Facilitating pyramid to horizontal oriens-alveus interneurone inputs: dual intracellular recordings in slices of rat hippocampus

Abstract

1. In adult rat hippocampal slices, simultaneous intracellular recordings from pyramidal cells in CA1 and interneurones near the stratum oriens-alveus border revealed excitatory connections that displayed facilitation on repetitive activation in twelve of thirty-six pairs tested. 2. Postsynaptic interneurones were classified as horizontal oriens-alveus interneurones by the pronounced 'sag' in response to hyperpolarizing current injection, high levels of spontaneous synaptic activity and by the morphology of their somata and dendrites, which were confined to stratum oriens-alveus and their axons which projected to stratum lacunosum-moleculare where they ramified extensively, in the region of entorhinal cortex input to CA1. 3. Excitatory postsynaptic potentials (EPSPs) elicited by single pyramidal cells were 0 to 12 mV in amplitude. Mean EPSP amplitude (single spikes) was 0.93 +/- 1. 06 mV at -70 +/- 2.3 mV (n = 10). The rise time was 1.2 +/- 0.5 ms and the width at half-amplitude was 7.5 +/- 4.7 ms. 4. EPSPs fluctuated greatly in amplitude; the mean coefficient of variation was 0.84 +/- 0.37 for the first EPSP and 0.47 +/- 0.24 for the second. Apparent failures of transmission frequently occurred after first presynaptic spikes but less frequently after the second or subsequent spikes in brief trains. 5. EPSPs displayed facilitation at membrane potentials between -80 mV and spike threshold. Second EPSPs within 20 ms of the first were 253 +/- 48 % (range, 152-324 %) of the mean first EPSP amplitude. Third EPSPs within 60 ms were 266 +/- 70 % (range, 169-389 %) and fourth EPSPs within 60-120 ms were 288 +/- 71 % (range, 188-393 %). Both proportions of apparent failures of transmission and coefficient of variation analysis indicated a presynaptic locus for this facilitation.

Figure 1. Raw data from a pyramid to horizontal O-A interneurone connection

A, single sweep EPSPs at -72 mV. Presynaptic action potentials (one sweep) are shown above. Due to the variation in the size of the EPSPs from sweep to sweep (lower records in A), an average was computed. Most of the EPSPs illustrated in subsequent figures are composite averages triggered from the rising phase of the first presynaptic spike for the first EPSP and the rising phase of the second spike for the second EPSP and so on. B shows the computed average. The spike artifacts visible here are removed graphically from other figures.

Figure 2. Electrophysiological characteristics of two horizontal O-A interneurones (A and B) and a pyramidal cell (C)

Voltage responses to depolarizing and hyperpolarizing current pulses are shown. The amplitude of the largest negative pulse and the largest positive pulse are given on each trace. Intervening current pulses are -0.8, -0.6, -0.4 and -0.2 nA, respectively (ascending order in A and C). For the cell in B the intervening pulses are -0.6 and -0.2 nA. Horizontal O-A interneurones displayed a pronounced ‘sag’ in the response to hyperpolarizing current injection, rebound depolarization after the pulse and little spike accommodation or frequency adaptation. A single pyramidal cell spike superimposed on a single horizontal O-A interneurone spike is shown in D. The interneurone spikes were faster than pyramidal spikes and were terminated by a larger after-hyperpolariztion (AHP). The scale bar for the superimposed spikes is shown on the right-hand side. The scale bars for C apply to A.

Figure 3. Morphology and single sweep EPSPs of a horizontal O-A interneurone

A and B, full reconstruction of a horizontal O-A interneurone drawn from three 60 μm transverse sections. The cell body was located in the O-A border, with horizontally oriented sparsely spiny dendrites confined to the oriens and the alveus (Alv). The axon projected from the cell body to s. lacunosum-moleculare, where it ramified extensively. SO, s. oriens; SP, s. pyramidale; SR, s. radiatum; SLM, s. lacunosum-moleculare. For simplicity the interneurone axon from only one of three 60 μm sections containing the axon is drawn in A, the remaining axon is drawn in B where the dotted line illustrates the relative position of the main axon. The current-voltage relation for this cell is shown in Fig. 2A. Three single sweeps (raw data) of the EPSPs elicited in this cell by a simultaneously recorded pyramid are shown in C. The single sweeps illustrate the variation in the size of the EPSPs from sweep to sweep and an apparent failure of transmission after the first presynaptic spike (third trace, *). The composite averaged EPSP (60-120 sweeps) and the SDTC (standard deviation time course) are shown in D.

Figure 4. Morphology and synaptic responses of two horizontal O-A interneurones in different planes of section

A and B, two horizontal O-A interneurones are shown reconstructed from 2 different planes of section. These were the largest (B) and the smallest (D) first spike EPSPs recorded to date. The interneurone in A was reconstructed from a more longitudinally oriented section of hippocampus and the interneurone in C from a traditional transverse section. There was a difference in extent of the axonal and dendritic arborization in the two cells. Dendritic arborization is greater in the cell recorded in a more longitudinally oriented slice and axonal arborization greater in a transverse slice. The EPSPs elicited in both of these interneurones are shown below. The scale bar applies to both B and D.

Figure 5. An interneurone that was postsynaptic to two consecutively recorded pyramidal cells

A, the axonal arborization was slightly different from the other 7 reconstructed interneurones in that it innervated s. oriens, albeit sparsely, as well as projecting to s. lacunosum-moleculare where it ramified extensively. The axon was reconstructed from four 60 μm thick sections. B and C, the amplitude and duration of the EPSPs differed between the two connections.

Figure 6. EPSP amplitude distributions for four pyramid to horizontal O-A interneurone connections

Single sweep EPSP amplitudes were measured under direct manual control, binned and plotted as probability distributions. Where no apparent postsynaptic response was elicited by a presynaptic spike a zero was entered. The first bin therefore represents the apparent failures of transmission in each case. Where second, third or fourth EPSPs summed with preceding EPSPs, an averaged first, second or third EPSP was scaled to match the amplitude of the equivalent single sweep event and the amplitude measured as the difference between the peak of the EPSP and the equivalent point on the decay phase of the preceding EPSP. For first EPSPs and later EPSPs that were well separated in time, the amplitude was measured as the difference between the baseline preceding the EPSP and the peak. Because these measures were necessarily controlled by hand to eliminate obvious artifacts or spontaneous events and the longest reasonable samples were used, the durations of the baseline and peak samples chosen to represent the most accurate estimates varied from event to event. Estimates of equivalent noise distributions were therefore not feasible. Because relatively few measurements are included in each histogram (70-200 for first and second EPSPs, 50-180 for third EPSPs, 20-40 for fourth and fifth EPSPs) data are coarsely binned and histograms included simply as a description of the data. The proportion of failures in response to the first presynaptic spike is greatest with the smallest EPSP (931026A, > 50 %) and least with the largest EPSP (960307, < 40 %). The proportion of failures decreases and the proportion of larger events increases when second, third, fourth and fifth EPSPs are compared with first EPSPs for all data sets collected at one spike burst per 3 s (0.33 Hz). At 1 Hz, however, (931006A1) second EPSPs were on average slightly smaller than first EPSPs and the proportion of failures was similar.

Figure 7. Normalized second, third and fourth EPSP amplitudes plotted against the time interval following the first spike in the train

Data subsets used for these points each include EPSPs recorded from a single pair, at one membrane potential and with a relatively narrow range of interspike intervals. The mean interval for each data set is plotted. The s.d.s about the mean were < 4 ms for intervals < 20 ms and up to 10 ms for the longest intervals. Normalized mean EPSP amplitude is plotted for each subset (n, 20-60), e.g. the mean amplitude of the second EPSP divided by the mean amplitude of the first EPSP for that subset. c.v.s for these EPSPs were large, 0.84 ± 0.37 for the first EPSPs and 0.47 ± 0.24 for second EPSPs. Double log regression lines are indicated by the dotted lines. Correlation coefficients were, however, low (0.7 > r > 0.6) and these lines simply represent the trend.

Figure 8. Normalized c.v.⁻² plotted against normalized mean EPSP amplitude for second and third EPSPs in brief trains

For each data subset (see Fig. 7 legend) mean second (•) or third (□) EPSP amplitude and c.v.⁻² were normalized using the mean and c.v.⁻² of the first EPSP for that data subset. The majority of points lie above the line of slope 1, indicating that the facilitation was due to an increase in the probability of release.

Figure 9. Comparison of two EPSPs elicited in a single horizontal O-A interneurone by two consecutively recorded pyramidal cells (A and B)

Both EPSPs increased in amplitude and duration with postsynaptic depolarization, despite their different time course at any one membrane potential. Paired pulse facilitation was apparent in both connections at all 3 membrane potentials.

Figure 10. EPSPs in a pyramid to horizontal O-A interneurone connection generated by brief spike trains of different durations and recorded at different postsynaptic membrane potentials

The averaged EPSPs each include between 60 and 120 single sweeps. EPSPs elicited by brief trains of 3 presynaptic spikes were recorded at 3 different postsynaptic membrane potentials (-56, -61 and -76 mV). At -76 mV averaged responses to spike trains of 3 different durations are compared. The facilitation of the second and third EPSPs appears to decline with increasing interspike interval. A similar time dependence is apparent at -61 mV, where averaged responses to spike trains of 2 durations are compared. At -56 mV, maximal second and third EPSP facilitation at the briefest interspike intervals resulted in postsynaptic action potentials, so only the averaged response to the longest (slowest) train is illustrated. Comparison of EPSPs elicited by spike trains of similar duration indicates that the EPSPs increased in amplitude with postsynaptic depolarization.

Figure 11. EPSPs in a pyramid to horizontal O-A interneurone connection generated by brief spike trains of different durations and recorded at different postsynaptic membrane potentials

The averaged EPSPs each include between 60 and 120 single sweeps. On the left, EPSPs elicited by similar, brief trains of 3 presynaptic spikes were recorded at 4 different postsynaptic membrane potentials (-61, -69, -72 and -78 mV). Averaged EPSPs decreased in amplitude and duration with hyperpolarization to -78 mV and with depolarization to -69 mV from -72 mV. This latter decrease in amplitude may, however, be an overestimate as at -69 and -61 mV the largest single sweep events elicited postsynaptic action potentials (see Fig. 12) and were excluded from the analysis. Paired pulse and brief train facilitation are, however, apparent and relatively similar at all membrane potentials. On the right, responses to brief 3 and 4 spike trains of different durations are compared at two membrane potentials (-61 and -78 mV). At -61 mV averaged responses to 3 spike trains of two different durations are compared. Facilitation of second and third EPSPs appear to decline with increasing interspike interval. At -78 mV averaged responses to trains of 4 spikes in which the timing of the third and fourth (but not the second) presynaptic spikes are different, are compared. Facilitation of the third and fourth EPSPs appears to decline with increasing interspike interval.

Figure 12. Postsynaptic spikes

At membrane potentials less negative than -69 mV, second, third and fourth EPSPs (but very rarely first EPSPs) in brief trains could trigger postsynaptic spikes in this interneurone. This figure illustrates the short and constant latencies of these postsynaptic spikes, although the spike AHP varied from sweep to sweep. On the left, 3 single sweep responses to pairs of presynaptic spikes are superimposed. On the right a single sweep response to a train of 4 presynaptic spikes is shown. Sweeps including postsynaptic spikes were excluded from the averaged EPSPs illustrated in other figures.

Figure 13. Summary diagram of the dendritic and axonal arborization and possible synaptic contacts made and received by a horizontal O-A interneurone

A pyramidal cell directly excites a horizontal O-A interneurone, which might then suppress inputs from entorhinal cortex to that pyramidal cell by shunting current in distal dendrites.