Differences in synaptic transmission between medial and lateral components of the perforant path

Brain Research, 303 (1984) 251-260

251

Elsevier BRE 10102

Differences in Synaptic Transmission Between Medial and Lateral Components of the Perforant Path WICKLIFFE C. ABRAHAM and NEIL McNAUGHTON

Department of Psychology, Universityof Otago, Dunedin (New Zealand) (Accepted November 15th, 1983)

Key words: medial perforant path - - lateral perforant path - - dentate gyrus - - evoked potentials - synaptic transmission - - cable theory

The differences between the potentials recorded in the hilus of the dentate gyrus following test shocks applied separately to the medial perforant path (MPP) and the lateral perforant path (LPP) have been ascribed to the greater length of dendrite over which the LPP potentials are electrotonically conducted to the somata of the granule cells. We tested this hypothesis by recording MPP and LPP evoked potentials in the hilus and in the molecular layer of both in vivo and in vitro preparations. Analysis of field potential and current source density depth profiles in vivo indicated that different waveshapes occurred not only in the hilus but at the sites of synaptic contact in the molecular layer as well. In the in vitro study, paired stimulating and recording electrodes were stepped through the molecular layer and revealed a relatively sudden waveshape change around 225/~m from the cell layer, where the transitional zone between MPP and LPP terminal fields was expected to be located. Quantitative analysis of the differences between the potentials recorded in the molecular layer and the hilus revealed that electrotonic decay accounts for approximately 20% of the difference seen in the hilus between the MPP and LPP potentials. Our data therefore suggest that the differences between MPP and LPP hilar potentials are due mostly to differences between the two pathways in their properties of synaptic transmission and are due relatively little to the different sites of synaptic contact on the dendritic tree. INTRODUCTION The medial and lateral areas of the entorhinal cortex in the rat p r o j e c t via the angular bundle to the dentate gyrus ( D G ) . Fibers from the medial entorhinal cortex travel in the medial perforant path (MPP) and t e r m i n a t e on the granule cell dendrites largely within the middle third of the molecular layer. Fibers from the lateral entorhinal cortex travel in the lateral p e r f o r a n t path (LPP) and terminate on the distal third of the granule cell dendrites9,12,15. In addition to their different loci of termination these two pathways differ histochemically 8 and physiologically. One p r o m i n e n t physiological difference r e c o r d e d in the dentate hilus is that L P P shocks e v o k e field EPSPs with longer p e a k latencies, slower risetimes (RT) and greater width at half-amplitude (halfwidth) than do M P P stimuliTA2,13. These differences in waveshape have been i n t e r p r e t e d in terms of Rail's cable mode114 of the electrotonic spread of

m e m b r a n e potentials 12. A c c o r d i n g to this interpretation, the LPP potentials show a greater attenuation of waveshape via electrotonic decay than do M P P potentials since the L P P terminates m o r e distally on the granule cell dendrites. The square-root of the RT, measured in the hilus, has been taken by others as an index of the distance b e t w e e n the active synapses and the somata H. W e question the above explanation on two accounts. First, it requires the previously untested assumption that the M P P and L P P e v o k e potentials of similar shape at the site of origin in the molecular layer. Preliminary evidence from our l a b o r a t o r y has shown this not to be the case t. Second, since the M P P and LPP terminal fields are adjacent9,12, cable theory predicts a smooth transition in evoked potential waveshape as a stimulating electrode within the angular bundle passes from M P P to LPP fibers. In fact a step transition has been r e p o r t e d 12. F u r t h e r m o r e , when a stimulating electrode was advanced within the bor-

Correspondence: W. C. Abraham, Department of Psychology, Box 56, University of Otago, Dunedin, New Zealand. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

252 ders of a particular pathway, no gradual change in response shape was reported12, as might be expected if the cable model hypothesis were correct and the terminal fields were proximodistally distributed in the regions of the molecular layer 15. The present study was designed to address these issues directly by comparing MPP and LPP evoked potentials recorded in the hilus to the MPP and LPP potentials recorded at each pathway's terminal zone in the molecular layer of both in vivo and in vitro preparations. METHODS

In vivo procedures Male Sprague-Dawley rats (300-500 g) were anesthetized with sodium pentobarbital (60 mg/kg i.p.) and placed in a stereotaxic instrument. Supplemental doses of anesthetic (12 mg/kg) were administered as required. A heating lamp was used to maintain rectal temperature at 37 _+ 0.5 °C. The skull was exposed and leveled between lambda and bregma. Each rat was provided with one 75/am teflon-coated stainless steel wire recording electrode (exposed at the crosssectional area of the tip) in the D G and two ipsilateral monopolar 125,um nichrome wire stimulating electrodes (insulated with formvar except at the crosssectional area of the tip), one each in the MPP and LPP. The coordinates for electrode placement were (relative to bregma): DG, 3.8 mm posterior and 2.5 mm lateral; MPP, 7.7 mm posterior and 4.0 mm lateral; LPP, 7.7 mm posterior and 5.0 mm lateral. The depth of each electrode was determined electrophysiologically as described below. Skull screws served as recording and stimulating indifferent electrodes. Stimulus timing and evoked potential sampling were controlled by a DEC PDP 11/34 laboratory computer. Field potentials were amplified and filtered (1-3 kHz) by a Grass P15 preamplifier, sampled and digitized at 100-/as intervals and stored on magnetic disks for later analysis. Electrical stimulation typically consisted of diphasic square-wave pulses (125-500/~A, 0.1 ms half-wave duration) delivered through WPI constant current stimulus isolators.

Experimental protocol The recording electrode was lowered vertically

into the hippocampus by a Frederick Haer hydraulic microdrive to the depth of the dentate hilus as determined by recorded cellular discharge. In 7 animals stimulating electrodes were then placed stereotaxically in the angular bundle at locations which gave good separation of the MPP and LPP waveforms. This was achieved by adjusting the stimulating electrode depth until the RT > of the short-latency positive evoked responses to single test shocks fell within the range previously described for the MPP and LPP (MPP = 1.43 _+ 0.03; LPP = 1.76 + 0.10 ms~/~)1~. These measures were taken when the field EPSP was about 2 mV in amplitude, well below threshold for a population spike. The MPP electrode placements were always dorsomedial to the LPP electrode sites. These electrophysiologically defined electrode placements give good isolation of MPP and LPP fibers as revealed by the pattern of terminal degeneration in the molecular layer following lesions at the electrode sites 12 (Abraham and Mason, unpublished observations). In some animals convergence tests were used to confirm that the two pathways converged onto an overlapping set of granule cells ~2. Specifically, we required that nearly simultaneous stimulation of the MPP and LPP (MPP delayed by 3-5 ms) produce a hilar population spike at least two times larger than that observed following linear summation of the individually recorded MPP and LPP evoked potentials. MPP placements lateral to LPP placements gave poor convergence and were not used in the experiment. The recording electrode was then withdrawn through the hilus, the dorsal blade granule cell layer and the molecular layer in 50-/am steps. At each step 8 potentials were elicited at 0.1 Hz, 4 from each stimulating electrode. Each set of 4 was averaged. The laminar profile was continued until well into CA1. In a second experiment involving two animals, the recording electrode was fixed in the dentate hilus and the stimulating electrode was advanced in 200-/am steps across the angular bundle at an angle of 25-30 ° from the midline and 35 ° anterior-posterior. At each step the stimulation current and half-wave duration was adjusted to elicit a positive synaptic potential of 0.3-0.5 mV in amplitude. Eight field potentials were sampled at 0.1 Hz and averaged for each stimulus site.

253 Field Potentials

CSO

o[Fig. 1. In vivo MPP laminar profile with drawing of a granule cell and distance across the molecular layer indicated. B: field potential profile with 4 responses averaged at each 25-/~mstep of the recording electrode. Notice the peak negative response is close to the middle of the molecular layer. C: current densities calculated from the field potentials using a 3-point difference formula5. This analysis gives enhanced spatial resolution of the sink/source relationships. A small source is found near the hippocampalfissure and a larger one in the granule cell layer. A major sink, representing the MPP synaptic zone, occurs in the middle of the molecular layer. Calibration bar: 1 mV (field potentials) or 200 mV/mm2(CSD), 2 ms.

Data analysis The laminar profiles generated in Experiment 1 were subjected to one-dimensional current source density (CSD) analysis. These double-differentiation calculations sharpen the spatial resolution of local membrane currents by removing spatially slow-changing potentials, e.g. volume-conducted potentials generated in CA1, which may distort waveshape measurements. The double-differentiation was achieved using the 3-point finite difference formula (D1) of Freeman and Nicholson 5 which gave maximal resolution of the primary sources and sinks. High frequency spatial noise was avoided by using relatively large sampling steps of 50/~m. Since we did not measure tissue conductivity the results are expressed as mV/mmL One-dimensional CSD analysis of perforant path-DG field potentials has been previously used in both in vivo and in vitro experiments2,10. Fig. 1B and C illustrate a field potential laminar profile and associated CSD analysis for MPP stimulation. Both the field potential and CSD waveforms were analyzed at the sites where maximal amplitude positive and negative responses were recorded for each of the LPP and MPP stimulus conditions. As illustrated in Fig. 2B, the responses were measured for onset latency (at 10% of peak amplitude), peak latency (from onset), peak amplitude, RT in (10-90% of peak amplitude) and width at half-amplitude (half-

width). The waveforms in Fig. 2C and D were taken from one animal's depth profile and show representative field potentials and CSDs for both the LPP and MPP.

In vitro procedures Male Sprague-Dawley rats (300--600 g) were decapitated and the brains swiftly removed and placed on filter paper moistened with medium resting on an icecold petri dish. The hippocampus was dissected out and placed on moistened filter paper on the stage of a Sorval tissue chopper. Transverse slices of hippocampus were taken at 500/~m from the middle twothirds of the hippocampus and were placed in ice-cold oxygenated medium. They were subsequently transferred to the stage of a chamber which had previously been filled with ice-cold medium. Oxygen mixed with 5% CO 2 was bubbled into the chamber and escaped over the slices at a rate of 0.5 l/min throughout the experiment. Medium entered the chamber at a rate of approximately 10 ml/h and drained out of the chamber via the filter paper on the stage. The composition of the medium was 10 mM glucose, 124 mM NaCI, 5 mM KCI, 1.25 mM KH2PO 4, 2 mM MgSO4, 2 mM CaCI2, 26 mM NaHCO 3. The chamber was brought up to its working temperature of 34.0 + 0.5 °C over the initial 20 min of incubation. The slices were then left to equilibrate for at least 1 h before recording began. The experiment was usually terminated within 8 h. Two active recording and two bipolar stimulating electrodes were used. The active recording electrodes were glass micropipettes filled with 4 M NaC1 having a tip resistance in the range 0.5-2.0 Mr2. The bipolar stimulating electrodes were twisted insulated stainless steel wires, 25 ,um in diameter, with a resistance of approximately 150 KQ. One stimulating and one recording electrode were mounted together in a single electrode carrier with a horizontal tip separation of approximately 700/~m; these will be referred to as the 'moving' electrodes. The other stimulating and recording electrodes were mounted in separate carriers and will be referred to as the 'fixed' electrodes. The ground and indifferent electrodes were placed in contact with the stage of the recording chamber.

254 Experimental protocol and data analysis

The slice chamber was viewed through a microscope and the electrodes were positioned in the tissue under visual guidance (Fig. 2A). The fixed recording electrode was placed in or just below a minimally curved portion of the granule cell layer of the dentate upper blade where the largest amplitude positive responses were recorded. (For comparison purposes with the in vivo experiment, however, this will be referred to as the 'hilar' placement). The fixed stimulating electrode was then placed in the middle third of the molecular layer. The moving electrodes were oriented such that the line between their tips was parallel to the granule cell layer and the evoked response was near maximal. The position of the moving recording electrode was adjusted so that movement normal to the cell layer would bring it close to the position of the fixed recording electrode. The moving stimulating electrode was placed on the opposite side of the moving recording electrode from the fixed stimulating electrode (Fig. 2A). After these adjustments the moving electrodes were moved only normal to the granule cell layer, penetrating the slice for 75-100/~m at each placement. Responses were recorded as the moving electrodes were stepped across the molecular layer in either direction between the hippocampal fissure and the granule cell layer. At each step 4 combinations of

stimulation and recording were sampled 8 times at 0.1 Hz and averaged: fixed stimulating-fixed recording (FS-FR); fixed stimulating-moving recording (FS-MR); moving stimulating-fixed recording (MSFR); moving stimulating-moving recording (MSMR). FS-FR provided an indication of the health of the slice; only those slices showing stable FS-FR responses over the course of the experiment were included in the study. One-dimensional CSD calculations on the FS-MR data provided an estimate of the bandwidth of fibers activated by a typical stimulus by resolving the spatial extent of synaptic-related inward membrane current. A weighted 5-point smoothing formula (D3) was employed to reduce high frequency spatial noise accompanying the smaller (10-25 ktm) sampling intervals 5. The other two electrode combinations provided data for the comparison of hilar and molecular layer evoked potentials. Two experimental paradigms were employed. In the first, 15 slices from 7 animals received relatively high intensity diphasic pulses (5.5-12.7 V, 50-100/~s half-wave duration) and data were collected at 25 ktm intervals. These intensities were below threshold for a granule cell population spike, but activated a rather large band of fibers as shown by CSD analysis. In the second paradigm, 4 slices from 3 animals were stimulated at the lowest intensities producing reliable potentials (4.0-9.0 V, 50~s half-wave duration) and

TABLE I In vivo field potential (FP) and CSD waveshape differences between MPP and LPP as a function of recording site

A, mean peak latency and half-width values in ms; mean RT v2values in ms~': (n = 7); B, F ratios and significance levels for the ANOVAs. Evoked response shape characteristic Peak latency FP

Half-width CSD

FP

RT ~/~ CSD

FP

CSD

A. Electrode positions Stimulating

Recording

LPP MPP LPP MPP

Hilus Hilus Molecular layer Molecular layer

4.3 3.0 3,3 1.9

3.6 2.3 3.0 1.9

5.5 5.0 4.9 3.2

4.5 4.4 4.8 3.3

1.76 1.43 1.50 1.17

1.61 1.29 1.51 1.17

111.3"** 133.5"** <0.1

205.6*** 7.6* 0.!

B. Analysis of variance

Stimulation site Recording site Stim x Record * P < 0.05; ** P < 0 . 0 1 ;

94.1 *** 82.1"** 0.1 *** P < 0.001 ( d f =

1,12).

53.2*** 9.5** 0.3

69.0*** 46.0*** 12.3"*

6.8* 2.1 9.8**

255

C. Field Potentials

D. CSDs

(lower half of Fig. 2C). These differences were similar to those observed between the corresponding positive potentials recorded in the hilus (upper half of Fig. 2C). Similar correspondence was observed following CSD analysis (Fig. 2D), It can be seen in Table IA that the mean values of 3 waveshape measures (peak latency, half-width and RT 1/2) for the group of 7 animals show differences between the LPP and MPP waveshapes in the molecular layer that mirror the waveshape differences recorded in the hilus. The only exception was half-width, which was often attenuated in LPP positive potentials by the appear7.0'- A

Fig. 2. A: schematic diagram of the DG dorsal blade and the relative positions of the two recording (FR, MR) and two stimulating (FS, MS) electrodes for the in vitro preparation. The MS and MR electrodes were held 700-1000/~m apart in a single electrode carrier and moved along the trajectory indicated by the dotted lines. Either 25 Hm (Experiment 1) or 10/~m (Experiment 2) steps across the molecular layer were used. B: sample waveform showing the measures used to quantify responses. C: sample MPP (solid lines) and LPP (dotted lines) evoked potentials recorded in viva from either the hilus (upper half) or the molecular layer (lower half) at the point of maximal amplitude for each pathway. D: current densities calculated from the profile which included the potentials shown in C. Responses shown are the maximal source (upper half) and sink (lower half) currents obtained for that profile. Note that the relationship between MPP and LPP responses is maintained. Calibration bar: 1 mV (field potentials) or 80 mV/mm 2 (CSD), 2 ms.

data were collected at 10~tm intervals between 100 #m from the cell layer and the hippocampal fissure. The evoked potentials were sampled and quantified in much the same way as for the in viva data. However, despite averaging 8 responses, the waveforms collected at 10/~m steps remained somewhat distorted by noise. Thus, prior to waveshape analysis, we performed a running 9-point (representing 0.9 ms) unweighted average to smooth the potentials. RESULTS

In viva Experiment 1: recording electrode depth profile The LPP and MPP peak negative field potentials were recorded in the molecular layer an average of 130/~m apart and differed in both shape and latency

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Fig. 3. A: Plot of peak latency and half-width data for in viva Experiment 2, Responses were recorded at a fixed hilar site while the stimulating electrode advanced through the angular bundle in 200/zm steps. A step in each measure is observed for both animals. B: relationship between half-width and peak latency. The data are taken from both Fig. 3A of the present experiment (filled symbols) and Fig. 5 of reference 12 (open symbols). A single linear regression line has been fitted to the data. The overall correlation between the two variables in 0.945. Circles and triangles indicate data from individual animals in the two studies.

256 ance of a late negative potential (Fig. 2C and D). For this measure, the molecular layer difference was even larger than that observed in the hilus. CSD analysis did not alter this overall pattern of results. A 2 x 2 analysis of variance (ANOVA) on each measure revealed that the peak latencies and RT ~,~of both the field potentials and the CSDs showed highly significant main effects of stimulating site and recording site with no interaction (see Table IB for results of the statistical analyses). The significant effect of stimulation site indicates that the MPP and LPP responses differed both in the hilus and in the molecular layer. The absence of a significant interaction term indicates that this difference was equally large at both locations. These data suggest that the synaptic potentials differ between the two pathways at their sites of origin. Furthermore, the waveshape measures of the hilar responses were always greater than those found in the molecular layer as shown by the significant effect of recording site. This indicates attenuation of the response waveshape over the distance between recording sites. The lack of a statistically significant interaction between this attenuation and stimulating site implies that the LPP and MPP show similar degrees of attenuation. In contrast, A N O V A of the half-width measures did reveal a significant interaction of recording site with stimulating site. This finding was due to a reduced MPP/LPP difference in the hilus but not in the molecular layer. As mentioned above, the LPP half-width reduction in the hilus appeared to be due to the presence of a late negative potential at this recording site.

plotting the half-width and peak latency data against each other (see their Figs. 5 and 6) to compare with the theoretical relationship between these shape indices described by Rail TM. The error resulted in a function spuriously similar to the function predicted by cable theory. We have replotted the data from their original experiment with the data from the present experiment to re-examine the issue. Fig. 3B shows no evidence of a curvilinear relationship between half-width and peak latency, even at long peak latencies. In fact a single linear regression accounts for 89.5% of the variance with no significant quadratic component in the data (linear F = 465.0, df = 1/58, P < 0.0001; quadratic F = 0.01, n.s.). Since the LPP terminates on the distal portions of the granule cell dendrites where a curvilinear relationship is predicted by a simple cable modeU 4, the data do not appear to support the notion that electrotonic decay accounts for the difference in LPP and MPP potential waveshapes recorded in the hilus. However, these data do not necessarily count against the hypothesis since the topographic projection of angular bundle fibers to the D G molecular layer is not precisely known.

In vitro Experiment 1: high voltage profile, 25-#m steps Fig. 4 shows the mean MS-MR profiles obtained peok

latency

holf-widlh

onsel Iotency

riSetime~

fissure 300

In vivo Experiment 2: stimulating electrode depth profile Fig. 3A plots the peak latency and half-width of hilar potentials for two animals while the stimulating electrode tracked through the angular bundle. These data confirm the previously reported observation 12 that a step function in field potential shape occurs as the stimulating electrode progresses from a dorsomedial position to a ventrolateral one. A plot of RT 1/~ against electrode depth showed a similar step function (data not shown). The step is presumed to have occurred as the stimulating electrode moved from MPP to LPP components of the angular bundle. Unfortunately the authors of the original study 12 appear to have mistakenly reversed the axes when

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Fig. 4. Mean waveform measures (MS-MR) from in vitro Experiment 1 plotted in 25 ,um steps from cell layer to fissure. Note the 3 separate zones of responses: 50 # m , 175 # m and 275 Mm from the cell layer. The two distal zones correspond to the sites of synaptic contacts for the M P P and LPP. Error bars indicate _+2 standard error limits.

257 from 15 slices when a relatively high level of stimulation was used. The negative synaptic potentials did not change monotonically with distance from the cell layer in either p e a k latency, half-width or R T 1/~. Instead there were 3 distinct zones, centering on 50, 175 and 275/~m from the cell layer, which gave different waveshape values: lowest at 175 ~ m from the cell layer, higher at 275 ktm and highest at 50 k~m. These zones c o r r e s p o n d well to the previously described MPP, LPP and commissural/associational terminal fields, respectively6,9. A l t h o u g h these d a t a confirm the in vivo findings that different potential waveshapes are p r o d u c e d in different synaptic zones of the D G molecular layer, they do not allow us to conclude w h e t h e r or not some fraction of the differences normally r e c o r d e d in the hilus are also due to the cable p r o p e r t i e s of the dendrites. F o r this we n e e d to c o m p a r e the waveforms obtained from the moving and fixed recording electrodes ( M S - M R vs M S - F R ) . This comparison can be m a d e only in the following e x p e r i m e n t where current spread to the inner third of the dendrites and distortion of the positive EPSPs r e c o r d e d by the F R electrode was eliminated by the use of very w e a k test pulses.

In vitro Experiment 2: low voltage profile, lO-[tm steps Stimulation was delivered at 10-tzm steps from 150 to 330 ~ m from the cell b o d y layer. Fig. 5 shows all 3 p a r a m e t e r s of the M S - M R waveforms with the characteristic step function shape change at the assumed junction of M P P and LPP terminal zones. The data varied within a t w o - s t a n d a r d - e r r o r range from 150 to 220 p m and again within a t w o - s t a n d a r d - e r r o r range from 270 to 330 ktm. A N O V A of the M S - M R and M S - F R d a t a together shows that in each case the d a t a followed a c o m b i n e d linear and cubic polynomial function (see Fig. 5) which was highly significant. F o r p e a k latency, half-width and R T 1/~ the linear trend F ratios were 149.7, 335.6 and 126.1, respectively, and the cubic t r e n d F ratios were 13.5, 10.3 and 15.2, respectively. A l l of these F ratios were significant with df = 1/94 at P < 0.0025. The presence of these highly significant trends allows us to view the s m o o t h curves drawn in Fig. 5 as a more accurate r e p r e s e n t a t i o n of the underlying data than the m e a n values o b t a i n e d at each depth. H a d

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Fig. 5. A: comparison of MS-MR (left profile) and MS-FR (right profile) responses obtained at 10-ktmintervals from a single hippocampal slice. Responses have been averaged over two consecutive recording sites but have not been further smoothed. Notice the step in waveshape occurring for both profiles around 250/~m from the cell layer. Dotted lines have been drawn corresponding to the MPP average peak latency. Calibration bar: 0.1 mV, 2 ms. B: peak latency, half-width and RT w data averaged over 4 slices for responses recorded 150--330 pm from the cell layer. For each of these measures, the absolute value obtained from MS-MR is plotted on the left. These data describe the EPSP shape characteristics at the point of termination of the stimulated fibers (synaptic zone). For each measure also, the difference between the absolute values obtained from MS-MR and those obtained from MS-FR (hilar deviation) is plotted on the right. The hilar deviation estimates the contribution to hilar waveforms of factors other than those producing variations in the synaptic zone data. ANOVA showed that there were highly significant trends in these data which are represented by the fitted polynomial regression functions drawn as continuous lines in the figure. The parallel dotted lines indicate 2 standard error limits. See text for explanation of these results.

the M S - M R results c o n f o r m e d with the assumption of similarity between medial and lateral EPSPs at their sites of origin, neither linear nor cubic trends should have been present. Equally, had the results been due solely to some steady change in the nature of the EPSPs with distance from the cell layer, no cubic c o m p o n e n t would have b e e n present. These statistics show, therefore, that the differences b e t w e e n EPSPs r e c o r d e d in the medial and lateral path termination zones are discrete. Fig. 5 also reveals small differences b e t w e e n the

258 MS-MR and MS-FR data (termed hilar deviation). However, in no case did this deviation involve a cubic component (F -- 1.2, 0.6, 0.6, respectively, for the 3 measures), but was due to a small linear (peak latency: F = 4.4, df = 1/94, P < 0.05) or a linear plus quadratic (half-width: linear F = 21.5, quadratic F = 30.1, df = 1/94, P < 0.0001) effect. In the case of RT 1/2,no significant deviations were observed at all. The deviation of MS-FR values from MS-MR values should reflect, at least in part, electrotonic decay. As such, it would be expected to show a relatively steady change with respect to distance from the cell layer. This is borne out by the presence of linear but not cubic deviations. These trends, while small, are in the direction predicted by cable theory and, in the cases of peak latency and half-width, are highly significant. These data suggest that the differences between medial and lateral responses recorded at the cell layer are due to a large difference in synaptic transmission (linear plus cubic components of the synaptic zone data) and a much smaller contribution of differential electrotonic decay (linear hilar deviations). Using the least-squares estimates of the 3 shape indices, we quantitatively determined what part of the difference observed at the cell layer was attributable to differences in synaptic transmission and what part to electrotonic decay. The values of the curves in Fig. 5 at 200 #m and 300 #m were used as representative of the medial and lateral responses. The differences between these values are shown in Table II. As can be seen from Table II only about 20% of the differences at the cell layer are not attributable to the differences observed in the molecular layer. TABLE II

Components of variation in the hilar responses to MPP and LPP stimulation ST, differences in synaptic transmission; ED, differences attributable to electronic decay. Values are taken from the fitted curves in Fig. 5 and represent differences between the depths 200/~m and 300/~m. ED%, ED difference expressed as a percentage of the total variation

ST ED ED %

Peak latency

Half-width

(ms)

(ms)

+0.8 +0.2 20

+1.3 +0.35 21

RT~/2(ms1/2) +0.2 +0.05 20

Finally, to ensure that the stimulating electrodes were activating only a narrow band of fibers at the stimulus strengths used, we analyzed the responses recorded by the MR electrode as it tracked past the FS electrode. CSD analysis of the responses in 4 slices revealed that the average width of the FS-generated current sink was 30/~m (range 20-40/~m). Since the currents delivered to the FS and MS electrodes were similar, this analysis estimates also the bandwidth of fibers activated by the MS electrode. The result indicates a sufficiently confined stimulus field to differentially activate MPP and LPP fibers at points beyond _+ 15/~m of the MPP-LPP border, and is compatible with the 20-30/~m transition zone between the two areas giving differently sloped potentials shown in Fig. 5. DISCUSSION

The main question addressed by the present series of experiments was whether, and to what extent, the differences between MPP and LPP evoked potentials recorded in the hilus of the D G reflect electrotonic conduction along differing lengths of granule cell dendrite. The use of cable theory in this situation assumes, however, that identical synaptic currents occur at each of the two input locations. Recording the synaptic potentials at their site of origin in the molecular layer revealed that the differences in waveshape recorded in the hilus in fact were very similar to those at the dendritic recording sites. This similarity was observed both in vivo while stimulating the MPP and LPP at the level of the angular bundle and in vitro when stimulating the MPP and LPP fibers directly in the dentate molecular layer. CSD analysis of the in vivo responses confirmed that the evoked response differences originated primarily in the molecular layer, implying that the synaptic currents generated by the two pathways differ in their temporal characteristics. As previously reported 12, a step function change in waveshape was observed as the stimulating electrode progressed through the angular bundle. Such a step could arise from identical synaptic input from the two pathways if the group of fibers activated at any one stimulus site diverge to terminate throughout the entire terminal field of that particular pathway. However, in the in vitro preparation, we found a similar step

259 function in the hilus that was equally well represented in the molecular layer. In this case we were using a low intensity stimulus that activated only a 30 pm band of molecular layer fibers at any one time. Thus the step observed in the hilus in vivo is unlikely to be explained by divergence of MPP and LPP fibers from the angular bundle onto their respective areas of dendrite. Analysis of the difference between simultaneously recorded molecular layer and hilar potentials showed significant linear but not cubic variation with depth for both peak latency and half-width. This variation between hilar and molecular layer potentials indicates that some attenuation over distance does occur, presumably as a function of electrotonic decay. Our best estimates indicate that only 20% of the difference observed in the hilus between MPP and LPP waveshapes is explained by the apparent electrotonic decay (Table II). The lack of a cubic trend in the deviations indicates that the decay did not contribute to the step function. It is reasonable that electrotonic decay only weakly differentiates the MPP and LPP since at least one estimate of the granule cell electrotonic length (0.94 L) indicates that these cells are electrically compact 4. The RT~ measure of hilar potentials has been used to estimate the distance from the cell body to the site of dendritic activation 11. Our data demonstrate that risetime differences between pathways originate primarily in the molecular layer where they are separated by a sharp transition zone. Furthermore, of the 3 waveshape measures, RT ~ was the only one for which the difference between MS-MR and MS-FR potentials, our estimate of electrotonic decay, was not statistically significant. We conclude that hilar potentials alone are insufficient to describe either the electrotonic properties of granule cells or the dendritic site of synaptic potentials. Hence we question their application in the absence of molecular layer recordings to other problems such as the effects of aging on granule cell membrane properties 3. A recent study reports that the fibers in the MPP and LPP have different conduction velocities 16, which would affect the relative timing of the synaptic potentials observed between the pathways. However, in 5 slices in which we were able to record both MPP and LPP fiber volleys, the MPP fiber volley peak latencies averaged only 0.15 ms (6%) less than

the LPP fiber volley latencies. The risetime of the fiber potentials showed a similar but smaller (4%) difference between pathways. Since the equivalent risetimes of the MPP synaptic potentials were approximately 0.7 ms (24%) less than those of the LPP potentials, differences in afferent synchrony can account for only a small part of the differences in synaptic potential waveshape. The underlying basis of the observed difference in MPP and LPP synaptic potentials remains unclear. There is evidence from a study of paired and repetitive stimulation that the medial pathway releases a larger fraction of its available transmitter per impulse than the lateral pathway 11. It has been suggested that this is due to differences in calcium mobilization inside the terminal u, a mechanism which could also explain differences in the time course of synaptic transmission. On the other hand, different transmitter or post-synaptic receptor species could be important factors in determining the timing of MPP and LPP synaptic events. There is as yet little evidence of such differences17. The present experiments confirm previous studies that the potentials evoked by stimulation of the MPP and LPP have different waveshapes. We have demonstrated that no more than 20% of the differences recorded in the hilus are explained by electrotonic conduction along different lengths of granule cell dendrites, and that the primary difference is one of synaptic transmission in each pathway's terminal field. ACKNOWLEDGEMENTS This work was conducted in the laboratory of Professor G. V. Goddard. We gratefully acknowledge his support during the course of the experiment and his comments on earlier versions of this manuscript. B. Dingwall and S. E. Mason provided excellent technical assistance. The laboratory computer software was written by Dr. R. M. Douglas. This work was supported by funds from a N.Z. Neurological Foundation grant to G.V.G. and N.Z. MRC grant 83/55 to N.McN. W.C.A. was supported by postdoctoral fellowships from the University of Otago, N.Z. and NINCDS, U.S.A.

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