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

Abstract

1. A new preparation of the in 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 microM) and the remaining component could be enhanced in Mg(2+)-free Ringer solution and blocked by AP5 (50 microM). In 70% of the dendrites, the EPSPs were followed by a prolonged after-hyperpolarization (AHP) which could be blocked by a selective and potent GABAB antagonist, CGP55845A (2 microM). 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 microM) 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 Ca2+ 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.

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 (A/O, alveus/oriens; 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 A/O (Stim. 1) and afferent fibres near the SR/L-M border (Stim. 2). At all locations, stimulation of the A/O 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.

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 μA, interpulse interval 50 ms) recorded about 275 μm 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 μA are shown superimposed while the other traces are means. D, the spike generating tEPSPs in response to 250 μA 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.

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 (10 μm), CGP 55845A (2 μm) and AP5 (50 μm). 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 μA and 500 μA, 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.

Figure 6. Properties of distally evoked dendritic spikes

Aa, dendritic recordings of individual threshold responses from two different dendrites. With threshold-straddling 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.

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 (> = 1 mV) recorded in standard Ringer solution. 129 individual cEPSPs were included in the above plots.

Figure 4. The glutamatergic component of the dendritic synaptic potential

A, the non-NMDA receptor antagonist CNQX (10 μm) 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 Mg²⁺-free Ringer solution. C, addition of both CNQX (10 μm) and the NMDA-receptor antagonist AP5 (50 μm) completely blocked the dendritic EPSP. RMP: in A, -74 mV; in B, -66 mV; and in C, -71 mV.

Figure 5. The GABAergic component of the dendritic synaptic potential

A, the voltage dependency of the distally evoked dendritic response. Before afferent stimulation V_m 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 GABA_B-receptor antagonist CGP 55845A (2 μm) 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.

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 (t_p) and time-to-spike (t_s) were both measured from the first inflection at the beginning of the EPSP. V_m at the peak of the EPSP (V_Thr) was also measured and represents the threshold level. B, the ratio between t_s and t_p plotted as a function of V_Thr. 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 t_s was measured. D, the relationship between t_s and t_p of subthreshold EPSPs (n = 11). The dotted line shows a correlation of 1.0 between t_s and t_p. Except for one case, the points are clustered around the dotted line. RMP: in A and C, -68 mV.

Figure 7. Voltage-dependency of distally evoked dendritic spikes

A, individual dendritic recordings showing the effect of changing the dendritic V_m 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.

Figure 8. Distally evoked dendritic spikes are blocked by somatic application of TTX

A, the experimental setup for local application of TTX (10 μm) 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, 10–30 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.

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 dV/dt as a function of the predicted dV/dt. The dashed line indicates a correlation of 1.0 between the observed and predicted dV/dt with a limit of +10 % (dotted line). •: correlation > 1.1; ○: correlation < = 1.1. D, superimposition of three dendritic responses to distal stimulation: threshold response (1), submaximal response (2), maximal response (3). The predicted and observed dV/dt 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.