significantly reduced and it became possible to distinguish the relative origins of (Received 28 September 1992)

Transcription

1 Journal of Physiology (1993), 468, pp With 6 figures Printed in Great Britain MODULATION OF EPSP SHAPE AND EFFICACY BY INTRINSIC MEMBRANE CONDUCTANCES IN RAT NEOCORTICAL PYRAMIDAL NEURONS IN VITRO BY ANDREW NICOLL*, ALAN LARKMAN AND COLIN BLAKEMORE From the University Laboratory of Physiology, Parks Road, Oxford OXI 3PT (Received 28 September 1992) SUMMARY 1. Intracellular recordings were made from pyramidal neurons in layers II/III and V of rat visual cortical slices. Distal and proximal excitatory postsynaptic potentials (EPSPs) were evoked using extracellular bipolar electrodes placed on the slice horizontal to each cell, near the apical and basal dendrites respectively. Experiments were conducted in the presence of 2-amino-5-phosphonopentanoate, picrotoxin and, in most cases, 2-hydroxy-saclofen. 2. For layer II/III pyramidal neurons, voltage undershoots following distal and proximal EPSPs (n = 7 pairs) and injected somatic pulses were rarely apparent. In layer V pyramidal neurons substantial voltage undershoots were recorded following distal and proximal EPSPs (n = 27 pairs) and injected somatic pulses, with undershoot being greatest for apical inputs (P = -1). The greater undershoots following apical EPSPs were also apparent in semilogarithmic plots of voltage decay where the slope of decay for apical EPSPs was quicker than the voltage decay following pulses of current injected at the soma. There was no significant difference in the shapes of distal and proximal EPSPs in layer II/III or layer V pyramidal cells under control conditions. 3. Pharmacological agents were used to reduce voltage undershoots. The most successful of these was alinidine, a putative blocker of the slow inward rectifier (IH) conductance. In the presence of bath-applied 1,UM alinidine, undershoots were significantly reduced and it became possible to distinguish the relative origins of EPSPs on the basis of their shape. Distally generated EPSPs (n = 14) had rise times and half-widths that were 2-8 and 1-5 times longer respectively than those evoked proximally (n = 1; P = -1 for both parameters). 4. These results confirm previous theoretical simulations of somatic recordings in passive model neurons where distal EPSPs display slower rise times and longer halfwidths than proximal EPSPs. The present results suggest that, at least in pyramidal neurons of layer V, distal synaptic inputs can be specifically modulated by intrinsic membrane conductances. * Present address and address for correspondence: Department of Physiology, School of Medical Sciences, University Walk, Bristol, BS8 ITD. MS PHY 468

2 694 A. NICOLL, A. LARKMAN AND C. BLAKEMORE INTRODUCTION The efficacy or weight of a synaptic input depends on some combination of the peak amplitude, rise time and area of the postsynaptic potential (PSP) at the soma (Stratford, Mason, Larkman, Major & Jack, 1989). Assuming the synaptic current is brief and PSP propagation is passive, theoretical simulation of the shapes of PSPs in a model neuron predict that inputs originating distal to the cell body will be of a smaller peak amplitude and longer duration than proximally generated ones, owing to dendritic decrement of the distal PSPs (Rall, 1967; Jack & Redman, 1971; Stratford et al. 1989; Cauller & Connors, 1992). This has been found to be the case experimentally in the spinal motoneuron which, with some qualifications, can be modelled as a simple collapsed cable (Jack, Miller, Porter & Redman, 1971; Clements & Redman, 1989). The current at the Ia afferent synapse is brief and voltageindependent (Finkel & Redman, 1983) and because excitatory postsynaptic potentials (EPSPs) are, at least to a first approximation, propagated passively down the dendritic tree (Jack et al. 1971), it is possible to determine the relative origin of the synaptic input from simple measurement of the time course of PSPs at the soma. Furthermore, there is an excellent correspondence between the electrotonic distance and anatomical location of synapses in the motoneurone (Redman & Walmsley, 1983). Unfortunately, a similar analysis has not been possible in cortical pyramidal cells. In these cells, synaptic currents may include N-methyl-D-aspartate (NMDA) as well as non-nmda components, and the NMDA receptor-activated synaptic current is prolonged and voltage dependent (e.g. Forsythe & Westbrook, 1988; Lester, Clements, Westbrook & Jahr, 199). The dendritic and electrotonic geometries of pyramidal cells are complicated (Stratford et al. 1989; Larkman & Mason, 199), with dendrites of many different electrical lengths, possessing numerous spines which might be excitable (Rall & Segev, 1988). Lastly, hippocampal and neocortical pyramidal cells have numerous voltage- and ligand-gated conductances, some of which are probably located on their dendrites and which might be activated by synaptic potentials (see Hounsgaard & Midtgaard, 1989). Because of the complexity of pyramidal cells, interpretation of PSP shape may be difficult and consistent models are not yet available. In hippocampal CAl pyramidal cells, Andersen, Silfvenius, Sundberg & Sveen (198) found little difference in time course of distal and proximal EPSPs. However, in a similar study, Turner (1988) did find that, on average, distally evoked EPSPs had longer rise times and half-widths than proximally evoked ones. For 'minimal' (ca. -45 mv) EPSPs, he found the ratio of distal- to proximal-normalized 1-9% rise time and half-width to be 2-1 and 1-5 respectively. A passive model of the dendritic geometry would account for the observed differences with reasonable accuracy (Walmsley & Stuklis, 1989). Equivalent experiments have not been attempted in neocortical pyramidal neurons. Therefore, the aim of this work was to attempt to distinguish the relative origin of EPSPs in neocortical pyramidal neurons on the basis of their time course recorded at the soma, as a first step in elucidating the factors influencing PSP shape and synaptic efficacy in neocortical pyramidal cells. EPSPs were evoked distal ('apical') and proximal ('basal') to the cell bodies, mostly located in layer V.

3 EPSP SHAPE IN NEOCORTICAL PYRAMIDAL NEURONS However, the membranes of layer V pyramidal cells display considerable nonlinearity and, in particular, voltage undershoots following EPSPs and injected pulses are prevalent, distorting their shapes. Therefore, channel blockers were used to reduced the influence of intrinsic membrane conductances on EPSP shape, but no attempt was made to investigate or identify the currents themselves. We found that intrinsic membrane conductances have a considerable effect on EPSP shape, particularly for apical inputs. Some of these results have been reported in abstract form (Nicoll, Larkman & Blakemore, 1991 a; Nicoll, Major, Larkman & Blakemore, 1991 b). METHODS Slice preparation and maintenance Brain slices were prepared from young adult albino rats (Sprague-Dawley, 12-2 g) using conventional methods (Larkman, Mason & Blakemore, 1988). Each animal was anaesthetized with halothane gas (Fluothane, ICI), decapitated and the brain rapidly removed. Under cold (3 C) artificial cerebrospinal fluid (ACSF), six coronal slices of 4,m thickness were cut from one hemisphere of visual cortex on a vibrating microtome. Each slice was trimmed and then transferred to an interface-type recording chamber where they were allowed to recover at 34 C for 2 h before recording was attempted. The ACSF used during the slice preparation and recovery contained (mm): NaCl, 124; KCl, 2-3; MgSO4, 1 ; KH2PO4, 1M3; CaCl2, 2-5; NaHCO3, 26; glucose, 1; ph, 7*4. The composition of the ACSF used during neuronal recording was modified to contain: (1) the NMDA receptor antagonist, D,L-2-amino-5-phosphonpentanoate (D,L-AP5, Tocris Neuramin; 5 or 1/tM); (2) higher concentrations of Mg2+ (4-8 mm) to reduce excitability; (3) the y-aminobutyric acid (GABAA) receptor antagonist, picrotoxin (Sigma; 5 or 1,M); and (4) the GABAB receptor antagonist, 2-hydroxy-saclofen (Sigma; 1 or 2 #M) was also added in most experiments. Neuronal impalement and data collection and analysis Microelectrodes were filled with 2 M methyl potassium sulphate and 5 mm KCl. Their DC resistances, measured in ACSF, were 6-9 MQ. Pyramidal neurons were impaled in the lower part of layer II/III and throughout layer V. Where possible, layer V pyramids were classified as either burst firing or non-burst firing on the basis of their responses to long (3 ms) pulses of depolarizing current (Mason & Larkman, 199). Distal and proximal EPSPs were evoked at X2-2 Hz (8-3 V, -2 ms) using an extracellular bipolar electrode placed close to the apical or basal dendrites respectively of the impaled pyramidal cell, i.e. in layers I/II or III/IV for layer 11/111 pyramids and in layers I/II or V/VI for layer V pyramids; in most experiments, EPSPs were evoked alternately from two bipoles placed on the slice surface together. The EPSPs were matched in peak amplitude and were interleaved with 2 ms somatic current pulses whose voltage responses were also matched to the amplitude of the EPSPs. Data were filtered (2 khz), digitized (5 or 1 khz) and averaged (generally 1-25 trials) directly onto computer disk via signal averaging software (SIGAVG, Cambridge Electronic Design) processed through a laboratory interface (CED 141, Cambridge Electronic Design). The effects of the following on EPSP shape were examined: (1) the site (i.e. the cortical layer) of the stimulating bipolar electrode; (2) the putative slow inward rectifier (IH) conductance blocker alinidine (2-(N-allyl-N-[2,6-dichlorophenyl]-amino-2-imidazoline bromide, relative molecular mass = 351; Boehringer Ingelheim) applied extracellularly; (3) caesium applied extracellularly (Cs+); and (4) the quaternary lignocaine derivative QX-314 (N-(2,6- dimethyl-phenylcarbamoylmethyl)triethylammonium bromide, relative molecular mass = 314; Astra) and Cs+ applied together intracellularly (QX-314, + Cs+). EPSP shape indices were measured from the averaged records and, where possible, were normalized by dividing by the membrane time constants (see below) derived from the interleaved pulse responses. Measurement of voltage decay The voltage decays following EPSPs and current pulses were displayed as semilogarithmic plots. The amplitudes of 'fellow' EPSPs and pulses were usually matched during the experiment, but in the few cases where there was a large difference between the two, voltages were normalized by their peak amplitudes to facilitate comparison. For layer II/III cells, time constants were calculated

4 696 A. NICOLL, A. LARKMAN AND C. BLAKEMORE from the slope of a straight line fitted by least-squares regression through the latter, log-linear parts of decay for each EPSP and pulse response. As the 'final' exponential time constant was probably not encountered, the slopes of the voltage decay following EPSPs and injected current pulses were referred to as T. and T, respectively (rather than 8 and r). In layer V cells, the slopes of the voltage decay following EPSPs and current pulses at late times (estimated as for layer II/III cells) were called S. and Sp, respectively, as decay was usually very non-linear throughout large portions of the semilogarithmic plots, and the calculation was performed for semi-quantitative comparison only. Corresponding time intervals were used to estimate the 'time constants' (the term is used loosely) of synaptic and pulse response decays, beginning the regression at the same time after the peak of the EPSP or pulse offset. RESULTS All cells recorded displayed characteristics typical of pyramidal neurons (Connors, Gutnick & Prince, 1982; Mason & Larkman, 199) with resting membrane potentials (RMPs) ranging from -65 to -85 mv. EPSP shape in layer II/III pyramidal neurons Seven pairs of apical and basal EPSPs were recorded from three layer II/III pyramidal neurons (Table 1). EPSP shape indices were normalized by dividing by Tp of the cells in which they were recorded. Normalized EPSP rise times and half-widths were not correlated within the apical and basal groups separately, but were correlated at the 5% significance level when taken together (r2 = -38, P = X2, n = 14). The mean 1-9 % rise times of apical and basal EPSPs were significantly different at the 5% level but there was no difference in their normalized half-widths (Table 1). None of the EPSP waveforms displayed voltage undershoots (Fig. 1) although some of their semilogarithmic plots did display a slight downward deviation from log-linear decay ('sag') at late times (see Nicoll, Larkman & Blakemore, 1992). Proximal and distal EPSPs in layer V pyramidal neurons In order to resolve better any differences in apical and basal EPSPs, all further experiments were conducted on layer V pyramidal cells which are geometrically, and possibly electrically, longer than those in layer II/III (see Discussion). Seven pyramidal neurons were impaled in layer V. For each cell, a bipole was placed successively in each neocortical layer at about 4 um horizontal to the apical dendritic 'axis' of the neuron. Small EPSPs were evoked at each location, interleaved with 2 ms current pulses. Thirty-six EPSPs (-5--8 mv) were recorded (about 6 evoked from each layer). On average, there was little trend in EPSP shape index with the bipole location (Fig. 2). although for both rise time and half-width, there were individual EPSPs which could be selected to show large differences in shape index depending on the layer of stimulation. However, the voltage undershoot following EPSPs was related to the layer in which the bipolar electrode was placed (Fig. 2): when it was measured at its most negative point and expressed as a percentage of the peak EPSP amplitude, undershoot was greatest following the ' most apical' inputs and decreased progressively as the stimulation site was moved from layer I to layer VI, the 'most basal inputs'. Voltage undershoots following layer I/II-evoked EPSPs were significantly greater than those following EPSPs evoked from bipoles in layer V or VI (P < -1).

5 EPSP SHAPE IN NEOCORTICAL PYRAMIDAL NEURONS E.8 Apical Basal.6- E '~.4- D( Time (ms) Fig. 1. Superimposed individual apical and basal EPSP waveforms recorded from the same layer II/III pyramidal cell. In these examples, the 1-9 % rise time and half-width of the apical EPSP were 2-2 and 1-3 times that of the basal EPSP respectively. However, over the population of EPSPs recorded, the differences in apical and basal EPSPs were smaller than for layer V neurons. TABLE 1. Characteristics of apically and basally evoked EPSPs in three layer II/III pyramidal cells Apical Basal (n= 7) (n= 7) P Peak amplitude (mv) n.s. 1-9% rise time (ms) Half-width (ms) n.s. Normalized 1-9% rise time Normalized half-width n.s. Tp (ms) n.s. J:/Tp ratio n.s. Data shown as means + standard deviations. P determined from paired t tests (n.s., not significant). T., time constant of the voltage decay following a synaptic potential; Rp, time constant of the voltage decay following a pulse of current injected at the soma. There was little difference in EPSP shape index between apical and basal inputs, perhaps because layer 11/111 pyramidal neurons are too short (geometrically and electrically) to resolve any effects. Voltage undershoot It was decided to investigate voltage undershoot further using only the most separated bipole locations. Fourteen layer V pyramidal neurones were impaled and twenty-seven pairs of apical and basal EPSPs were evoked (Table 2; Fig. 3). Again there was no significant difference in EPSP 1-9 % rise time or half-width between apical and basal inputs and rise time and half-width were not correlated for apical or basal inputs (Fig. 4A). Voltage undershoot was correlated to EPSP peak amplitude for both apical and basal inputs (r2 = -68, P = < 1 and r2 = -67, P < 1 respectively); undershoot following injected pulses was also correlated with the amplitude of the pulse (r2 = -7, P < -1; see Fig. 4B and C). However, when it was expressed as a percentage of the EPSP peak amplitude (or pulse response

6 698 A. NICOLL, A. LARKMAN AND C. BLAKEMORE *E X 6@ \. 5,.- 1* E 6.- X 5@ ~_.8 n 4S C ~~~~~~~~~~~~~ ~~~~~~~~~~~~-6-4 Y.- X 2.4 z 3. G- _ II III IV V VI Bipole location (layer number) Fig. 2. Characteristics of EPSPs recorded in layer V pyramidal cells in control ACSF. EPSPs were evoked by a bipole placed in different layers of visual cortex. There was a consistent trend in the amount of voltage undershoot following EPSPs, but not in rise time or half-width. Each point represents the mean of 5-7 EPSPs. Continuous line, percentage voltage undershoot following EPSPs; dashed line, normalized 1-9% rise time; dotted line, normalized half-width. TABLE 2. Voltage undershoot following EPSPs recorded in fourteen layer V pyramidal neurones Apical Basal (n = 27) (n = 27) Pulse Peak amplitude (mv) % rise time (ms) Half-width (ms) Undershoot (mv) * * * Percentage undershoot (%) * * 2* * Sp (ms) SI/SP * * Data shown as means + standard deviations. Significance determined by paired t tests, *P < 1 between all groups (n.s., not significant). Sp, slope of voltage decay following a pulse of injected current; S., slope of voltage decay of a synaptic potential. Undershoot following distal EPSPs was significantly greater than that following proximal EPSPs or following somatic current pulses. Under these conditions, there was no difference in apical and basal EPSP shapes. amplitude), undershoot was not correlated (as shown by simple regression) to the EPSP peal amplitude (Fig. 4D), or to the amplitude of the pulse response. Percentage undershoot was significantly greater in apical inputs than in basal inputs, which in turn displayed greater undershoot than followed injected pulses (Table 2). There was no correlation between percentage undershoot and rise time or half-width for either apical or basal EPSPs. The semilogarithmic plots of voltage decay following injected current pulses (as well as following EPSPs) were extremely non-linear in layer V cells, sometimes showing considerable 'sag' (Fig. 3C). EPSP shape indices were therefore not normalized, but it was possible to compare EPSPs and pulses because they had been interleaved. S. for apical inputs at late times was significantly quicker than for basal EPSPs (P < 1, Table 2). Surprisingly, undershoot was not correlated to any

7 EPSP SHAPE IN NEOCORTICAL PYRAMIDAL NEURONS decay slope but there was a strong correlation between S. and Sp (r2 = 86, P = 1 for apical inputs and r2 = 94, P = -1 for basal inputs). Despite the prevalence of undershoot, half-widths were strongly correlated to S. and Sp for both apical and basal EPSPs (r2 = and P = 11 for all 4 correlations). In a :.6 A. Apical Basal.1 - g.5 E a) E.3 E Pulse B Time (ms) e2 el eo > e-1 Ef -2 c e C e4 e5 e lime (ms) e Time (ms) Fig. 3. Voltage undershoot in recordings from layer V pyramidal neurons. A, apical and basal EPSPs interleaved with the membrane response to injected current pulses. Interleaved data can be directly compared without normalizing by membrane time constants. The order of apical and basal stimulation was reversed in some experiments. In this example, stimulation frequency was 3 Hz; bipole voltage was adjusted to give EPSPs of similar peak amplitude. B, superimposed individual waveforms of the apical and basal EPSPs shown in A, highlighting the greater undershoot following the apical EPSP with little other difference in shape. C, semilogarithmic plots of the voltage decay following EPSPs (apical, trace 1; basal, trace 2) and an injected current pulse (trace 3) in a layer V pyramidal neuron when voltage undershoot was prevalent. The plots display considerable non-linearity, even in the current pulse response. The plots were aligned in time so that the peaks of the EPSPs corresponded to the pulse offset. further analysis, all EPSPs recorded were separated into two groups: small (peak amplitude mv, n = 1) and large ( mv, n = 17) inputs but no differences resulted from analysing these groups separately (see Turner, 1988). Included in this sample of pyramidal neurons impaled were six pairs of EPSPs

8 7 A. NICOLL, A. LARKMAN AND C. BLAKEMORE recorded from pyramidal cells which displayed burst firing characteristics. No significant difference was found in the amount of voltage undershoot or in other subthreshold parameters between non-burst and burst firing cells here, or in other experiments described below. m 4r A *8r B 351 E : 4L 2 L- 15 w in O ie~ O o.. E.6 4.~4 '.2. >: 1.. *.* *&O@ * EPSP 1-9 % rise time (ms) EPSP peak amplitude (mv) I-O.3 = 2 U) n ~ Q a) 4) C m B M3N S u _, IK._ x n.nl AI %pu Current pulse amplitude (mv) o 2 n c 15 U) ) c X 1 25r D 't. to EPSP peak amplitude (mv) Fig. 4. Some relationships between voltage undershoot and other parameters., apical EPSPs;, basal EPSPs. A, shape index plot of EPSPs recorded in control ACSF, when undershoot and sag were prevalent. Rise time and half-width were not correlated for either apical or basal EPSPs. Compare with Fig. 5B. B, EPSP peak amplitude versus voltage undershoot following EPSPs. Undershoot was correlated to amplitude (apical: r2 = -68; basal: r-2= -67, P < 1 in both). C, voltage undershoots following injected current pulses. Undershoot was correlated to the amplitude of the pulse (r2 = -7, P < -1). D, percentage undershoot plotted against EPSP peak amplitude. When undershoot was expressed as a percentage of peak amplitude, it was not correlated (by simple regression) with EPSP peak amplitude. Effects of blockers of intrinsic conductances Alinidine In the circumstances encountered above, no difference could be found in the shapes of EPSPs evoked apically and basally. In an attempt to reduce voltage undershoots, alinidine was added to the bathing medium. Alinidine is a putative blocker of the 'H.

9 EPSP SHAPE IN NEOCORTICAL PYRAMIDAL NEURONS current (Lillie & Kobinger, 1984) and is also thought to affect other K+ conductances non-specifically (see Discussion). Six layer V pyramidal neurons were impaled and twenty-four EPSPs of peak amplitude mv were recorded. There was a significant difference in the shape of the waveforms of apical and basal EPSPs with 71 ra 2-5r B E.6 X ' Cu ~.N 2. F 1.5 go ol.2.1 MIn z 1.F o* e Time (ms) Normalized 1-9 % rise time C-3 Ce e Time (ms) Fig. 5. Results of recordings made in the presence of bath-applied 1 SM alinidine, a putative blocker of the IH conductance. A, superimposed individual waveforms of an apical and basal EPSP recorded in the presence of alinidine. The 1-9 % rise time and half-width of this apical EPSP were 3.7 and 1-8 times that of the basal EPSP. B, shape index plot of EPSPs recorded in alinidine. Rise time and half-width (and when normalized as shown here) were correlated for basal EPSPs and when apical and basal EPSPs were grouped together (r2 = -77, P < 1)., basal EPSPs; *, apical EPSPs. Compare this with Fig. 4A. C, semilogarithmic plots of voltage decay following EPSPs (apical, trace 1; basal, trace 2) and injected current pulses (trace 3) in 1 #M alinidine. In this example, all three traces display sag. The greater sag following the apical EPSP in alinidine is emphasized with the thicker line in trace 1. alinidine treatment such that voltage undershoots were reduced and semilogarithmic plots were fairly linear. Every apical EPSP except one had a longer 1-9% rise time than any basal EPSP (Fig. 5A). Half-widths in apical EPSPs were also significantly longer than in basal EPSPs and the difference between parameters for apical and basal EPSPs increased when the data were normalized by Sp (Table 3). Normalized

10 72 A. NICOLL, A. LARKMAN AND C. BLAKEMORE 1-9% rise time and normalized half-width were not correlated for apical EPSPs but were for basal EPSPs and for apical and basal EPSPs together (r2 =.77, P = '1). The shape index plot for EPSPs in alinidine was clearly different than in its absence (cf. Figs 4A and 5B). For basal EPSPs, half-width was correlated to S. (r2 = -52, P = 19 and Sp (r2 = -78, P = 1) but this was not true for apical EPSPs. S. for basal EPSP decay TABLE 3. The shapes of apical and basal EPSPs recorded in 1,UM alinidine from six layer V pyramidal neurons Apical Basal (n = 14) (n = 1) P Peak amplitude (mv) % rise time (ms) 7< < 1 Half-width (ms) < 1 Normalized 1-9% rise time < 1 Normalized half-width 1P < 1 88/Sp 1P n.s. Data shown as means + standard deviations. Significance determined from unpaired t tests (n.s., not significant). There was a clear difference in the shapes of apical and basal EPSPs in the presence of bath-applied 1,UM alinidine, such that the rise times and half-widths were much longer in apical EPSPs than in basal ones. Voltage undershoots were reduced, but not eliminated, with alinidine (cf. Table 2). was weakly correlated with Sp (r2 = -52, P = -19) but there was no similar correlation for apical EPSPs (P = -88). Two of the apical EPSP semilogarithmic plots (both from burst firing cells) displayed much more sag than the others with SJ/SP ratios of -86 and 77, indicating a significant shortening of decay. When these were removed from the analysis, however, there was no change in any of the above conclusions. With alinidine, virtually no undershoot was visible in the EPSP or pulse waveforms but sag was often obvious in semilogarithmic plots. For semilogarithmic plots of voltage decay in the presence of alinidine, there was very little downward deflection at late times associated with pulse responses (hence Sp was more reliable), some associated with basal EPSPs but significant amounts of sag associated with apical EPSPs (Fig. 5C). Extracellular caesium Apical and basal EPSPs could be distinguished on the basis of shape with alinidine treatment, although significant non-linear membrane behaviour remained. Unfortunately, higher alinidine concentrations were found to impair slice health quite dramatically so it was decided to use an alternative channel blocker. Cs+ has been shown in other studies to be effective in reducing voltage undershoots mediated by outward K+ conductances (e.g. Miles & Wong, 1986; Storm, 199). Fourteen pairs of interleaved apical and basal EPSPs were recorded from ten layer V pyramidal neurones in the presence of 3 mm CsCl.. Bath application of Cs+ resulted in a difference between the 1-9% rise time of apical and basal EPSPs which was weakly significant (Table 4); apical EPSP half-width was also longer than basal halfwidth but the difference was barely significant. Rise times and half-widths were not correlated for either group in Cs+.

11 EPSP SHAPE IN NEOCORTICAL PYRAMIDAL NEURONS 73 ~el e' E > e -2 (.. e Time (ms) Fig. 6. Semilogarithmic plots of the voltage decay following EPSPs (apical, trace 1; basal, trace 2) and injected current pulses (trace 3) when both 5 mm Cs' and 5 mm QX-314 had been included in the electrode filling solution. These averages of 1 records were taken after the cell had been impaled for 5 min, by which time all three plots appeared relatively linear, although the signal-to-noise ratio was poor. TABLE 4. Effect of 3 mm extracellular Cs+ on EPSP shape recorded in ten layer V pyramidal neurons Apical Basal (n= 14) (n= 14) P Peak amplitude (mv) n.s. 1-9% rise time (ms) Half-width (ms) SS/SP n.s Data shown as means + standard deviations. Significance determined by paired t tests (* n = 8; n.s., not significant). S,, slope of voltage decay of a synaptic potential; Sp, slope of voltage decay following a pulse of injected current. Three millimolar Cs+ reduced non-linear membrane behaviour but was not as effective as alinidine in revealing distal and proximal EPSPs on the basis of their shapes. Intracellular QX-314 and caesium In a further attempt to improve on the effects of alinidine, four layer V pyramidal neurons were impaled with microelectrodes containing 5 mm QX-314 and 5 mm CsCl dissolved in 2 M methyl potassium sulphate. QX-314 blocks all Na+ currents (Connors & Prince, 1982), and Cst is thought to block many of the K+mediated intrinsic conductances (Miles & Wong, 1986; Storm, 199). In two cells, interleaved apical and basal EPSPs were evoked and the impalements were maintained for 25 and 5 min. In both cells, the EPSP waveforms showed little undershoot initially but their semilogarithmic plots were non-linear, particularly for the apical inputs. After 45 min the semilogarithmic plots were linear when displayed (Fig. 6) but the EPSP waveforms still did not show any difference in rise time or halfwidth.

12 74 A. NICOLL, A. LARKMAN AND C. BLAKEMORE DISCUSSION These results show that it is possible to determine the relative origin of synaptic inputs (apical or basal) in neocortical pyramidal neurons from the shape of EPSPs recorded at the soma: (1) The time course of EPSPs was significantly affected by intrinsic membrane conductances, which caused voltage undershoot and sag. (2) When non-linear membrane behaviour was reduced pharmacologically, it was possible to observe apical EPSPs which had rise times and half-widths which were on average 2-8 and 1-5 times longer respectively than basal EPSPs in layer V pyramidal cells. These results are comparable to those of Turner (1988) in hippocampal CAI pyramidal neurons (but where channel blockers were not needed to resolve apical and basal EPSPs). (3) The greater voltage undershoot and sag in apical inputs, which has also been observed to occur in CAI hippocampal pyramidal cells (Major, 1992), may represent a mechanism for selectively altering the influence of distal synaptic inputs. EPSP shape in layer II/III pyramidal neurons It was easier to observe differences in EPSP shape index between apical and basal EPSPs in layer V cells which, for any one set of assumptions, may be electrically longer than layer II/III pyramidal neurones (Larkman, Major, Stratford & Jack, 1992). In addition, intrinsic membrane conductances may have distorted EPSP shape in layer II/III neurons, as was found for layer V pyramidal cells. Indeed, Thomson, Girdlestone & West (1988) found that Cs+ had marked effects on membrane properties of pyramidal cells in layer II/III of sensorimotor cortex. EPSPs in layer V pyramidal neurons Of the sixty or so cells impaled in layer V in this study, most were classified as either non-burst firing or burst firing, although seven cells could not be definitely placed in either category. In agreement with Connors et al. (1982) we also found little difference in subthreshold properties in vitro between non-burst firing and burst firing cells, although Mason & Larkman (199) have reported that bursters display greater 'sag' than non-bursters. A major consideration in this work was obtaining stimulation that was sufficiently 'apical' or 'basal'. However, the fact that undershoot generally decreased with bipole location from layer I to layer VI is good evidence for a consistent relationship between relative 'synapse position' with the site of stimulation. EPSP shape and voltage undershoot Compared to cells of layer II/III, voltage undershoot and sag in semilogarithmic plots were more prevalent in cells of layer V where, in addition, there was more undershoot associated with apical (distal) inputs than with basal (proximal) ones. Without the use of channel blockers, no trend in apical and basal EPSP shape index could be seen and EPSP rise time was not correlated with half-width. Using simple linear regression, undershoot and EPSP amplitude were correlated for both apical and basal inputs. It might be expected that any distinction between apical and basal inputs would blur as peak amplitude increased and the number of presynaptic

13 EPSP SHAPE IN NEOCORTICAL PYRAMIDAL NEURONS afferents activated increased (as was found in CAI pyramids by Turner, 1988). However, the correlation between undershoot and EPSP amplitude was similar when small (< 1 mv) and large EPSPs were analysed separately. This suggests that even the 'large' inputs used here were still reliably 'apical' or 'basal' (see above) and the fact that no correlation was found between rise time and half-width was not a result of increased EPSP amplitude. A possible explanation to account for the finding that undershoot following apical EPSPs was more prevalent than that following basal EPSPs is that the channels mediating the conductance(s) responsible are denser on distal (or the apical) dendrites. It is now clear that dendrites are more than just passive propagaters of synaptic potentials, many cell types possessing dendritic voltage-dependent conductances (Miles & Wong, 1986; Hounsgaard & Midtgaard, 1989). Calcium may have a special role in dendritic excitability (e.g., Llina's & Sugimori, 198; Jahnsen & Llina's, 1984; Stafstrom, Schwindt, Chubb & Crill, 1985) and some electrophysiological evidence suggests that the largest Ca2+ currents in neocortical neurons may be generated distally (Stafstrom et al. 1985). One attractive hypothesis to explain the greater undershoot following apical EPSPs is to suppose a higher density of Ca2+ channels located on the apical or distal dendrites, resulting in greater distal Ca2+-dependent K+ current activation. For example, the fast outward calciumdependent potassium conductance IK(Ca) may contribute to a medium duration (5-1 ms) after-hyperpolarization in some cell types (Storm, 1989; Schwindt, Spain & Crill, 1992), the time course of which is similar to the time course of voltage undershoot following EPSPs in this study. Alternatively, there is some evidence to suggest that distal dendrites rest at less negative potentials than do the proximal ones or cell bodies of pyramidal cells (Holmes & Woody, 1989; Bernander, Douglas, Martin & Koch, 1991; Holmes & Rall, 1992; Traub, Miles & Buzsaiki, 1992). This might also result in greater current activation distally and possibly explain the greater apical undershoots found here (but see below). Effects of blockers of intrinsic conductances Alinidine and extracellular caesium Alinidine is known to affect pace-maker potentials in the cardiac sinoatrial node where it blocks the IH current (Lillie & Kobinger, 1984) in a voltage-dependent manner. It might also affect other K+ conductances non-specifically, especially at the higher concentrations used here (H. F. Brown, personal communication), and a presynaptic effect of alinidine cannot be ruled out. Compared with control experiments, the presence of 1 fm alinidine in the ACSF resulted in a significant difference in the shape of apical and basal EPSPs whose mean rise times and halfwidths were correlated. Voltage undershoots following EPSPs and pulses of injected current were also reduced, although alinidine was not successful in linearizing semilogarithmic plots from many of the EPSPs evoked from apically placed bipoles. Cs+ is also a blocker of the IH conductance (e.g. McCormick & Pape, 199; Spain, Schwindt & Crill, 1991): undershoots were reduced yet its addition to the ACSF was hardly effective in revealing a distinction between apical and basal EPSP shape indices, and there was no correlation between rise time and half-width. One 75

14 76 A. NICOLL, A. LARKMAN AND C. BLAKEMORE explanation for this could have been that the concentrations of Cs+ used here (3 mm) were insufficient to block IH (but see McCormick & Pape, 199; Spain et al. 1991). Alternatively, IH-generated undershoot may have been blocked by Cs+ but another conductance was still causing some non-linear decay, the result of which was only visible in the more sensitive semilogarithmic plots. Sag in apical inputs was still stubbornly present in Cs+ (as it was with alinidine); this presumably accounts for the fact that apical EPSP rise times in Cs+ were half those achieved with alinidine, although basal EPSP rise times were similar. The rise times of basal EPSPs recorded when undershoot was prevalent, and in the presence of bath-applied alinidine and Cs+ were all similar (see Tables 2, 3 and 4). Basal EPSP half-widths with undershoot/sag present and in Cs+ were similar but the half-widths for EPSPs recorded in alinidine were slightly longer. For apical EPSPs, both rise times and half-widths were shortest when undershoot was present, longer in the presence of Cs+ and longest in alinidine. Cs+ and alinidine were successful in blocking undershoot associated with basal EPSPs and injected somatic pulses but even alinidine could not produce linear semilogarithmic plots of apical EPSP decay. Attempting to reduce IH resulted in a reduction in most of the undershoot/sag associated with basal EPSPs and injected pulses but the greater effects of alinidine must have been due to the blockade of other conductances in addition to IH. Thus, the ratio found here in alinidine of apical to basal indices was 2-8 and 1-5 for 1-9% rise time and half-width respectively, which compares well with the 2-1 and 1P5 (rise time and half-width respectively) found by Turner (1988) in hippocampal CAI pyramids. For most cell types, IH is activated by hyperpolarization beyond about -6 mv, causing time-dependent rectification between threshold (-8 mv for CAl cells; Halliwell & Adams, 1982) and maximum activation at -11 mv. IH contributes to RMP in neocortical pyramidal neurones (Spain, Schwindt & Crill, 1987; Spain et al. 1991) and thalamocortical relay neurons (McCormick & Pape, 199) but not in hippocampal CAI cells (Halliwell & Adams, 1982). Variation in levels ofih activation are though to contribute to voltage undershoots in other cell types (Storm, 1989; McCormick & Pape, 199) and this slow (inward) rectifier may also be operating here. The greater apical undershoot would be accounted for by a greater IH channel density distally. However, more rigorous experiments are required to confirm the identity of the conductance(s) involved if, as may be the case, currents additional to IHare involved in the phenomena described in this study. Effect of QX-314 and intracellular caesium The quaternary lidocaine derivative, QX-314, blocks Na+ channels with a similar effect to tetrodotoxin (Connors & Prince, 1982), and may also affect outward K+ conductances (Hwa & Avoli, 1991). The inward-going Na+-dependent currents may contribute to prolongation of EPSP decay and as Cst blocks most K+-dependent outward currents, QX-314 and Cst together should be an effective combination in blocking conductances causing non-linear membrane behaviour. It was noticed though that the QX-314-Cs+ combination used appeared to require a relatively long period of time to diffuse into the cells merely to produce a rather modest change. Connors & Prince (1982) used QX-314 concentrations as high as 2 mm in CAl pyramids; Johnston, Hablitz & Wilson (198) used microelectrodes containing

15 EPSP SHAPE IN NEOCORTICAL PYRAMIDAL NEURONS 2 M CsCl which was actively iontophoresed into CA3 cells. The two cases of linear semilogarithmic plots eventually observed with QX-314-Cs+ were not manifested in differential apical and basal EPSP shape indices. Concluding remarks Intrinsic membrane conductances, as well as synaptic conductances (Nicoll et al. 1992) can be activated by synaptic potentials and influence EPSP shape, at least in layer V pyramidal neurons. In this study, no attempt was made to conclusively identify the voltage-dependent conductances that smeared the difference in shape between apical and basal EPSPs (cf. Turner, 1988). Alinidine may block other K+ conductances in addition to IH, so its exclusive involvement in the greater apical EPSP undershoot cannot be stated with certainty. It is possible that more than one conductance is involved in shaping EPSP decay and the voltage undershoot following pulses of injected current at the soma may also be mediated by different currents from those affecting EPSPs in the dendrites. Furthermore, IH is activated by hyperpolarization at levels which may not be encountered if distal dendrites did, in fact, rest at the less negative membrane potentials as suggested above. The overall aim of this study was to obtain reliable recordings of EPSP shapes so that simple measurement of the time course of an EPSP recorded at the soma could be used to determine the relative origin of the synaptic input. Ultimately it would be desirable to incorporate these data in a model so that the electrotonic location and relative efficacies of synaptic inputs could be determined. Unfortunately, pyramidal neurons, especially in neocortex, cannot be represented as equivalent cylinders (Stratford et al. 1989; Larkman et al. 1992). It is still not possible to compare the electrical lengths of neocortical pyramids with certainty and both equivalent cylinder and compartmental models of neocortical pyramidal neurones suffer from significant nonuniqueness (Stratford et al. 1989; Cauller & Connors, 1992; Larkman et al. 1992). In the present study, when voltage undershoots and sag were reduced by alinidine, 1-9 % rise times and half-widths were on average 2-8 and 1-5 times longer in apical EPSPs than in basal EPSPs. Using a compartmental model, Stratford et al. (1989) simulated the waveforms of EPSPs originating proximally and distally in a layer V non-burst-firing neuron, assuming there was no somatic shunt. They found EPSP 1-9% rise time- and half-widths-ratios (apical: basal) were 1 and 2-5 respectively when a 'high' membrane resistivity was incorporated into the model and 5 and 2 respectively using a 'low' value for that parameter. Hence, the longest rise times for apical EPSPs recorded here experimentally (when channel blockers were used) were still shorter than those of simulated EPSPs using the model and parameters in Stratford et al. (1989). The determination of the effectiveness of a PSP at the soma is complicated and the relative importance of peak amplitude, rise time and the area of the voltage response at the soma depends on the particular situation (Fetz & Gustafsson, 1983). In this study, the amplitudes of EPSPs at distal sites of input were unknown, so it was not possible to quantify the effects of distance or intrinsic conductances specifically on EPSP amplitude. However, the shortened apical EPSP rise times and half-widths (which are not correlated with amplitude) in layer V pyramidal neurons in conditions of prevalent undershoot presumably reduces the chances of temporal integration occurring for those distal inputs and hence their effect at the cell body. 77

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