 260–280 �m.In such prepared animals we were able to record simultaneouslyboth the field-EPSP and the population spike in the CA1 in freelymoving rats at the place of their generation.

3.2. Stable recordings of the field-EPSP and the population spikein freely moving rats and induction of an early-LTP in bothrecorded field potentials by primed burst stimulation

To verify, whether rats with such implanted “double”-recordingelectrodes were suitable for long-lasting, stable recordings of fieldpotentials, two separate stimulus intensities for the field-EPSP andthe population spike were determined. For this, an I/O curve wasrecorded by stimulating the cCA3 by using rising stimulus intensi-ties (50 �A up to 600 �A) (see recorded field potentials at distinctstimulus intensities in Fig. 2B). From this I/O relation two separatestimulus intensities were determined, one to induce a potential of60% of the maximal slope of the field-EPSP (measured between thetwo markers presented in Fig. 1D) and one for 25% of the maximal

population spike amplitude (measured between the two markerspresented in Fig. 1E).

As shown in Fig. 3, stable recordings in both parameters, thefield-EPSP (Fig. 3B, open circles) and population spike (Fig. 3D,open circles) were observed for at least 24 h by test stimulation

e slope of the field-EPSP (B) and the population spike (D) in CA1. (A) Analogous traceaseline; middle: 65 min after beginning the experiment; right: 9 h after beginningne; middle: 5 min after first train of weak tetanus; right: 8 h after weak tetanus)ured at its place of generation after test stimulations (open circles, n = 10) and forrcles, n = 9). Mean values (±SEM) of the slope of field-EPSP (% field-EPSP, relative toation spike obtained by test stimulation of the cCA3 (left: baseline; middle: 65 minpulation spike after the application of a weak tetanization protocol (left: baseline;(D) Represents the time course of the amplitude of the population spike measuredand for the population spike after weak tetanization at time point “0” symbolized

n spike (% population spike, relative to baseline) are shown. (E) E–S relationship ofexperiment with applying test stimuli to the cCA3. (F) E–S relationship of recorded

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f the cCA3 (n = 10). Fig. 3A and C, upper panels, represents anal-gous traces for the field-EPSP and population spike, respectivelyleft: baseline, middle: 65 min after beginning of the experiment,ight: 9 h after beginning of the experiment). When comparing the–S relation (Fig. 3E) calculated the day before test stimulation andfter 26 h, no differences were detected. This means that the long-asting stimulation of the cCA3 did not induce any changes in thenput–output relations of the pyramidal cell population of the CA1.his is an important prerequisite for investigations of changes inynaptic efficacy instead of changes in excitability during differentorms of synaptic plasticity such as LTP.

In a second set of experiments it was investigated, how a primedurst stimulation applied to the cCA3, known to be an inductionrotocol for an early-LTP in the CA1, changes the time course ofoth recorded field potentials. The stimulus intensity for the primedurst stimulation was about 25% of the maximal population spikemplitude. Fig. 3B (filled circles) and D (filled circles) shows theime course of an early-LTP in CA1 for the slope of the field-EPSPnd the amplitude of the population spike, respectively, inducedy primed burst stimulation of the cCA3 (n = 9). The slope functionf the field-EPSP (Fig. 3B, filled circles) showed an initial potentia-ion after primed burst stimulation of about 134.7 ± 10.14% whichas significantly increased up to 3 h after primed burst stimulationhen compared to the control recordings with test stimuli (Fig. 3B,

pen circles) (p < 0.05, 2-way ANOVA). After 5 h the slope func-ion of the field-EPSP returned to the baseline level (108.0 ± 8.83%,< 0.05; Wilcoxon-signed rank test). The population spike ampli-

ude (Fig. 3C, filled circles) showed an initial potentiation afterrimed burst stimulation of about 238.6 ± 34.64% which was sig-ificantly increased up to 4 h after primed burst stimulation whenompared to the control recordings with test stimuli (Fig. 3D,pen circles; p < 0.05; 2-way ANOVA). After about 7 h, the popula-ion spike amplitude returned to the baseline level (127.2 ± 8.13%;< 0.05; Wilcoxon-signed rank test). When comparing the E–S rela-

ion (Fig. 3F) calculated the day before the induction of an early-LTPnd after 26 h, no differences were detected. An early-LTP seems noto induce any changes in excitability or the net balance betweenxcitation and inhibition.

Our results show, that in freely moving animals the simultane-us recording of both the field-EPSP and the population spike withfixed “double”-recording electrode implanted in the CA1 region

llowed stable recordings of both measured field potentials by testtimulation of the cCA3 over a period of at least 24 h. The appli-ation of a primed burst stimulation induced an early-LTP in both,he field-EPSP and the population spike with its characteristic timeourse.

. Discussion

The aim of our study was to develop and establish a method forhe simultaneous recording of both, the field-EPSP and the popula-ion spike of pyramidal neurons in the CA1 region in freely movingats by stimulation of the contralateral CA3. Recently, a similarpproach was introduced by our lab which allows the stable record-ng of the both parameters in the dentate gyrus in freely behavingnimals (Frey and Frey, 2009). This method allowed reliably long-erm recordings of the field-EPSP as well the population spike inheir places of generation. Like in the dentate gyrus, for electrophys-ological recordings in the CA1 a fixed bipolar, “double”-recordinglectrode was introduced. This bipolar electrode was made out

rom two single wires straightened close together to ensure toecord from the same neuronal population. The distance betweenhe both tips of such an electrode is given by the anatomical struc-ure of the CA1 region: the distance between the tips was about60–280 �m. During preparation the bipolar recording electrode

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was moved down at specified coordinates, so that the shorter tipof the electrode reached the stratum pyramidale of the CA1 forrecording the population spike and the longer tip automaticallyreached the stratum radiatum for recording the field-EPSP at theplace of their generations (Figs. 1 and 2). After preparation andrecovery for at least 10 days, an I/O relation was taken in freelymoving rats to determine stimulus intensities to evoke potentialsof 60% of the maximal slope of the field-EPSP and 25% of the max-imal amplitude of the population spike. In such prepared freelymoving animals with the fixed “double”-recording electrode, wewere able to record in a very reliable manner stable field potentialsfor a duration of 24 h at least (Fig. 3, open circles). In a series ofexperiments, an early-LTP was induced using a primed burst stim-ulation of the cCA3. This particular pattern of stimulation revealeda characteristic time course of both, the field-EPSP and the pop-ulation spike, recorded at their places of generation (Fig. 3, filledcircles). The used primed burst protocol (single priming pulse fol-lowed 190 ms later by a high-frequency burst of 10 pulses at afrequency of 100 HZ) was well described by Diamond et al. (1988)to evoke plasticity events in vitro as well as in the awake rat. Theseauthors tested different time intervals between the priming pulseand the subsequent high-frequency train as well the optimal num-ber of pulses within the high-frequency burst. A priming intervalof about 140–170 ms in combination with a high-frequency burstconsisting of 8–10 impulses at a frequency of 100 HZ seems to beoptimal for the induction of LTP measured as an increase in theamplitude of the population spike in CA1. In addition to time inter-vals, frequency and numbers of stimuli, the stimulus intensity is animportant factor to reliably induce a defined form of LTP in the CA1.Hassan et al. (2006) could show, that in dependency of the stimulusintensity, tetanization protocols can result in different time coursesof LTP. At higher stimulus intensities, i.e. 60% of the maximal slopeof the field-EPSP, a transient depression followed by late-LTP wasoften detected in these studies. Reduction of the stimulus inten-sity to 30% eliminated this transient depression and revealed animmediate LTP in the CA1. Thus, besides the applied stimulationprotocol pattern for induction of different forms of LTP, the stim-ulus intensity is a crucial factor which determines the plasticityoutcome.

Here, we also show, that the value of stimulus intensity caneven be better determined by simultaneously recording the spikeand the EPSP, if corresponding recording electrodes were posi-tioned at their place of signal generation. In contrast to the singlerecording of the field-EPSP, which does not reach an asymptoticmaximal value by a strong stimulus, the population spike follows anasymptotic individual characteristic, i.e. the amplitude of the pop-ulation spike reflexes the sum of the number of discharging cellsand an increased synchronity of firing which finds it asymptoticlevel at a given high stimulus per animal. Leung and Au showedin vitro a different potentiation level and a different time courseof field potentials after application of a primed burst protocol atvarious stimulus intensities according to the I/O relations for thefield-EPSP and population spike (Leung and Au, 1994). If a stim-ulus intensity for the primed burst at the threshold-level for thepopulation spike was used, only transient forms of potentiationwere obtained, whereas 50% of the maximal value resulted in along-lasting potentiation. A primed burst protocol applied with astimulus intensities of maximal levels for the population spike didnot result in any potentiation, while a primed burst protocol witha stimulus intensity of the maximal level for field-EPSP causeda small but long-lasting potentiation. The primed burst protocol

used in the present study, was applied with a stimulus intensity of25% of the maximal level of the amplitude of the population spike.This particular pattern at that particular intensity resulted in a reli-able early-LTP in both recorded parameters, the field-EPSP and thepopulation spike (Fig. 3, filled circles). Our method of preparation

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nd recording using the fixed “double”-recording electrode com-ined with our first results for the induction of an early-LTP by arimed burst stimulation protocol can now be used for the furtherevelopment of protocols useful to induce reliably different formsf synaptic plasticity such as late-LTP or long-term depression inippocampal CA1-neurons in freely moving animals. In future weill even improve the standardization of stimulation intensities

etween different animals, by determining a stimulation intensityer animal which induces the asymptotic level of the populationpike. As mentioned above, in contrast to the EPSP, each individuals characterized by this specific asymptotic level for the popula-ion spike. Taking this stimulation intensity, new I/O curves canhen be constructed, including test stimulation intensities of, fornstance, threshold-intensity for the spike, 25%, 50%, 75% and 100%f the maximal population spike. This way of I/O curves would allows to choose standardized test intensities individually determinednd thus, more specific and comparable results between differentnimals.

cknowledgements

This work was supported by the DFG FR1034-7 to JUF and SFB79 TP B4 to JUF and SF.

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