Evidence for two physiologically distinct perforant pathways to the fascia dentata

Brain Research, 199 (1980) 1-19

l

© Elsevier/North-Holland Biomedical Press

Research Reports

EVIDENCE F O R TWO P H Y S I O L O G I C A L L Y DISTINCT P E R F O R A N T PATHWAYS TO T H E FASCIA D E N T A T A

B. L. McNAUGHTON* Department of Psychology, Dalhousie University, Halifax, N.S. B3H 4JI (Canada) and Institute of Neurophysiology, University of Oslo, Karl Johans Gate 47, Oslo 1 (Norway)

(Accepted April 17th, 1980) Key words: perforant pathway -- fascia dentata - - hippocampal slice

SUMMARY The effects of paired and brief trains of stimuli were compared in the medial and lateral components of the perforant pathway to the rat fascia dentata, both in vivo, and in the in vitro transverse hippocampal slice. In the intact preparation a sharp transition in response properties occurred at the midpoint of the medial to lateral range of input. In vitro this transition was found to correspond exactly to the transition in Timm stainability which characterizes the border of the medial and lateral termination zones in the outer 2/3 of the molecular layer. These data indicate that there are two physiologically distinct subdivisions of the perforant path. Evidence is presented that the interpathway differences are at least partly due to differential quantal contents of the EPSP under resting conditions. INTRODUCTION The afferent fibre projection to the fascia dentata from the entorhinal cortex, known as the perforant pathway, has become the focus of considerable interest in recent years because of its remarkable capacity for both physiological and anatomical modification. In addition, the relatively simple laminar structure of this system makes it a particularly convenient model for the study of cortical function. Although the entorhinal cortex itself has been divided into distinct medial and lateral subfields on the basis of both cytoarchitecture and sources of afferent projections 2,3,14,2s,33, most studies of the physiology of the perforant path-granule cell projection have assumed it to comprise a single homogeneous system. A suggestion that this * Present address: Institute of Neurophysiology, University of Oslo, Karl Johans Gate 47, Oslo 1, Norway.

2 view might be oversimplified was made by Hjorth-Simonsen 8, who showed by degeneration techniques that fibres originating in the lateral division of the entorhinal area terminated in the distal one-third of the molecular layer, whereas fibres originating in the medial divison terminated in the middle third. In these studies, the initial trajectories of the fibres in the angular bundle were found to be anatomically separate. This laminar distribution of entorhinal terminals was confirmed by Steward 31 using autoradiographic tracing techniques. From an analysis of the relation between injection site and depth of termination, however, Steward concluded that there must be a continuously ordered mapping from the mediolateral axis of the entorhinal area onto the proximodistal axis of the outer two-thirds of the molecular layer. While the concept of a more or less continuously ordered mapping appears to be valid, there is good histochemical evidence that the middle and outer thirds of the molecular layer are biochemically distinct. This was shown by Hjorth-Simonsen 9 using the Haug modification of the Timm sulfide-silver method for heavy metals. The localization of these histochemical differences to the presynaptic terminals of the perforant path was indicated by Laurberg and Hjorth-Simonsen 15. Furthermore, lesions in the lateral entorhinal area result in a loss of Timm staining in the outer zone of the molecular layer coincident with synaptic terminal degeneration (Haug, personal communication). McNaughton and Barnes 22 found that, with an extracellular electrode located in the hilus of the fascia dentata, the evoked population EPSP thus recorded varied in waveform according to the location of the stimulating electrode along the medio-lateral axis of the perforant path trajectory. Laterally elicited responses, which were shown by terminal degeneration to involve synaptic activation in the outer molecular layer, exhibited greater rise times and half-amplitude widths than medially elicited responses (corresponding to synaptic activation in the middle third of the molecular layer). The separation of the initial trajectories of the medial and lateral components was also confirmed in these studies. In the present experiments, the assumption is made that the waveform variation of the field EPSP following stimulation of inputs terminating at different levels on the dendrites is a simple result of passive electrotonic spread. Similar effects of passive electrotonic spread of membrane currents on field potential peak latencies have been predicted on theoretical grounds 25 27,30, and demonstrated experimentally 24,25. Nelson and Frank 24 observed a gradual increase in the peak latency of the A-spike field potential of antidromically activated motoneurons as the recording electrode was moved away from the position of maximal response. Since it had been shown that the active currents of the A-spike were restricted to the initial segment of the axon it was concluded that the peak shift was due to electrotonic (decremental) spread of current within the dendrites. Nicholson and Liinas 26 showed that under certain atypical conditions the propagation of active spikes in alligator Purkinje cell dendrites was suppressed. Under these conditions, synaptic activation of the superficial dendritic regions resulted in purely passive field potentials which reversed polarity in depth. Both in their experimental records, and in the results of their theoretical model, one can see a shift in the rise time and an increase in half-width of the fully reversed field EPSPs with increasing distance from the locus of synaptic input.

While it is thus clear that passive propagation effects are to be expected in extracellular recordings, a more detailed discussion of the effects to be anticipated in the present context is nevertheless warranted here. There are several lines of evidence which indicate that the granule cell dendrite behaves passively following perforant path activation. Using current source density analysis in hippocampal slices Jefferys12 showed that while the proximal region of the dendrite did possess some electrical excitability, the threshold for active inward current was much higher than in the soma region. The only active dendritic responses following synaptic activation resulted from antidromic propagation of spikes initiated in the cell body region. McNaughton and Barnes 2z found that synaptic responses following simultaneous activation of two convergent subsets of perforant path fibres was always less than the linear sum of the independent responses, never greater as would be expected for an active process showing threshold behaviour. Finally, the spatial peak of extracellular negativity following synaptic activation shows no sign of propagation such as is seen with active dendritic responses in alligator Purkinje cells26. Stevens a°, Nicholson 25 and others have shown that, given certain not too unrealistic assumptions about the electrical properties of neural tissue, the extracellular potential due to spatially non-uniform synaptic activation of a neuron is proportional to the convoluted surface integral of the membrane current density divided by the distance from the recording site. In other words, as a first approximation, the extracellular potential close to a particular region of the neuron is roughly proportional to the local membrane current density, contributions from more remote current sources and sinks falling off rapidly with distance. This approximation will be even more accurate when recording in or below the granule cell layer in the fascia dentata since the neurons are organized in a curved, partially closed-field configuration. Klee and Rail la have shown that such a configuration can be expected to differ from a strictly parallel population of core conductors in that the equivalent source current density of the population will fall offwith radial distance from the center of curvature. Thus, when recording in the hilus, the extracellular potential following synaptic activation in the dendrites should be very nearly proportional to the membrane current transient near the granule cell bodies. In addition, the partially closed configuration should result in the hilar region being approximately isopotential with the cell body layer, so that accurate electrode placement within this region should be relatively unimportant. That this is indeed the case can be seen in Fig. 1. It can be shown from simple cable theory ix that the membrane current at the soma behaves in much the same way as the membrane voltage following synaptic input at various distances from the recording site. Specifically, the rise time of the current transient is an accelerating function of the distance and, for reasonably short distances, should be approximately proportional to the square of the distance. For these reasons, it is assumed here that the square root of the rise time of the synaptic field potential recorded below the granule layer is approximately proportional to the distance of active synapses from the soma layer. It will be shown that this assumption agrees reasonably well with experimental observation. The square root of the rise time of the field EPSP will be denoted by the Greek letter, Z, in the following. The present study concerns short-lasting changes in synaptic efficacy which follow

4 single or repetitive activation of relatively small subsets of the total perforant path population. As described recently 1~,zl,23, long-lasting enhancement of synaptic strength following tetanic stimulation of the type originally described by Bliss and Lomo 5 and Bliss and Gardner-Medwin 4, requires the concurrent high frequency activation of relatively large subsets of the afferent fibre population. With the low intensity stimulation used here, only transient changes are observed. These changes are phenomenologically very similar to effects of repetitive activation seen at neuromuscular junctions and a wide variety of other synapses. These have generally been referred to as 'facilitation', 'depression' (or depletion), 'augmentation', and 'potentiation'. In preliminary studies of'potentiation' and 'facilitation' at perforant path synapses, the data were extremely variable from preparation to preparation, or even within preparation, if the stimulating electrode was moved between trials. Such variability had previously been reported by Lomo 19. On the other hand, the effects were quite reproducible from trial to trial if the electrodes were not moved. It became apparent that there was some systematic relation between the amount of 'facilitation' and 'potentiation' observed and the waveform of the extracellular EPSP. This relationship accounts to a large extent for the variability among preparations and electrode positions, and is described in detail here. METHODS Two sets of experiments are described here using, in one case, the intact rat anaesthetized with sodium pentobarbital, and in the other, in vitro transverse slices of rat hippocampus. In vivo studies

These experiments were performed on male hooded rats of the Charles River strain ranging in weight from 350 to 650 g. An initial intraperitoneal injection of 0.55 ml sodium pentobarbital (Nembutal; 60 mg/ml) was given which was generally sufficient to produce surgical anaesthesia within 10-15 min. Occasionally, supplemental doses of 0.2 ml were necessary. Respiratory blockage was prevented by insertion of an endotracheal tube by tracheotomy prior to placing the animal into the stereotaxic frame. Rats were mounted in a Kopf stereotaxic frame with the top of the skull in the horizontal plane. The skull was exposed and cleared of connective tissue, and the temporal muscles de-inserted to allow access to the lateral aspect of the skull. Bone flaps were carefully removed over dorsal hippocampus and posterotemporal cortex. The dura was slit with a 23-gauge hypodermic needle just sufficient to allow stimulating and recording electrode penetration. Immediately following surgical preparation intraperitoneal infusion of Nembutal was begun at a rate of from 0.15 to 0.30 ml/h (60 mg/ml) depending on the animal's weight and the depth of anaesthesia already present. This method maintained the depth of anaesthesia relatively constant throughout the experiment. The animal's body temperature was maintained at 35 °C ( ± 1 °C) by an electrically shielded incandescent lamp (60 W) which was switched by feedback from a rectal thermoprobe.

Electrodes for recording extracellular field potentials in the fascia dentata consisted of a single strand of stainless steel wire insulated with Teflon (Medwire, 114 #m o.d.). These electrodes were quite durable, so that the same electrode could be used in different preparations throughout a particular study. While they had the disadvantage of providing relatively poor spatial resolution, this was not required in the in vivo studies, since the recording was carried out in the hilus where the spatial rate of change of potential following perforant path activation is quite small (see Fig. 1). Monopolar stimulating electrodes were constructed from a single strand of Teflon-coated platinumiridium wire (Medwire, 200 #m o.d.) or stainless steel wire (114 #m o.d.). Ground and reference electrodes consisted of nichrome wires soldered to stainless steel screws. These were screwed into the skull over the frontal sinus (recording referent and ground), and at the midline at the junction of the interparietal and occipital bones (stimulus referent). Primary amplification was by means of a Grass P51B AC differential preamplifier with low and high half-amplitude cut-off frequencies set at 10 Hz and 3 kHz respectively. Stimulation was provided via photon-coupled stimulus isolators (WPI Series 800). All stimuli consisted of constant current, balanced, diphasic pulses ranging from about 50/zA to about 150 ~A (each way) in different situations. Pulse durations in different experimental situations varied from 20 #sec to about 50/zsec each half-cycle. With optimal stimulus locations in the angular bundle, the threshold for observing a perforant path synaptic response was as low as 30/tA × 20 #sec. Stimulus patterns were generated using two WPI series 800 digital stimulators which were controlled by the digital output of an on-line PDP-11/34 computer. The recording electrode was placed in the hilus of the dorsal fascia dentata 3.8 mm posterior and 2.0 mm lateral to bregma. The optimal depth was located by monitoring multiple unit activity as illustrated in Fig. 1. Stimulating electrodes were located at various positions along the mediolateral axis of the perforant pathway as described by McNaughton and Barnes ~2. The specific stimulus configurations in different experiments are described in Results. In vitro studies

The in vitro experiments were carried out using methods similar to those described by Skrede and Westgaard 29. M611-Wistar rats weighing from 300 to 400 g were killed with an overdose of ether. The brains were rapidly but carefully removed and placed on filter paper moistened with the incubation medium (see below). The hippocampi were dissected from both hemispheres and sectioned in the plane approximately transverse to the septo-temporal axis on a Sorvall tissue chopper. The nominal section thickness was 440 #m. Sections were transferred immediately to a standard perfusion chamber 29 perfused at a rate of approximately 1 ml/min with an oxygenated (95 ~ 02: 5 % CO2) medium containing: (in mM) NaC1 124; CaCI2 2; KCI 2; KH2PO4 1.25; MgSOa 2; NaHCO3 26, glucose 10. The slice surfaces were covered only by a thin film ofperfusion medium. The atmosphere above the slices consisted of humidified 95 ~o 02:5 % CO2 at a temperature of 34 4- 1 °C. Although preparation of the slices was generally complete within 8 min of opening the skull, they were allowed to equilibrate for 1.5 h before starting the experiments. Slices examined earlier than 1.5 h generally gave poor responses

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Fig. l. Demonstration of the method used to locate the optimal recording depth according to multiple unit activity (MUA) density. For this demonstration, a recording electrode was first positioned in the hilus by auditory monitoring of MUA density. One stimulatingelectrode was then placed in each of the medial and lateral perforant paths. The recording electrode was then withdrawn and a second penetration made in 5-/tm steps using a hydraulic microdrive system. At each step samples were taken of the evoked synaptic potentials from the medial and lateral perforant paths, with the stimulus intensity set well below population spike threshold, and of the MUA density. The latter was measured by passing the EEG signal through a high pass filter (300 Hz half-amplitude cut-off), rectifying it, and finally, passing the rectified signal through an analogue integrator with a 3-sec time constant. In A is shown the amplitude of the extracellular EPSPs of the medial and lateral pathways as a function of recording depth. The laterally elicited response was maximally negative in the outer region of the molecular layer, whereas the medially elicited response was maximally negative at about the middle of that zone. Note also that the spatial rate of change of potential in the hilus is very small. This is consistent with the partially closed field configurationof the fascia dentata as discussed in the text. The MUA density is shown as a function of depth in B. The vertical scale is logarithmic. A reconstruction of the electrode penetration made from camera lucida drawings of histological sections is shown in C. Abbreviations: CC, corpus callosum; A, alveus; P, pyramidal cell body layer; M, molecular layer of fascia dentata; G, granule cell body layer; H, hilus of fascia dentata.

which clearly improved with time. I n these studies, extracellular stimulation a n d recording were carried out within the perforant path t e r m i n a t i o n zone in the outer 2/3 of the molecular layer (see Fig. 7). Glass pipettes filled with 4 M p o t a s s i u m acetate were used for recording. These had resistances (at 1 kHz) of between 1 a n d 5 MfrS. The stimulus electrode consisted of a tungsten wire with a tip electrolytically sharpened to a b o u t 10/~m. The recording amplifier was A C coupled with 3 dB filters set at 1 Hz a n d 2 kHz.

I n b o t h in vivo a n d in vitro studies stimulus i n d u c e d changes in the responses were expressed as a f r a c t i o n a l change relative to the c o n t r o l level: P o s t s t i m u l a t i o n value - - P r e s t i m u l a t i o n value P r e s t i m u l a t i o n value RESULTS

In vivo studies Relation between EPSP waveform and response following high frequency activation A c t i v a t i o n o f small subsets o f p e r f o r a n t p a t h fibres at high frequency resulted in an increase in the m a g n i t u d e o f the synaptic response to subsequent stimuli. This increased responsiveness d e c a y e d within several minutes. A typical example o f the transient P T P seen with such s t i m u l a t i o n in the lateral p a r t o f the p e r f o r a n t p a t h is shown in Fig. 2. W h e n the decay d a t a are expressed as a f r a c t i o n a l change relative to baseline, a n d p l o t t e d semilogarithmically, there a p p e a r to be two e x p o n e n t i a l c o m p o n e n t s present with time constants differing by a factor o f a b o u t 10 (Fig. 2D). These c o m p o nents are d e n o t e d here b y A a n d P because o f their similarity to ' a u g m e n t a t i o n ' a n d ' p o t e n t i a t i o n ' as defined by M a g l e b y a n d Zenge120 for n e u r o m u s c u l a r data. The present e x p e r i m e n t was carried o u t on 10 animals, d a t a being collected first f r o m one a n d then f r o m the o t h e r hemisphere. A f t e r p o s i t i o n i n g the r e c o r d i n g electrode

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Fig. 2. This example is representative of the effects seen when a stimulus train of 100 pulses at 250 Hz is delivered to the lateral component of the perforant pathway. The control response and the response recorded 3 sec following the train are denoted by A and B respectively. The inflection seen on the failing phase of the elevated response (B) is the beginning of a 'population spike' due to the discharge of some of the granule cells. This demonstrates that the response increment is a real increase in synaptic efficacy, since the probability of postsynaptic output is increased. The time course of the increased synaptic efficacy plotted relative to the mean baseline response is shown in linear coordinates in C and in semilogarithmic coordinates in D. From the latter plot it is apparent that the data are well described by a composite of two exponential functions. These are referred to in the text as components A and P because of their similarity to augmentation and potentiation described by Magleby and Zengel 2°. The line drawn for component A was derived by least-squares fit to the residuals after subtraction of component P (see text).

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Fig. 4. A : the mean fractional change in the amplitude of the field EPSP 3 sec following a stimulus train of 100 pulses at 250 Hz is plotted as a function of the square root of the EPSP rise time. B: the mean relation between the fractional change in a test response relative to a control response preceding it by 45 msec vs the square root of the field EPSP rise time, In both studies there was an abrupt transition in response properties at about the middle of the range of the abscissa which corresponds approximately to the border of the medial and lateral perforant path termination zones (see Discussion). While the smooth curves through the data were fitted by eye, the departures from the linear prediction represented by them were statistically significant.

for maximal positive response, repeated penetrations with a stimulating electrode were made at varying positions along the mediolateral axis of the angular bundle as described by McNaughton and Barnes 22 (see their Fig. 1A). On each penetration, the stimulus current was adjusted to give an extracellular EPSP amplitude of less than 2 mV. This was well below the threshold for observing long-lasting synaptic enhancement following high frequency stimulation 2a and also welt below the granule cell discharge threshold. Test stimuli were delivered at 0.5 Hz for 24 pulses, after which a conditioning train of 100 stimuli at 250 Hz was delivered at the same intensity. This was followed by an additional 50 test stimuli at 0.5 Hz. After an approximately 5-min rest period, the stimulating electrode was withdrawn and moved to a new location corresponding to a different subpopulation of perforant path fibres (as determined on the basis of EPSP rise time), and the stimulation procedure repeated. Approximately 18 such runs were carried out on each hemisphere of each animal, with the position of the stimulation along the mediolateral axis being varied in a random fashion while holding the recording site fixed. The average of the first 24 responses in each run and the peak amplitude values of all responses were stored on magnetic disc. At the end of the experiment, each averaged baseline response waveform was classified according to the square root of its rise time (Z) into one of 12 intervals ranging from 1.08 to 1.90 (msec) i. All responses within an interval were normalized about their peak amplitudes and averaged across hemispheres and animals. The averaged response waveforms for the odd numbered intervals are shown in Fig. 3. As an estimate of the magnitude of the increase in synaptic responsiveness, the mean of the first two responses following conditioning were expressed as a fractional difference relative to the preconditioning baseline. These data were classified according to the z-value of the EPSP into 12 intervals as described above. The mean fractional change versus X is plotted in Fig. 4A. From this plot, it is apparent that not only does the fractional change increase from about 0.35 to about 1.75 over the range of x, but also that the relationship between these two variables departs appreciably from linearity. This departure from the linear prediction was statistically significant (the residual after a linear fit gave variance ratio of F ---- 3.86 with df = 10, 288). The data for EPSP amplitude versus time were analyzed as follows. First, the EPSP amplitude data for each run were expressed as a fraction of the mean baseline response. The post-tetanic decay curves were then averaged within each interval. Using the average decay curves, linear regression parameters were calculated for the semi-logarithmic data between 30 and 80 sec (component P) after tetanization. These provided estimates of the initial value of P (P0) and the decay time onstant (rp). The calculated exponential functions were subtracted from the original data and the procedure was repeated on the logarithms of the residuals between 2 and 12 sec to obtain parameters for component A. As can be seen from Table I the initial magnitudes of both components increased over the range of Z and thus contributed to the net increase shown in Fig. 4A. The relative contributions of the increases in the two components were, however, clearly different. This may be seen by comparing the means of the first 4 and last 4 x-groups for each component. For component P, the relative increase was 181 ~o while for component

10 TABLE I The values derived by least-squares analysis for the initial magnitudes ( Ao, Po) and decay time constants (VA, rp) are shown with the corresponding input location parameters (Z) ]or the EPSP

These parameters were derived from the same data as the points shown in Fig. 3A. The equation fractional difference Ae t/VA + Pe t/vp accounts for approximately 93 ~ of the variance in a semilogarithmic plot of the decay time course. Component P

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A it was 708 ~ . It should be pointed out that the sums of the initial values of these two derived components are considerably higher than the observed values for the net fractional increase shown in Fig. 4A. This is partly due to the fact that the initial values refer to t = 0 whereas the data shown in Fig. 4A are the means of the data taken at 2 and 4 sec following high frequency stimulation. In addition, however, the magnitude of the synaptic response was markedly reduced during and immediately after tetanic stimulation. Thus, the early points o f the observed time course are superimposed on the recovery from tetanic depression. This recovery was essentially complete within about 5 sec. R e l a t i o n b e t w e e n E P S P w a v e f o r m a n d r e s p o n s e to p a i r e d s t i m u l i

This section concerns the relationship between the z-value o f the EPSP and the magnitude o f a test response following at various delays after a single conditioning stimulus. This was studied in 3 different ways. The relation between Z and per cent change o f EPSP amplitude 45 msec following a ccnditioning pulse was studied in both hemispheres o f 3 animals. The recording electrode was located in the standard dentate hilus position. A stimulating electrode was m o u n t e d on a hydraulic microdrive system (F. Haer) which was set to advance in 50/~m steps every 5 sec. Immediately prior to advancing the microdrive a pulse pair was delivered (45 msec interpulse interval), and the corresponding response pair recorded on the magnetic disc. Vertical penetrations were made along two tracks 5.5 and 5.0 m m lateral to the midline and extending from 9.6 m m to 7.5 m m posterior to bregma. Parallel penetrations were made along these lines at 0.3 m m intervals, records being taken from the depth of the first appearance of responses (usually a b o u t 2.0--2.5 m m below the

11 cortical surface) to the deepest point from which responses could still be elicited reliably. The z-value and fractional change were determined for each of the 1715 (total) stimulus locations in the 3 animals. The results, shown in Fig. 4B were consistent with the previous experiment (Fig. 4A). For the medial half of the range of Z (1.05-1.55) there was a net depression of the test response of about -4).03. On the lateral half of the z-range (1.55-2.05) there was net facilitation of about +0.33. The time course of the effect of paired pulses in the medial and lateral components (Z < 1.5 and Z > 1.6 respectively) was studied in two experiments. The first experiment involved 6 animals in which the response to medial perforant path stimulation was measured in one hemisphere simultaneously with the contralaterally elicited lateral perforant path response measured from a second recording electrode located symmetrically in the contralateral fascia dentata. A series of pulse pairs with interpulse intervals ranging from 20 to 310 msec was delivered through each stimulating electrode. The interval between the last pulse of each pair and the first pulse of the next pair was 2 sec. The entire series of 30 intervals was repeated 25 times. The fractional difference between the responses in each pair was calculated and the means for each interval were averaged across animals. A statistically significant difference was found between pathways at all intervals tested. The medial pathway showed an initial small facilitation of approximately +0.10 which gave way to a depression by 100 msec which attained a maximum of about--0.12 at the longest intervals observed (310 msec). The lateral pathway showed a facilitation of about +0.60 at 20 msec which decayed to around zero by 310 msec. Since the depression of the medial response in this study was still increasing at 310 msec, it appeared likely that with a pair repetition rate of 0.5 Hz the response pairs were

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Fig. 5. The fractional change in field EPSP amplitude in the paired pulse paradigm is shown as a function of interpulse interval for the medial (filled circles) and lateral (open circles) inputs in both the intact (A) and hippocampal slice (B) preparations. In both cases, the interval between stimulus pairs was 30 sec. Increasing the between pair frequency resulted in a reduction of the relative depression on the medial input, and an increase in the relative facilitation of the lateral input.

12 not statistically independent of one another. For this reason, a second study was carried out in which the pair repetition rate was reduced to 1/30 Hz. With the electrode configuration as just described, a randomized series of 30 interpulse intervals was delivered, ranging from 20 msec to 4 sec. The overall series was repeated 4 times and the per cent differences between corresponding response pairs averaged. This procedure was carried out on 2 animals. The shape of the time course (Fig. 5A) was essentially similar to the first experiment, with the exception that no net facilitation was observed on the medial pathway in either animal. There was, however, a relative facilitation superimposed on a prolonged depression. The net depression reached a maximum of about --0.20 at 300-400 msec, and recovered approximately exponentially thereafter with a time constant of about 4 sec. The lateral pathway showed net facilitation similar to the previous experiment but reduced somewhat in magnitude, followed by a small depressive phase which was much smaller than the results for the medial pathway, but similar in time course.

Response of medial and lateral synapses during repetitive stimulation This experiment examined the changes in the magnitude of the extracellular EPSP during repetitive activation and the extent to which these changes may be systematically related to changes in the size of the presynaptic fibre response 1. Two recording electrodes were used in this study, one in the standard dentate location and one in the angular bundle, located approximately at the point where perforant path fibres enter the fascia dentata. This second electrode was used to monitor the activity of stimulated axons, presumably those of the perforant pathway. Four animals were used in this study. Each experiment consisted of two parts. In the first part, either the medial or the lateral pathway was stimulated. In the second part the alternate pathway was stimulated. The order of study was counterbalanced between animals. Stimulus trains of 20 pulses were delivered at frequencies of 0.2, 0.5, 1.0, 2.0, 4.0, 6.7 and 12.5 Hz, with a 90-sec recovery period between trains. The entire procedure was repeated 6 times. The results, expressed as fractional change relative to the first response in each series, were averaged across the 6 repetitions and across the 4 animals. The average results for the field EPSP at 2 and 12.5 Hz are shown in Fig. 6A. There was a clear difference between pathways which became apparent at and above 1 Hz. As is consistent with the paired pulse data presented above, the lateral pathway showed only a mild EPSP depression at lower frequencies, and an initial facilitation followed by a return to baseline at 12.5 Hz. The medial pathway showed depression only, which increased with frequency to about --0.4 at 12.5 Hz. All responses above 1 Hz differed significantly between pathways with the medial showing the greatest response depression. The fibre responses on the lateral pathway were not affected by frequency. On the medial pathway there was a slight decline at 2 and 4 Hz and a slight increase at 7 and 12.5 Hz. Thus, the effects on the EPSP at these frequencies are not accounted for by changes in the fibre response. In order to assess the effects of higher frequencies on the fibre response, an additional test was made on each of the animals in the study just described. Following the low frequency tests, each pathway received (separately) a series of 15 trains of 33 pulses

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Fig. 6. A: the effectsof repetitive stimulation at 2 Hz and at 12.5 Hz on the field EPSP are compared for medial (filled circles) and the lateral (open circles) perforant pathways. The data are expressed as fractional change relative to the first response in each 20 stimulus train. Each data point represents the mean from 4 animals. All differences are statistically significant (P < 0.05). B: the average fibre responses from one animal of the first and 33rd stimuli in a 250 Hz train are shown for the medial and lateral pathways. There were no consistent differencesin the ability of the fibres from the two pathways to respond repetitivelyat this frequency.Nor were there any apparent differencesin conduction velocity.

delivered at 250 Hz. Since the flow of synaptic current during the first part of the train made fibre response measurement unreliable, only the means of the first and last (33rd) stimuli were compared. Examples of these are shown in Fig. 6B. Neither pathway showed any marked tendency towards fibre response attenuation at 250 Hz, although in some cases there was a slight broadening of the response. There were no consistent differences in the peak latency of the fibre responses between pathways, indicating that their conduction velocities are approximately equal.

In vitro studies Time course of field EPSP changes with paired pulse stimulation of proximal and distal synapses The following experiment was carried out in 16 slices from 5 rats. Stimulating and recording electrodes were placed approximately 500 Fm apart at the same depth in the molecular layer. Both the medial and lateral termination zones were tested in each slice, the order of testing being counterbalanced. At each zone, pulse pairs were delivered with intervals ranging from 30 to 2000 msec. The amplitude of the negative-going field EPSP following each stimulus was recorded and the fractional difference between within-pair responses calculated. The mean data from the 16 slices are shown in Fig. 5B. It can be seen that the results were rather similar to the in vivo study in which the location of the active synapses was inferred from the EPSP waveform. The synapses of the outer zone showed marked facilitation while those of the middle zone were depressed.

Correlation of transition in paired pulse response properties with histochemically defined termination fields In 5 experiments, the variation in facilitation properties at a fixed inter-pulse

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Fig. 7. A: photomicrograph of the fascia dentata from a hippocampal slice stained with the T i m m Haug sulphide-silver method following completion of the experiment illustrated in B. With this method, the molecular layer is sharply divided into 3 laminae. The outer, darkly staining lamina, and the middle pale lamina have been shown to correspond to the lateral and medial perforant path termination fields respectively. Abbreviations: FIS, hippocampal fissure; S, superficial lamina of stratum moleculare; M, middle lamina of stratum moleculare; D, deep lamina of stratum moleculare; G, stratum granulosum; H, hilus; LAC, stratum lacunosum moleculare of field CA1. B : plot of paired pulse facilitation as a function of stimulating and recording location in the hippocampal slice shown in A. The transition in response properties corresponds exactly to the transition in Timm stainability demarcating the border of the medial and lateral termination fields. C: similar plot of the ratio of field EPSP to afferent fibre potential. The ratio is lower in the lateral termination field and increases abruptly as the Timm stain transition is crossed. Data from a different slice than A and B. Dashed lines in B and C indicate the location of the borders of the Timm fields of the corresponding slices.

15 interval of 40 msec was determined as a function of depth in the outer 2/3 of the molecular layer. First, the depth of the molecular layer was measured with the micrometer on the electrode micromanipulator. Then, both the stimulating and recording electrodes were moved in successive 10/~m steps from the outer margin of the molecular layer towards the granular layer. At each step, the average responses to 10 stimulus pairs (delivered at 3-sec intervals) was measured. The mean fractional difference between responses within pairs was then plotted as a function of position in the molecular layer. In addition, the compound action potential due to the activation of the presynaptic fibres 1 was measured in 3 of the 5 experiments. The ratio of the amplitudes of the EPSP and fibre potentials was then similarly plotted as a function of depth in the molecular layer. Following each experiment, the slice was removed from the incubation chamber and processed by the Timm stain for heavy metals according to Haug 10 to reveal the boundaries of the medial and lateral perforant path termination fields. In all experiments, a well defined transition occurred in the paired pulse response properties at a depth in the molecular layer corresponding to the transition in Timm stain reactiveness which delineates the boundary of the medial and lateral termination zones. A similar transition occurred in the EPSP to fibre response ratio, which was low in the lateral termination zone and higher in the medial. An example of these data, and the corresponding Timm-stained fascia dentata is shown in Fig. 7. The role of extracellular Ca z+ concentration on paired pulse response At neuromuscular synapses, when the quantal content of the EPP is reduced by manipulation of the external divalent cation concentrations, the curve for fractional difference versus inter-pulse interval is shifted from one resembling the data presented here from the medial perforant path to one resembling the effects seen on the lateral pathway (compare Fig. 4 of this report with Fig. 4 of Lundberg and Quilisch TMand with Takeuchi32). At the neuromuscular junction the process of facilitation is thought to be superimposed on a depression due to a reduction in the number of quanta available for release when the initial release probability is high. At reduced Ca 2+ levels, the depletion of available quanta on the first response becomes negligible and facilitation predominates. The following experiment was designed to determine whether similar effects occur in the medial perforant path. In 4 experiments, paired stimuli with an interstimulus intervals of 150 msec were delivered once every 30 see, stimulation and recording electrodes being located in the middle zone of the molecular layer. After establishing that the baseline level of paired pulse depression was stable, the perfusion fluid was switched to one containing no calcium. Complete fluid exchange in the slice chamber required approximately 30 min, during which time the stimulation was continued. As the exchange progressed, the initial magnitude of the EPSP fell progressively while the fibre responses remained relatively constant. This was accompanied by a reduction in the magnitude of relative depression on the second response on each pulse pair until, with very small initial responses, the net depression was converted to net facilitation. Restoration of the calcium to the perfusion fluid restored the initial magnitude of the EPSP and the initial level of paired pulse depression. An example of these data is shown in Fig. 8.

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DISCUSSION The classical a n a t o m i c a l a n d physiological descriptions of the perforant pathway to the fascia d e n t a t a regarded it as a single h o m o g e n e o u s system3,6,17. As discussed in the i n t r o d u c t i o n , however, more recent work has shown that the terminals of the fibres arising in the medial a n d lateral parts of the e n t o r h i n a l area are topologically separated in the molecular layer, a n d that there are two biochemically distinct subfields in this region, the distinguishing characteristics of which are localized in the afferent fibres• The possibility was raised that although the m a p p i n g from medial to lateral e n t o r h i n a l area may be topologically c o n t i n u o u s , there m a y nevertheless be two physiologically discrete subsystems.

17 The waveform characteristics of extracellularly recorded EPSPs in the hilus of the fascia dentata were previously shown to correspond, generally, to the location on the dendritic axis of synapses whose fibres were lesioned at the site of stimulation2L Electrode locations yielding fast or slow rising responses yielded terminal degeneration in the zones shown by others to be occupied by medial or lateral entorhinal afferents respectively. It was suggested above that the waveform differences in the field EPSPs recorded from the hilar region probably represent the effects of passive spread of membrane current from the synaptic zones to the recording site near the granule cell somata. It was suggested also that the square root of the rise time of the field EPSP (Z) might be approximately related to the distance of the activated synapses from the granule cell layer. In two in vivo experiments, it was found that the observable range of X was from about 1.1 to about 2.1. The corresponding ratio of that portion of the granule cells occupied by perforant path terminals to their total length is also about 0.5. Thus, the actual correspondence between the distribution of synaptic locations and the range of Z appears to be reasonably good. Since the conduction velocities and synchrony of the afferent fibre responses were not found to differ between medial and lateral pathways, and since the duration of the synaptic current, as estimated by the duration of the negative-going field EPSP, was similarly unrelated to input location, it appears that the parameter, Z, is a good approximation of the relative location of active synapses on the granule cell dendrites. The short-term changes in synaptic efficacy referred to as facilitation, depression, component A, and component P, were significantly related to the z-value of the particular population of synapses under study. In general, there was a large increase in the post-activation growth of EPSPs with increased Z- Furthermore, there was a step-like transition in the synaptic modification properties at about the mid-range of Z. By interpolation, the middle of the z-range corresponds to the middle of the perforant path termination zone, which is where the histochemical staining pattern of entorhinal afferents exhibits a sharp transition. This was confirmed directly in the hippocampal slice preparation by the demonstration that the location of the transition in synaptic properties corresponds exactly to the histochemical transition in the same material. The possibility that the inter-pathway differences reported here are due to differential changes in either resting potential or input impedance (for example an interneuronally induced shunt restricted to the medial zone) is ruled out by the demonstration 22that activation of the medial pathway has no effect on the field EPSP of the lateral pathway over the intervals used in the present study. Furthermore, the differences were found to remain relatively constant over a broad range of stimulus strength, down to the smallest detectable responses. This makes the involvement of interneurons rather unlikely. On the basis of these arguments and the evidence just summarized, it is reasonable to conclude that the perforant path projection to the granule cells is composed of two functionally discrete subsystems. Although this conclusion is contrary to a recent report by Harris, Lasher, and Steward 7 that the perforant pathway is functionally homogeneous, their conclusion was based on the responses to stimulation at frequencies at or

18 below 1 Hz. As shown here, the differences between the two inputs do not become very striking until higher frequency s t i m u l a t i o n is e m p l o y e d . It is p r o b a b l e that with the small n u m b e r o f animals used in the study o f Harris et al. 7 the small difference at I Hz would n o t be detected. The ratio o f EPSP to fibre response was f o u n d to be greater in the medial p a t h w a y . F u r t h e r m o r e , the depression seen there converts to facilitation upon reduction o f external Ca ~+. These results suggest that, u n d e r resting conditions, the medial p e r f o r a n t p a t h releases a larger fraction o f its available t r a n s m i t t e r per impulse than the lateral pathway. ACKNOWLEDGEMENTS The in vivo studies f o r m e d p a r t o f a d o c t o r a l dissertation presented to the F a c u l t y o f G r a d u a t e Studies, D a l h o u s i e University, in 1978. I a m grateful to Prof. G. V, G o d d a r d for n u m e r o u s critical discussions concerning the in vivo work, a n d to Dr. R. M. D o u g l a s for p r o v i d i n g the PDP-11 software. The in vitro w o r k was carried o u t in the l a b o r a t o r y o f Prof. P. A n d e r s e n to w h o m I a m also grateful a n d I t h a n k Dr. F . - M . S. H a u g for assistance with the histochemical work. I particularly t h a n k Dr. C. A. Barnes for assistance in all phases o f the w o r k presented here. This w o r k was s u p p o r t e d by G r a n t s A0365 f r o m N R C o f C a n a d a a n d 576-0967 f r o m the C a n a d a Council to G. V. G o d d a r d , a n d by p o s t g r a d u a t e a n d p o s t d o c t o r a l scholarships f r o m N R C o f C a n a d a to the a u t h o r .

REFERENCES l Andersen, P., Holmqvist, B. and Voorhoeve, P. E., Entorhinal activation of dentate granule cells, Acta physiol, scand., 66 (1966) 448-460. 2 Beckstead, R. M., Afferent connections to the entorhinal area in the rat as studied by retrograde cell-labelling with horesradish peroxidase, Brain Research, 152 (1978) 249-264. 3 Blackstad, T. W., On the termination of some afferents to the hippocampus and fascia dentata, Acta anat. (Basel), 35 (1958) 202-214. 4 Bliss, T. V. P. and Gardner-Medwin, A. R., Long-lasting potentiation of synaptic transmission in the dentate area of unanesthetized rabbit following stimulation of the perforant path, J. Physiol. (Lond.), 232 (1973) 357-374. 5 Bliss, T. V. P. and Lomo, T., Long-lasting potentiation of synaptic transmission on the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. PhysioL (Lond.), 232 (1973) 334-356. 6 Cajal, S. R., Histologie du Systdme Nerveaux de l'Homme et des Vert~bres, A. Maline, Paris, 1911. 7 Harris, E. W., Lasher, S. and Steward, O. V., Analysis of the habituation-like changes in transmission in temporo-dentate pathway of the rat, Brain Research, 162 (1979) 21-32. 8 Hjorth-Simonsen, A., Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata, J. comp. Neurol., 147 (1972) 219-232. 9 Hjorth-Sirnonsen, A., Some intrinsic connections of the hippocampus in the rat : an experimental analysis, J. comp. Neurol., 147 (1973) 145-162. 10 Haug, F.-M. S., Heavy metals in the brain, Advanc. Anat. Embryol. Cell Biol., 47 (1973) 4. 11 Jack, J. J. B., Nobel, D. and Tsien, R. W., Electric Current Flow in Excitable Cells, Oxford University Press, London, 1975. 12 Jefferys, J. G. R., Initiation and spread of action potentials in granule cells maintained in vitro in slices of guinea-pig hippocampus, J. Physiol. (Lond.), 289 (1979) 375-388.

19 13 Klee, M. and Rall, W., Computed potentials of cortically arranged populations of neurons, J. Neurophysiol., 40 (1977) 647-666. 14 Krettek, J. E. and Price, J. L., Projections from the amygdaloid complex and adjacent olfactory structures to the entorhinal cortex and to the subiculum in the rat and cat, J. comp. Neurol., 172 (1977) 723-752. 15 Laurberg, S. and Hjorth-Simonsen, A., Growing central axons deprived of normal target neurones by neonatal X-ray irradiation still terminate in precisely laminated fashion, Nature (Lond.), 269 (1977) 158-160. 16 Levy, W. B. and Steward, O., Synapses as associative memory elements in the hippocampal formation, Brain Research, 175 (1979) 233-245. 17 Lorente de N6, R., Studies on the structure of the cerebral cortex. III. Continuation of the study of the Amonic system, J. Psychol. NeuroL, 46 (1934) 113-177. 18 Lundberg, A. and Quilisch, H., On the effect of calcium on presynaptic potentiation and depression at the neuromuscular junction, Acta physiol, scand., 30 (1952) 121-129. 19 Lomo, T., Potentiation of monosynaptic EPSP's in the perforant path-dentate granule cell synapse, Exp. Brain Res., 12 (1971) 46-63. 20 Magleby, K. L. and Zengel, J. E., Augmentation: a process that acts to increase transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.), 257 (1976) 449~,70. 21 McNaughton, B. L., Dissociation of short- and long-lasting modification of synaptic efficacy at the terminals of the perforant path, Neurosci. Abstr., 7 (1977). 22 McNaughton, B. L. and Barnes, C. A., Physiological identification and analysis of dentate granule cell response to stimulation of the medial and lateral perforant pathways in the rat, J. comp. Neurol., 175 (1977) 439-454. 23 McNaughton, B. L., Douglas, R. M. and Goddard, G. V., Synaptic enhancement in fascia dentata: cooperativity among co-active afferents, Brain Research, 157 (1978) 277-293. 24 Nelson, P. G. and Frank, K., Extracellular potential fields of single spinal motoneurons, J. Neurophysiol., 27 (1964) 913-927. 25 Nicholson, C., Theoretical analysis of field potentials in anisotropic ensembles of neuronal elements, IEEE Trans. Biomed. Engng, 20 (1973) 278-288. 26 Nicholson, C. and Linas, R., Field potentials in the alligator cerebellum and the theory of their relationship to Purkinje cell dendritic spikes, J. Neurophysiol., 34 (1971) 509-531. 27 Rail, W., Electrophysiology of a dendritic neuron model, Biophys. J., 2 (1962) 145-167. 28 Segal, M., Afferents to the entorhinal cortex ofthe rat studied by the method ofretrograde transport of horseradish peroxidase, Exp. Neurol., 57 (1977) 750-765. 29 Skrede, K. K. and Westgaard, R. D., The transverse hippocampal slice: a well-defined cortical structure maintained in vitro, Brain Research, 35 (1971) 589-593. 30 Stevens, C. S., Neurophysiology: A Primer, Wiley, New York, 1966. 31 Steward, O. V., Topographic organization of the projections from the entorhinal area to the hippocampal formation of the rat, J. comp. Neurol., 167 (1976) 285-314. 32 Takeuchi, A., The long-lasting depression in neuromuscular transmission of frog, Jap. J. Physiol., 8 (1958) 102-113. 33 Van Hoesen, G. W., Pandya, D. N. and Butters, N., Some connections of the entorhinal (area 28) and peripheral (area 35) cortices of the rhesus monkey. II. Frontal lobe afferents, Brain Research, 95 (1975) 25-35.