Factors determining the efficacy of distal excitatory synapses in rat hippocampal CA1 pyramidal neurones

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1 Keywords: Pyramidal neurone, Dendrite, Synaptic transmission 7254 Journal of Physiology (1998), 507.2, pp Factors determining the efficacy of distal excitatory synapses in rat hippocampal CA1 pyramidal neurones Mogens Andreasen and John D. C. Lambert Department of Physiology, University of Aarhus, DK-8000 Arhus C, Denmark (Received 6 August 1997; accepted after revision 29 October 1997) 1. Anewpreparation ofthein vitro rat hippocampal slice has been developed in which the synaptic input to the distal apical dendrites of CA1 pyramidal neurones is isolated. This has been used to investigate the properties of distally evoked synaptic potentials. 2. Distal paired-pulse stimulation (0 1 Hz) evoked a dendritic response consisting of a pair of EPSPs, which showed facilitation. The first EPSP had a rise time (10 90 %) of 2 2 ± 0 05 ms and a half-width of 9 1 ± 0 13 ms. The EPSPs were greatly reduced by CNQX (10 ìò) and the remaining component could be enhanced in Mg -free Ringer solution and blocked by AP5 (50 ìò). In 70 % of the dendrites, the EPSPs were followed by a prolonged afterhyperpolarizarion (AHP) which could be blocked by a selective and potent GABAB antagonist, CGP 55845A (2 ìò). These results indicate that the EPSPs are primarily mediated by non- NMDA receptors with a small contribution from NMDA receptors, whereas the AHP is a GABAB receptor-mediated slow IPSP. 3. With intrasomatic recordings, the rise time of proximally generated EPSPs (3 4 ± 0 1 ms) was half that of distally generated EPSPs (6 7 ± 0 5 ms), whereas the half-widths were similar (19 6 ± 0 8 ms and 23 8 ± 1 ms, respectively). These results indicate that propagation through the proximal apical dendrites slows the time-to-peak of distally generated EPSPs. 4. Distal stimulation evoked spikes in 60 % of pyramidal neurones. At threshold, the distally evoked spike always appeared on the decaying phase of the dendritic EPSP, indicating that the spike is initiated at some distance proximal to the dendritic recording site. Furthermore, distally and proximally generated threshold spikes had a similar voltage dependency. These results therefore suggest that distally generated threshold spikes are primarily initiated at the initial segment. 5. At threshold, spikes generated by stimulation of distal synapses arose from the decaying phase of the dendritic EPSPs with a latency determined by the time course of the EPSP at the spike initiation zone. With maximal stimulation, however, the spikes arose directly from the peak of the EPSPs with a time-to-spike similar to the time-to-peak of subthreshold dendritic EPSPs. Functionally, this means that the effect of dendritic propagation can be overcome by recruiting more synapses, thereby ensuring a faster response time to distal synaptic inputs. 6. In 42 % of the neurones in which distal EPSPs evoked spikes, the relationship between EPSP amplitude and spike latency could be accounted for by a constant dendritic modulation of the EPSP. In the remaining 58 %, the change in latency was greater than can be accounted for by a constant dendritic influence. This additional change in latency is best explained by a sudden shift in the spike initiation zone to the proximal dendrites. This would explain the delay observed between the action of somatic application of TTX (10 ìò) on antidromically evoked spikes and distally evoked suprathreshold spikes. 7. The present results indicate that full compensation for the electrotonic properties of the main proximal dendrites is not achieved despite the presence of Na and Ca currents. Nevertheless, distal excitatory synapses are capable of initiating spiking in most pyramidal neurones, and changes in EPSP amplitude can modulate the spike latency. Furthermore, even though the primary spike initiation zone is in the initial segment, the results suggest that it can move into the proximal apical dendrites under physiological conditions, which has the effect of further shortening the response time to distal excitatory synaptic inputs.

2 442 M. Andreasen and J. D. C. Lambert J. Physiol The excitatory synaptic activation of hippocampal CA1 pyramidal neurones is primarily mediated by pyramidal neurones in the ipsilateral and contralateral CA3 region, axons of which form the Schaffer collateral and commissural pathways, respectively. Terminals of these fibres make synaptic contacts on most of the highly arborized dendritic tree of the CA1 pyramidal neurones (Andersen & Lœmo, 1966). A second minor excitatory input comes from neurones in layer III of the entorhinal cortex via the entorhinal pathway, which is thought to form synaptic contacts exclusively on the distal apical dendrites (Colbert & Levy, 1992). Theoretical analyses have indicated that the anatomical and electrotonic structure of the dendritic tree results in a marked attenuation of distally generated excitatory postsynaptic potentials (EPSPs) and alters their time course and efficacy, in terms of their ability to evoke spikes (Turner & Schwartzkroin, 1980; Mainen, Carnevale, Zador, Claiborne & Brown, 1996). However, experimental investigations of the influence of dendritic propagation on EPSP efficacy have so far not been conclusive. In one study, no difference was found between distally and proximally generated EPSPs, indicating that dendritic propagation has no effect on distal EPSPs (Andersen, Silfvenius, Sundberg & Sveen, 1980). In another study, distally generated EPSPs were found to be about twice as slow as proximally generated EPSPs (Turner, 1988). This difference was largest for small EPSPs (< 0 5 mv) and decreased as the amplitude of the EPSPs was increased, indicating that there may be an amplitude-dependent compensation for dendritic propagation. Recently, however, dual patch-clamp recordings from the soma and apical dendrites of the same pyramidal neurone have indicated that dendritic propagation also affects threshold EPSPs (see Fig. 1A in Spruston, Schiller, Stuart & Sakmann, 1995). The reasons for these different findings and discrepancies between experimental and theoretical investigations are presently not clear. One explanation could be that the theoretical analyses are usually based on the assumption that the dendrites are passive structures, which is now known not to be the case (for reviews see Johnston, Magee, Colbert & Christie, 1996; Stuart, Spruston, Sakmann & H ausser, 1997). There is now evidence that dendritic currents are involved in the amplification of distally evoked EPSPs (Magee & Johnston, 1995a; Lipowsky, Gillessen & Alzheimer, 1996; Gillessen & Alzheimer, 1997). However, the activation of these currents seems to enhance the amplitude of the EPSPs without changing the overall time course of distally evoked EPSPs at the somatic level (Lipowsky et al. 1996; Gillessen & Alzheimer, 1997). It therefore seems unlikely that the activation of dendritic currents can fully explain the different experimental findings and the discrepancies between theory and experimental observations. Another possibility concerns the dendritic location of the synapses. It is generally assumed that the Schaffer collateral fibres follow a laminar course parallel to the stratum pyramidale (SP; Andersen et al. 1980). However, Turner (1988) suggested that the observed unification of distally and proximally generated EPSPs when the stimulation was increased could be the result of a progressive activation of non-laminar afferents which form synapses in overlapping regions of the apical dendrites. Recent morphological studies of single CA3 pyramidal neurones have supported this by showing that Schaffer collateral fibres deviate substantially from a laminar course and that they often have an extensive system of collaterals within the CA1 region (Li, Somogyi, Ylinen & Buzs aki, 1994). It is therefore possible that the similar time course and efficacy of distally and proximally generated EPSPs reported by Andersen et al. (1980) results from stimulation of non-laminar fibres which activate synapses at more proximal locations, resulting in an overestimation of the efficacy of distally generated EPSPs. Another unresolved issue concerns the initiation of spikes by synaptic activation. Intradendritic recordings have indicated that apical dendrites of CA1 pyramidal neurones are capable of generating Na - and Ca -dependent spikes (for review see Stuart et al. 1997). Extracellular analyses have suggested that spikes evoked by distally generated synaptic potentials are primarily initiated in the proximal apical dendrites (Andersen & Lœmo, 1966; Herreras, 1990). However, intracellular and patch-clamp recordings have indicated that the site of initiation of synaptically evoked spikes is located primarily in the initial segment, although it can shift to the proximal dendrites under extreme conditions (Turner, Meyers, Richardson & Barker, 1991; Spruston et al. 1995). Therefore, some basic questions concerning the impact of dendritic propagation and synaptic spike initiation in CA1 pyramidal neurones still remain unresolved. (1) How does dendritic propagation affect distally generated EPSPs? (2) Is there a relationship between EPSP amplitude and dendritic modulation? (3) Where is the spike initiated? (4) Is there a shift in the initiation zone and, if so, under what conditions does it occur? To address these questions, we have developed a new experimental preparation, which provides a more complete isolation of the synaptic input to the distal parts of the hippocampal CA1 apical dendrites. This preparation has enabled us to assess the effect of dendritic propagation on distally evoked EPSPs. Moreover, the preparation has allowed us to gain insight into the spike generating capabilities of distally located excitatory synapses in mature animals. METHODS Experiments were performed on hippocampal slices prepared from sixty-four male Wistar rats ( g). Each rat was anaesthetized with chloroform, after which it was decapitated and the brain quickly removed and placed in a standard Ringer solution (see below) at 4 C. The hippocampus was dissected free and slices (400 ìm thick) were cut on a McIlwain tissue chopper. The slices were immediately transferred to the recording chamber, where they were placed on a nylon-mesh grid at the interface between warm

3 J. Physiol The efficacy of distal excitatory synapses 443 (31 33 C) standard Ringer solution (ph 7 3) and warm humidified Carbogen (95 % Oµ 5 % COµ). Perfusion flow rate was 1 ml min. Isolation of the distal synaptic input to CA1 pyramidal neurones The afferent input to the distal third of the apical dendrites of a group of CA1 pyramidal neurones was isolated by a double cut in stratum radiatum (SR) ìm from the superior border of SP. The cuts left a small bridge approximately 200 ìm wide at right angles to the dendritic axis (Fig. 1A and B). The cut was made with a custom-made knife consisting of two razor blade chips mounted together in the same plane but separated by a gap of 200 ìm. The knife was mounted on a micromanipulator and lowered into the slice parallel to a small region of SP in area CA1b. Because this procedure always left some uncut fibres, the incisions on each side of the bridge were completed using a microdissection knife (Fine Science Tools Inc., Heidelberg, Germany). This procedure ensured that only the apical dendrites of pyramidal neurones whose somata were located directly above the bridge will extend into the superficial part of SR and stratum lacunosum-moleculare (L-M) (Fig. 1B).Structuresneartheedgesofthe bridge arelikelytohave been damaged by compression with the knife thereby decreasing the functional width of the bridge to less than 200 ìm. However, the lateral extension of the dendritic tree at the level of the bridge is usually less than 200 ìm (Bannister & Larkman, 1995), so the dendritic tree of a large number of pyramidal neurones would be expected to be intact. Following the microdissection, the slice was allowed to rest for at least 1 h before recordings were started. Stimulation and recording procedure Intracellular recordings from CA1 pyramidal neurones were obtained using borosilicate glass microelectrodes (1 2 mm o.d., Clark Electromedical) filled with 4 Ò K acetate (tip resistances: MÙ). Penetrations of the distal apical dendrites were made at the centre of the bridge as indicated in Fig. 2A. Intradendritic recordings were identified by their similarity to those reported previously from histochemically verified dendritic recordings (Andreasen & Lambert, 1995). Dendritic penetrations were accepted for analysis if the resting membrane potential (RMP) was stable and more negative than 50 mv and the membrane input resistance (Rin) was ü 10 MÙ. Intrasomatic recordings were obtained from SP directly above the bridge as indicated in Fig. 9A. The criteria for accepting intrasomatic recordings were similar to those used earlier (Andreasen, Lambert & Jensen, 1989). Extracellular recordings were obtained using 1 Ò NaCl-filled glass microelectrodes (tip resistances: MÙ). Bipolar Teflon-insulated platinum electrodes (50 ìm in diameter) were used for orthodromic and antidromic stimulation of the CA1 pyramidal cells with constant-current pulses ( ìa; 50 ìs duration; 0 1 Hz). In order to activate the distal afferent fibres, a stimulation electrode was placed on the slice close to the border between SR and L_M on the subicular side of the bridge (Stim. 2 in Fig. 1A). This placement minimized the activation of non-laminar afferents. For stimulation of the proximal afferent fibres close to the superficial border of SP, a monopolar electrode consisting of a blunt glass pipette (tip diameter ìm) filled with 1 % agar dissolved in 0 9 % NaCl was used. For antidromic activation, a bipolar stimulation electrode was placed in SP (Fig. 6C). Unless otherwise noted, a paired-pulse stimulation protocol was used with an interpulse interval of 50 or 100 ms. The two responses to pairedpulse stimulation are termed the conditioning (c) and test (t) response, respectively. Conventional recording techniques were employed, using a high input impedance amplifier (Axoclamp 2A, Axon Instruments Inc.) with bridge-balance and current injection facilities. Results were digitized on-line using a Labmaster AÏD converter and pclamp acquisition software (Axon Instruments Inc.) on a 486 PC computer and recorded for off-line analysis using a modified digital audio processor (Sony PCM-701es) and a video tape recorder. EPSP amplitude, rise time (10 90 %) and half-width were measured with respect to the pre-stimulus baseline. Time-to-peak and timeto-spike were measured from the beginning of the EPSP (i.e. the first deviation of the voltage from the baseline following the stimulation). Spike amplitude was measured from the first inflection point to the peak. All analyses were performed using pclamp analyses software. Values are given as means ± s.e.m. unless otherwise indicated. For statistical analyses, Student s t test was used as appropriate. Drugs and solutions The composition of the standard Ringer solution was (mò): NaCl, 124; KCl, 3 25; NaHµPOÚ, 1 25; NaHCO, 20; CaClµ, 2; MgSOÚ, 2; ª_glucose, 10; bubbled with Carbogen (ph 7 3). Some of the experiments were performed in the presence of dl-2-amino-5-phosphonovaleric acid (AP5, 50 ìò), bicuculline methobromide (BIC, 10 ìò) and CGP 55845A (2 ìò) in order to block N-methyl- ª_aspartate (NMDA), ã_aminobutyric acid (GABA)A and GABAB receptors, respectively. In some experiments, nominally Mg -free Ringer solution was used. For local application of tetrodotoxin (TTX), a blunt glass pipette (o.d. 10 ìm), was filled with standard Ringer solution containing 10 ìò TTX and connected to a pressure injection system (PV830 Pneumatic PicoPump, WPI). The tip of the pipette was placed on the surface of the slice close to, and upstream to, the site of interest, thereby ensuring that most of the TTX would be washed away by the continuous flow of Ringer solution. TTX was applied by a short pressure pulse of ms (10 20 psi) after which the TTXcontaining pipette was withdrawn. All pharmacological compounds were made up in aqueous stock solutions of times the required final concentration and diluted in standard Ringer solution as appropriate. TTX and BIC were purchased from Sigma, AP5 and 6-cyano-7-nitroquinoxaline- 2,3-dione (CNQX) were purchased from Tocris and CGP 55845A was provided by Ciba Geigy. RESULTS Isolation of the distal synaptic input to CA1 pyramidal neurones The synaptic input to the distal part of the apical dendrites was isolated as described in Methods and the efficacy of this isolation was tested experimentally in twenty-two slices as outlined in Fig. 1A. A bipolar stimulation electrode (Stim. 2) was placed close to the border between SR and L_M on the subicular side of CA1. Stimulation here will activate the Schaffer collaterals in the superficial portion of SR, and, to a certain extent, the entorhinal pathway in L_M. Responses were recorded extracellularly in SP at three different locations: I, located approximately 200 ìm from the subicular edge of the bridge ; II, corresponded to the middle of the bridge ; III, approximately 200 ìm from the CA3 edge of the bridge. Stimulation of the alveusïoriens (AÏO) area

4 444 M. Andreasen and J. D. C. Lambert J. Physiol (Stim. 1) was used to control for the viability of the CA1 region. At all recording sites, stimulation of the AÏO evoked a complex field potential (Fig. 1C) consisting of an initial antidromic population spike (PS) followed by a field EPSP on which an orthodromic PS of variable amplitude was riding. This shows that there were viable pyramidal neurones in all three areas. In the example shown in Fig. 1C, high intensity distal stimulation evoked a response at location II consisting of a field EPSP on top of which is a small PS. The lack of response at locations I and III indicates that the cuts had effectively separated the superficial SR and L_M from the rest of area CA1, and that few, if any, non-laminar fibres run through the bridge into the deeper portion of SR. Although this was the case for the majority of slices tested, in a few instances very high intensity stimulation ( ìa) could evoke a small response at location III. The passive membrane properties of CA1 pyramidal neurones Intracellular recordings were obtained from seventy-six apical dendrites (see Methods). The mean RMP was 69 1 ± 0 3 mv (range 74 to 63 5 mv, n = 76). The input resistance (Rin) and the membrane time constant (ôm) were determined from the response to a small hyperpolarizing current pulse as described earlier (Andreasen & Lambert, 1995). The mean Rin was 17 5 ± 0 5 MÙ (range 10 to 28 7 MÙ, n = 76) and ôm was 5 8 ± 0 6 ms (range 3 to 10 8 ms, n = 48). In the remaining twenty-eight dendrites the presence of an inwardly rectifying current prevented determination of ôm. All the basic membrane parameters of Figure 1. Isolation of the distal synaptic inputs A, schematic illustration of the slice preparation and the electrode placement for testing the effectiveness of the isolation of distal synaptic inputs. In each slice, two cuts were made which left a small bridge in SR. B shows a drawing of the region containing the bridge with a schematic drawing of a CA1 pyramidal neurone superimposed (AÏO, alveusïoriens; SP, stratum pyramidale; SR, stratum radiatum; L_M, stratum lacunosum-moleculare). C, extracellular field responses, recorded at locations I III in A, to stimulation of AÏO (Stim. 1) and afferent fibres near the SRÏL-M border (Stim. 2). At all locations, stimulation of the AÏO evoked an antidromic spike (arrow) followed by an orthodromic field EPSP and a population spike. However, stimulation of the distal afferent fibres (Stim. 2) only evoked a response at location II opposite the bridge. The response consisted of a field EPSP with a small inflection indicative of a population spike.

5 J. Physiol The efficacy of distal excitatory synapses 445 the apical dendrites were similar to those we have reported previously (Andreasen & Lambert, 1995). Intracellular recordings were obtained from twenty-nine somata in SP, with a mean RMP of 69 4 ± 0 3 mv (range 73 to 64 mv, n = 29) and Rin of 33 5 ± 1 3 MÙ (range 21 to 50 MÙ, n = 29). The somatic voltage response to a hyperpolarizing current pulse was well described by a single exponential function giving a mean ôm of 14 7 ± 1 1ms (range 6 8 to 32 9 ms), which was nearly threefold slower than the dendritic ôm. Distally generated synaptic potentials recorded in the apical dendrites In order to investigate the effect of dendritic propagation on distally generated EPSPs, it is necessary to characterize these at the dendritic level. Dendritically recorded synaptic potentials (Fig. 2A) result from the summation of unitary synaptic potentials generated on parts of the apical dendrites which are distal to the bridge (Fig. 1B). In standard Ringer solution, distal paired-pulse stimulation evoked two fast EPSPs with marked paired-pulse facilitation (PPF) of the tepsp (Fig. 2B).Althoughnodetailedanalysis Figure 2. Dendritic responses to distal synaptic activation A, schematic illustration of the experimental setup for intradendritic recordings of synaptic responses. B, example of a typical dendritic response to distal paired-pulse stimulation (300 ìa, interpulse interval 50 ms) recorded about 275 ìm distally to the superficial border of SP. The response, consists of a cepsp and a tepsp, the latter being facilitation and followed by a prolonged AHP. Unless otherwise noted, the responses in this and the following figures are the mean of 4 to 5 individual recordings. C shows the dendritic response to distal stimulation of increasing intensity. PPF is present at each of the intensities illustrated and there is a progressive increase in peak amplitude of the EPSPs until spikes are initiated. Note that in this dendrite, the EPSPs were not followed by an AHP. Four individual responses to 250 ìa are shown superimposed while the other traces are means. D, the spike generating tepsps in response to 250 ìa on an expanded time scale. Note the spikes are evoked on the decaying phase of the EPSP at variable latency. RMP: in B, 64 mv; and in C and D, 71 mv.

6 446 M. Andreasen and J. D. C. Lambert J. Physiol of PPF was carried out, maximal PPF was seen to occur with interpulse intervals between 25 and 100 ms and decreased towards zero at intervals of 150 to 200 ms (n = 13, of which 8 were in the presence of 10 ìò BIC, which did not affect the time course of PPF). Increasing the stimulating intensity resulted in a progressive increase in the amplitude of both the c- and tepsp until a maximal amplitude was reached (Fig. 2C). When spikes were evoked, they initially appeared on the decaying phase of the EPSP and at a variable latency from the peak (Fig. 2C and D).In most cases, it was not possible to obtain acceptable exponential fits of the decaying phase of the EPSPs. The time course of the EPSPs ( ü 1 mv) was therefore represented by measuring the half-width and the rise time (10 90 %). Because there was no correlation between peak amplitude and rise time or half-width of the cepsps (or the tepsp) (Fig. 3) all control data were analysed collectively, irrespective of their amplitude. The mean rise time of the cepsps and tepsps were 2 2 ± 0 05 and 2 1 ± 0 04 ms, respectively, (n = 23) while the half-widths were 9 1 ± 0 13 and 9 6 ± 0 13 ms, respectively, (n = 23). The dendritic EPSP was greatly reduced by 10 ìò CNQX (n = 5), leaving only a small residual EPSP of a few millivolts (Fig. 4A), which was enhanced in nominally Mg free medium (Fig. 4B, n = 4). Note that the residual EPSP also showed PPF, particularly in Mg -free medium. In the presence of both CNQX and AP5 (50 ìò), the EPSP was totally blocked (Fig. 4C). None of the dendritic recordings showed any evidence of a fast inhibitory postsynaptic potential (IPSP) at RMP (Fig. 2B and C). Furthermore, changing the membrane potential (Vm) did not disclose a fast IPSP (n = 12, Fig. 5A). In the presence of BIC, however, the rise time (2 6 ± 0 1 ms, n = 22) and half-width (11 7 ± 0 3 ms) were significantly different from control values (P < and P < 0 001, respectively), indicating that GABAA receptors are activated to some extent. In 70 % (16Ï23) of dendritic recordings the EPSPs were followed by a prolonged afterhyperpolarization (AHP) of a few millivolts in amplitude and lasting several hundred milleseconds (Fig. 2B). The amplitude of the AHP increased with stimulating intensity (Fig. 5B). The AHP was completely blocked by 2 ìò CGP 55845A (n = 6, Fig. 5C and D), a selective and potent GABAB receptor antagonist, which has been shown to completely block both pre- and postsynaptic GABAB receptors at concentrations below 2 ìò (Davies, Pozza & Collingridge, 1993). The effect of CGP 55845A was, however, complex. In three cases, the EPSP decayed back to baseline following the block of the AHP. In the remaining three cases, the EPSP was followed by a prolonged depolarizing afterpotential (Fig. 5D). Furthermore, the amplitude of the EPSP was reduced in four cases, with the greatest effect being on the tepsp, resulting in a concomitant reduction in PPF (Fig. 5C). In the remaining two cases, CGP 55845A induced a large increase in the amplitude of both c- and tepsps without any significant change in half-width (Fig. 5D). In the presence of both BIC and CGP 55845A, the mean rise time and half-width of the cepsp was 2 6 ± 0 09 ms and 11 1 ± 0 3 ms, respectively (n = 16), which is similar to EPSPs in the presence of BIC alone. The spike generating properties of distally evoked EPSPs The spike generating properties of the distal dendrites were unaltered in the presence of BIC, CGP 55845A and AP5, either individually or in different combinations. Therefore, Figure 3. Relationship between peak amplitude and time course There is no correlation between rise time (10 90 %) and peak amplitude (A), or between half-width and peak amplitude (B) of dendritic EPSPs ( ü 1 mv) recorded in standard Ringer solution. 129 individual cepsps were included in the above plots.

7 J. Physiol The efficacy of distal excitatory synapses 447 all data concerning spike generation have been analysed collectively. Of the sixty-five dendrites in which the stimulus response correlation was investigated, 60 % (39Ï65) showed spike firing. In twenty-two of these cases, spikes were only evoked by the tepsp, even though the amplitude of the cepsp in many instances exceeded the threshold level for spiking. In the remaining seventeen cases, spikes were also evoked by the cepsp. In most cases the spikes evoked were single or double fast spikes, with a mean amplitude of 51 5 ± 0 6 mv (range 34 6 to 84 mv, n = 35) and were similar in all respects to the Na -dependent spikes evoked by intradendritic current injection (Andreasen & Lambert, 1995). Compound spiking, which consists of an initial fast spike followed by one or more broad Ca -dependent spikes (Andreasen & Lambert, 1995) was only evoked in four cases (Fig. 10). The threshold, defined here as the absolute Vm at the peak of EPSPs which intermittently evoked spikes, was 54 1±1mV(range 61 7 to 44 mv, n = 21). There was no correlation between the dendritic RMP and the threshold level (not shown). In the 40 % (26Ï65) of dendrites in which stimulation of the distal synapses did not evoke spikes, Vm at the peak of the tepsp nevertheless reached 55 9 ± 1 1 mv (range 66 8 to 44 8 mv, n = 26). In all cases, however, spikes could be evoked by intradendritic current injection. At threshold, spikes always appeared on the decaying phase of the EPSPs (Figs 2D and 6Aa). Spike latency, from the peak of the EPSP, was found to be independent of the threshold level, and could be the same whether this was, for example, 62 or 44 mv (Fig. 10B). However, at a given threshold, there was some variation in spike latency between different dendrites. This is illustrated in Fig. 6Aa, which shows a typical (left) and an extreme example (right). In some dendrites, small variations in amplitude of suprathreshold EPSPs were associated with a marked change in Figure 4. The glutamatergic component of the dendritic synaptic potential A, the non-nmda receptor antagonist CNQX (10 ìò) greatly reduced the dendritic EPSPs leaving only a small component of 1 2 mv in amplitude. B, in another dendrite the CNQX-resistant component was greatly potentiated by perfusion with nominally Mg -free Ringer solution. C, addition of both CNQX (10 ìò) and the NMDA-receptor antagonist AP5 (50 ìò) completely blocked the dendritic EPSP. RMP: in A, 74 mv; in B, 66 mv; and in C, 71 mv.

8 448 M. Andreasen and J. D. C. Lambert J. Physiol the spike latency (Fig. 6B) while in others, the spike latency varied noticably, even though the peak amplitude was unchanged (Fig. 2D). In eight dendrites, the peaks of some of the EPSPs evoked by threshold-straddling stimulation were markedly prolonged (Fig. 6Ca). This prolongation of the EPSP was associated with a reduction in threshold. This is highlighted in Fig. 6Cb, in which a subthreshold EPSP is superimposed on a prolonged threshold EPSP. EPSPs with prolonged peaks were also seen in the presence of AP5 (not shown), indicating that the prolongation was not caused by activation of NMDA receptors. The efficacy of distally evoked EPSPs in generating spikes was very sensitive to changes in the dendritic Vm. At threshold, a hyperpolarization of only 3 7 ± 0 37 mv (n = 10) was sufficient to completely block spike generation. Figure 5. The GABAergic component of the dendritic synaptic potential A, the voltage dependency of the distally evoked dendritic response. Before afferent stimulation Vm was stepped to different potentials by injecting 200 ms duration current pulses (0, ±0 4, ±0 8 na). The amplitude of the EPSPs decreased with depolarization and increased with hyperpolarization, but there was no indication of a fast IPSP. B, response to paired-pulse stimulation at two intensities. C, in the same dendrite, application of the selective GABAB-receptor antagonist CGP 55845A (2 ìò) completely blocked the AHP and, in this case, also reduced the peak amplitude of the EPSPs. D,in another dendrite, the AHP was replaced by a prolonged depolarizing afterpotential in the presence of CGP 55845A. Note the very marked increase in peak amplitude of both EPSPs in the presence of CGP 55845A. RMP: in A, 71 mv; in B and C, 71 mv; and in D, 63 mv.

9 J. Physiol The efficacy of distal excitatory synapses 449 For suprathreshold responses, hyperpolarization of the dendritic membrane by a few millivolts increased the spike latency and changed the EPSP to a threshold response. Further hyperpolarization completely blocked spike generation (Fig. 7A). The amplitude of the EPSPs was relatively insensitive to the small changes in dendritic Vm. This could be due to the small space constant of the distal part of the apical dendrites (Andreasen & Nedergaard, 1996) which would limit current spread in the distal direction from the recording site. Threshold spikes evoked by distal and proximal stimulation (see Methods) showed similar sensitivity to dendritic hyperpolarization (Fig. 7B). In contrast to distally evoked spikes, dendritic recordings of proximally evoked spikes showed that spikes always arose from the peak of the EPSPs (Figs 6Ab and 7B). The spike initiation zone for proximally evoked spikes is likely to be the initial segment, which is only a short distance from the active synapses. That distally evoked threshold Figure 6. Properties of distally evoked dendritic spikes Aa, dendritic recordings of individual threshold responses from two different dendrites. With thresholdstraddling distal stimulation, spikes always appeared on the decaying phase of the EPSP. A typical example is shown to the left (spike latency from peak: 3 3 ms) and an extreme example to the right (spike latency: 18 ms). Ab, dendritic recording of a proximally evoked threshold spike which rides on the peak of the EPSP. B, superimposed dendritic responses to distal stimulation at the same intensity. Note the variation in peak amplitude of the EPSP and spike latency. C, recordings from the same dendrite as in B. Ca, superimposition of two dendritic responses evoked with the same stimulating intensity. In each case a single fast spike was initiated, though one appeared on the decaying phase whereas the other arose from what appeared to be a prolonged peak. Cb, the prolongation of the EPSP peak (arrow) becomes more evident when superimposed on a subthreshold EPSP of similar amplitude. RMP: in Aa, 71 and 68 mv; in Ab, 70 mv; and in B and C, 68 mv.

10 450 M. Andreasen and J. D. C. Lambert J. Physiol spikes appeared after the peak of the EPSP would indicate that they are initiated at some distance proximal to the active synapses. Simultaneous patch-clamp recordings from dendrites and somata from young animals have recently led to the suggestion that synaptically evoked spikes are primarily initiated in the initial segment and then propagated back into the dendrites (Stuart & Sakmann, 1994; Spruston et al. 1995). To test whether the distally evoked dendritic spikes are somatic in origin, we conducted a series of experiments (n = 5) using local application of TTX (10 ìò) to the somatic region (see Methods). Recordings were made from the distal dendrites and paired-pulse stimulation was used to activate the distal afferent fibres (Fig. 8A). The intensity was adjusted so that spikes were only evoked consistently by the tepsp. The cepsp therefore served as a control for diffusion of TTX into the area of active terminals. Antidromic stimulation of SP served as a control that TTX had reached the pyramidal neurone whose apical dendrite had been impaled. After several stable controls, a 300 ms pulse of TTX was applied onto the surface of the slice, following which the TTXpipette was withdrawn. After 20 s, the antidromic spike had been blocked, but spikes could still be evoked by distal stimulation (Fig. 8B). After 180 s, however, the distally Figure 7. Voltage-dependency of distally evoked dendritic spikes A, individual dendritic recordings showing the effect of changing the dendritic Vm on the spike generating properties of a distally evoked EPSP. RMP is the top trace. Modest hyperpolarization ( 2 5 mv) increased the spike latency while larger hyperpolarization ( 5 3 mv) blocked spike generation. Note that the amplitude of the EPSP is little effected by the hyperpolarization. B, the effect of hyperpolarization on distally (D) and proximally (P) evoked threshold responses in a different dendrite. A small hyperpolarization of only 1 5 mv was sufficient to prevent both EPSPs from generating spikes.

11 J. Physiol The efficacy of distal excitatory synapses 451 evoked spike was also blocked, revealing a tepsp with an amplitude substantially greater than the spike threshold (Fig. 8B). At the same time, the cepsp was unchanged, indicating that blockade of the distally evoked spike was not due to TTX diffusing into the recording area. Similar results were obtained in four other experiments. In all cases there was a delay (32 ± 11 1 s; n = 5) before the antidromic spike was blocked, which was about three times shorter than the delay between the block of the antidromic spike and the distally evoked spike (92 ± 24 6 s, range 20 to 160 s). Somatic recordings of distally generated EPSPs We then used intrasomatic recordings to investigate how propagation through the proximal apical dendrites influences the time course and efficacy of distally generated EPSPs. To eliminate distortion due to variable activation of GABA andïor NMDA receptors, the experiments were carried out in the presence of CGP 55845A (2 ìò), BIC (10 ìò) and AP5 (50 ìò). Under these conditions, distally generated EPSPs recorded at the somatic level had a very slow time course (Fig. 9A), which is in marked contrast to EPSPs Figure 8. Distally evoked dendritic spikes are blocked by somatic application of TTX A, the experimental setup for local application of TTX (10 ìò) during intradendritic recordings. One stimulation electrode was used to activate the distal afferent fibres (stim. D) and another was placed over SP to activate the pyramidal neurones antidromically (stim. A). The tip of the TTX-containing pipette was placed on the surface of the slice close to and downstream from the antidromic stimulation electrode. Flow direction is indicated to the right. Ba, control responses to paired-pulse stimulation of the distal afferent fibres (closed arrows) with an intensity that consistently generated spikes on the tepsp. This was followed, 100 ms later, by a single antidromic stimulation (open arrow) at suprathreshold intensity. All spikes have been truncated. Eight control responses were collected to ensure their consistency. A single pulse (300 ms, psi) of TTX was applied under visual control and the pipette immediately withdrawn from the slice. Bb, after 20 s, the antidromic response was completely blocked while the orthodromic response was still unaffected. Bc, after 180 s, the distally evoked spike was also blocked. Superimposition of the responses in Ba and Bc to the right shows that the cepsp is unchanged while the tepsp following TTX has an amplitude which clearly exceeds the pre-ttx threshold (dotted line). RMP: 67 mv.

12 452 M. Andreasen and J. D. C. Lambert J. Physiol recorded in dendrites under similar conditions (Fig. 9B). The mean rise time and half-width of the somatic EPSPs were 7 06 ± 0 4 and 25 ± 0 8 ms, respectively, (n = 18), which are significantly different from the rise time (2 4 ± 0 07 ms, n = 20, P < 0 001) and half-width (11 3 ± 0 3 ms, P < 0 001) of the dendritic EPSPs. As with the dendritic EPSPs, the amplitude of the somatically recorded EPSPs increased with the stimulating intensity (Fig. 9A). However,thechangeinamplitudewasusuallynotvery marked and reached a maximum value of 4 1 ± 0 5 mv (range 1 3 to 12 3 mv). Furthermore, at RMP, distal EPSPs evoked spikes in only 22 % (4Ï18) of the somatic recordings (Fig. 9A) compared with 60 % of the dendritic recordings. The small amplitude, slower time course and decreased safety factor for spike initiation of the distally generated EPSPs recorded at the somatic level indicates that propagation through the main proximal dendrites does indeed have a substantial impact on the EPSPs. Penetration with a microelectrode will introduce a leak conductance which can have a substantial effect on the membrane properties (Spruston & Johnston, 1992) and therefore on the amplitude and time course of the EPSPs. Because the size of this leak conductance is likely to be different in dendritic and somatic recordings, a direct comparison between EPSPs recorded at the two sites is likely to be biased. Since the size of the leak conductance is not known, we cannot estimate the effect on the EPSPs or Figure 9. Somatic recordings of distally evoked EPSPs A, schematic illustration of the experimental setup for somatic recordings of distally and proximally evoked EPSPs. To the right is shown the responses to distal stimulation of increasing intensity in the presence of BIC (10ìÒ), CGP55845A (2ìÒ) and AP5 (50ìÒ). B, distally evoked EPSPs recorded in a dendrite (Distal dendritic, RMP 68 mv) and a soma (Distal somatic, RMP 69 mv) of two different pyramidal neurones. The responses are superimposed to the right, to highlight the difference in time course. C, somatic recordings in response to proximal (Proximal somatic) and distal (Distal somatic) threshold-straddling stimulation (95 ìa and 500 ìa, respectively). The subthreshold EPSPs (top) showed that the rising phase of the proximally evoked EPSP is faster than that of the distally evoked EPSP, whereas the decaying phases are similar. This was also the case for the threshold EPSP (bottom). The thresholds for the two spikes (marked by arrows) were, however, similar. All spikes have been truncated. The somatic recordings in A, B and C are from the same pyramidal neurone.

13 J. Physiol The efficacy of distal excitatory synapses 453 how it will bias the results. However, a somatic leak would be expected to affect synaptic potentials to a similar extent, irrespective of whether these are generated proximally or distally. Therefore, a direct comparison of EPSPs generated by proximal and distal synapses will provide a better estimate of the influence of dendritic propagation. One obvious, but important, assumption is that proximal and distal excitatory synapses are identical in all respects except for their location. Whether this is the case is currently unknown, but there is evidence that dendritic and somatic glutamate receptor ionophore complexes are identical with respect to their kinetics and conductance (Spruston, Jonas & Sakmann, 1995). This means that the rising phases of the EPSPs in the vicinity of the synapses should be comparable. This is important since changes in the rising phase, together with amplitude changes, are sensitive indicators of electrotonic attenuation (Spruston, Jaffe & Johnston, 1994). EPSPs evoked by very localized proximal stimulation (Fig. 9A, see Methods) were compared with distally evoked EPSPs recorded in the same pyramidal neurone. As shown in Fig. 9C, the rise time of proximally generated EPSPs (3 4 ± 0 1 ms, n = 8) was nearly twice as fast as that of the distally generated EPSPs (6 7 ± 0 5 ms, n = 8), while the half-width of the proximal EPSPs (19 6 ± 0 8 ms) was somewhat shorter than that of the distal EPSPs (23 8 ± 1 ms). These results confirm that distally generated EPSPs are affected by the dendritic propagation. Threshold spikes evoked by either distal or proximal EPSPs always appeared on the peak of the EPSPs recorded in the soma (Fig. 9C). This indicates that the spike initiation site for both proximally and distally generated EPSPs is close to the recording site. Furthermore, when tested in the same cell, the threshold level for somatic spikes was similar for proximally ( 58 7 ± 0 9 mv, n = 6) and distally ( 57 4 ± 1 1 mv, n =6, P> 0 025) generated EPSPs (Fig. 9C). Together with their similar sensitivity to hyperpolarization of the dendritic membrane, these results point to a similar site for threshold spike initiation. Analysis of dendritic threshold and suprathreshold responses The above analysis of somatic EPSPs was limited to subthreshold responses. However, voltage-dependent currents are present in the apical dendrites (Johnston et al. 1996), and it is likely that their influence will depend on the amplitude of the EPSPs. Because of the difficulties in evoking somatic spikes with distally generated EPSPs and the possible presence of a somatic leak, we used an alternative approach to analyse threshold and suprathreshold responses. This analysis is based on the reasonable assumption that synaptically generated spikes are primarily initiated at the initial segment (Stuart & Sakmann, 1994; Spruston et al. 1995) which, at least for threshold responses, is supported by the present study. Spikes evoked by distal EPSPs always arise from the peak of the somatically recorded EPSPs (Fig. 9C). This means that when a spike first appears on the dendritic EPSP, the peak of the EPSP at the spike initiation zone will have just reached the local threshold. The time-to-spike at threshold (ts, Fig. 10A) in the dendrites will therefore reflect the time-to-peak (tp) of the EPSP at the spike initiation zone. Because it takes time for the spike to propagate back to the dendritic recording site, ts will slightly overestimate tp at the spike initiation zone. However, since the velocity of back-propagating spikes in cortical pyramidal neurones has been estimated to be m s at room temperature and two to three times faster at 35 C (Stuart & Sakmann, 1994; Spruston et al. 1995), a back-propagating spike will take less than 1 ms to reach the dendritic recording site. We have chosen to ignore this small and (presumably) constant factor and set ts equal to tp of the EPSP at the spike initiation zone. A good estimate of the change in time course of the dendritic threshold EPSP at the spike initiation zone can therefore be obtained by expressing ts asaratiooftpof the dendritically recorded EPSP (Fig. 10A). A ratio of 1 0 indicates that the rising phase of the dendritic EPSP is unchanged at the spike initiation zone, while ratios > 1 0 indicate that the rising phase of the EPSP has become slower. All measured ratios were > 1 0, and none were < 1 0 (which would be consonant with dendritic amplification). The mean ratio (tsïtp) was 2 ± 0 2 (n = 21), which is very similar to the ratio between the rise time of distally and proximally generated EPSPs measured in the soma (6 7Ï3 4 = 1 97). This means that the rate-of-rise of the threshold dendritic EPSP is halved by the time it reaches the spike initiation zone. In Fig. 10B, the calculated ratio for each dendrite is plotted as a function of the threshold level (VThr, Fig. 10A). Except for one case, (which is the extreme example from Fig. 6Aa, where the ratio was 6 4), all the points are scattered between 1 and 3. Interestingly, the ratio is independent of the threshold level, even though the latter varied greatly from cell to cell (range: 61 7 to 44 mv) Increasing the stimulating intensity usually reduced the spike latency until, at maximum intensity, the spike arose from the peak of the dendritic EPSP (Fig. 10C). Measuring ts of these maximal responses and comparing them with the mean tp of subthreshold EPSPs (Fig. 10D) showed that they were very similar in most cases. We then examined the relationship between changes in amplitude and spike latency of distally evoked suprathreshold EPSPs. If propagation in the dendrites is a passive process, EPSPs of different amplitudes will experiencethesamedegreeofelectrotonicattenuationanda linear relationship between dendritic EPSP amplitude and spike latency should exist. We therefore made the following measurements: the time-to-spike for threshold responses (ts1); the amplitude of the EPSP just before the threshold spike (X); the time-to-spike for maximal responses (ts2); the difference in amplitude between the maximum EPSP and the peak of the threshold EPSP (d; Fig. 11A). The absolute Vm just before the dendritic threshold spike was taken to represent threshold at the initiation zone (ThrS). Although this is a reasonably accurate approximation (Stuart &

14 454 M. Andreasen and J. D. C. Lambert J. Physiol Sakmann, 1994; Spruston et al. 1995), as will be evident below, the precise value of ThrS is not important for the calculations. On the basis of these measurements, it is possible to make predictions about the rising phase of the maximal EPSP at the spike initiation zone if dendritic propagation is a passive process. In Fig. 11A, the predicted rising phase of the maximal EPSP is indicated by line Y, which is constructed by joining a point from the start of the threshold EPSP to a point (X+d) at the time of the threshold spike. The intersection between line Y and ThrS marks the time at which a passively propagating EPSP with an amplitude of X + d would be expected to reach threshold at the spike initiation zone. This should result in a reduction in ts1 such that the spike occurs at the time of the YÏThrS intersection. An estimate of the actual rising phase is obtained by joining a point from the start of the maximal EPSP to ThrS at the time of the maximal spike (line Z in Fig. 11A). We then calculated the slopes of lines Y (the predicted dvïdt) and Z (the observed dvïdt), respectively, using the following equations: Predicted dvïdt = (X + d)ïts1. Observed dvïdt = XÏt s2. If the predicted dvïdt is equal to or greater than the observed dvïdt, the relationship between changes in peak amplitude and spike latency can be fully accounted for by passive propagation of the distally generated EPSPs. If, on Figure 10. Analyses of dendritic spike latency A, the initial phase of a dendritic threshold response evoked by distal stimulation. The inset shows the full course of the response with compound spiking. The time-to-peak (tp) and time-to-spike (ts) wereboth measured from the first inflection at the beginning of the EPSP. Vm at the peak of the EPSP (VThr) was also measured and represents the threshold level. B, the ratio between ts and tp plotted as a function of VThr. A ratio of 1 0 (dashed line) indicates no change in the rising phase of the EPSP at the spike initiation zone. Values > 1 0 indicate a slowing of the rising phase at the spike initiation zone. Note that, with one exception (ratio 6 4), the ratios are independent of the threshold level. C, superimposition of two maximal responses with compound spiking from the same dendrite as in A. For maximal responses, only ts was measured. D, the relationship between ts and tp of subthreshold EPSPs (n = 11). The dotted line shows a correlation of 1 0 between ts and tp. Except for one case, the points are clustered around the dotted line. RMP: in A and C, 68 mv.

15 J. Physiol The efficacy of distal excitatory synapses 455 the other hand, the predicted dvïdt is smaller than the observed (e. g. Figure 11A), the EPSP must have reached threshold faster at the spike initiation zone than expected with passive propagation. The validity of these predictions and interpretations depends on two assumptions: (1) the time-to-peak of distally evoked EPSPs is independent of their amplitude; (2) Thrs is constant and independent of the rising phase of the EPSP. Regarding the first assumption, analysis of subthreshold EPSPs in individual dendrites showed this to be the case (Fig. 11B). Importantly, this means that on reaching the soma, the time-to-peak of passively propagating EPSPs is also independent of their amplitude and similar to that measured from threshold responses. Regarding the constancy of Thrs, somatic recordings of just-suprathreshold EPSPs or responses to small depolarizing current pulses have shown that even very marked variations in the rising phase of the responses do not affect Thrs to any great extent (Hu, Hvalby, Lacaille, Figure 11. Amplitude-induced changes in spike initiation A, superimposition of dendritic responses evoked by distal stimulation with threshold and maximal intensity. B, dendritic responses to distal stimulation of increasing intensity. Note that time-to-peak, indicated by the dotted line, is nearly constant. C, plot of the observed dvïdt as a function of the predicted dvïdt. The dashed line indicates a correlation of 1 0 between the observed and predicted dvïdt with a limit of +10 % (dotted line). 0: correlation > 1 1; 1: correlation û 1 1. D, superimposition of three dendritic responses to distal stimulation: threshold response (1), submaximal response (2), maximal response (3). The predicted and observed dvïdt for the submaximal response were 0 96 and 1 15, respectively, whereas they were 1 1 and 2 37 for the maximal response. RMP: in A, 68 mv; in B, 71 mv; and in D, 68 mv.