CA1 pyramidal to basket and bistratified cell EPSPs: dual intracellular recordings in rat hippocampal slices

Electrophysiological recordings

Paired recordings were performed using conventional sharp electrodes (resistance, 80-160 MΩ) containing 2 M potassium methylsulphate and 2 % biocytin (w/v) under current clamp conditions, using an Axoprobe amplifier (Axon Instruments). The search strategy for connected pairs involved first obtaining a stable intracellular recording from one pyramidal neurone located in the cell body layer. Then a search was made for an interneurone within or very close to stratum pyramidale. Single spikes or brief trains of action potentials were elicited in the pyramidal cell by injection of square wave current pulses delivered once every 3 s, and any voltage change in the interneurones recorded. Any possible connection from the interneurone to the pyramidal cell was also tested. If both tests were negative, a second pyramidal cell was penetrated and tested and so on. With connected pairs, continuous analog recordings from both neurones were made on magnetic tape (Racal Store 4). To ensure that any one data set included data collected only at one postsynaptic membrane potential (± 1 mV), postsynaptic membrane potential was held constant by continuous manual current clamp and then changed to another potential after sufficient data had been collected. Electrode balance was continuously monitored by observing voltage responses to small brief current pulses injected into the postsynaptic cell prior to each activation of presynaptic spikes. The electrophysiological characteristics of recorded cells were obtained from their voltage responses to 500 ms current pulses between -2 and +1 nA in amplitude (delivered from membrane potentials in the range -65 to -85 mV) using pCLAMP software (Axon Instruments).

Data analysis

Data that had been collected on analog tape were digitized, stored on optical disc and analysed off-line (using in-house software). Individual sweeps were observed and either accepted, edited, or rejected according to the trigger points that would trigger measurements and averaging of the EPSPs during subsequent data analysis. Averaging of EPSPs was triggered by the rising phase of the first presynaptic spike for the first EPSP, the rising phase of the second presynaptic spike for the second EPSP and so on. For the averages illustrated and measurements given, individual records in which second, third, fourth and fifth spikes fell within a narrow time window (e.g. 14-20 or 35-40 ms) following the first spike were selected into data subsets and averaged. Average EPSP amplitude was measured between the baseline and the peak of the EPSP. The 10-90 % rise time was measured as the time taken for the EPSP to rise from 10 to 90 % of its peak amplitude. The width at half-amplitude was measured as the time interval between the EPSP rising to 50 % and falling to 50 % of its peak amplitude. Numbers given in the text are means ±s.d.

Spontaneous EPSPs that were at least twice the baseline noise in amplitude and readily distinguishable by eye were counted and their amplitudes measured in 50 to 100 randomly selected sweeps each of 200 ms duration (equivalent to 10-20 s of recording). These measurements were not found to change significantly when longer analysis periods were used.

Histological processing

Following recording, cell pairs were filled with biocytin by passing 0.5 nA depolarizing current pulses in a 50 % duty cycle at 1 Hz for 5-15 min. The slices were incubated in the recording chamber for a further 5-15 min and were then fixed for at least 1 h in a solution of 0.1 M phosphate buffer containing 1.25 % glutaraldehyde-2.5 % paraformaldehyde or 3 % paraformaldehyde-0.5 % glutaraldehyde. After embedding the sections in gelatin, they were sectioned at 60 μm on a Vibratome. Injected biocytin was localized using the Vector stain ABC Elite kit (Vector Laboratories, Peterborough, UK), incubated with 0.1 % Triton X-100 overnight. The injected biocytin was then visualized using 3,3′5,5′-diaminobenzidine (DAB; Sigma) and the sections dehydrated and embedded in Durcupan resin (Fluka) on slides. Filled and recorded interneurones were fully reconstructed under a ×100 objective using a drawing tube or the Neurolucida neuron tracing system. Neurones were classified by their dendritic and axonal architecture.

Electrophysiological properties of CA1 cell body layer interneurones

Interneurones recorded in stratum pyramidale of CA1 could be subdivided into burst firing (BF), regular spiking (RS), classical fast spiking (CFS) interneurones and fast spiking interneurones with a rounded spike after-hyperpolarization (R-AHP) (see Table 1 for details and Fig. 1 for examples). However, interneurones in any one of three of these electrophysiologically defined groups could belong to either of the two main morphological cell types discussed here.

Burst firing interneurones responded to depolarizing current injections applied from membrane potentials negative to -60 to -65 mV with a high frequency burst of APs (Fig. 1). If held depolarized for a longer period they could fire tonically, but the threshold response was always a burst. One of the BF cells was identified as a basket cell (Table 1). The other was not fully recovered histologically. Another two basket cells (as well as two putative radial trilaminar interneurones that are not reported in any detail here) displayed properties intermediate between BF and RS cells. They could fire a single AP at threshold, but larger depolarizing pulses elicited a burst of APs superimposed upon a depolarizing envelope. These four cells had fast APs, relatively low input resistances and long time constants. Regular spiking interneurones were often difficult to distinguish from pyramidal neurones and could be either basket or bistratified cells according to their morphological features. They had the broadest APs and the longest spike AHPs, but similar input resistances and time constants to the BF cells. This broad group of RS cells also includes cells reminiscent of neocortical late spiking interneurones (Kawaguchi & Kubota, 1997; see Fig. 1 for an example). Four interneurones (1 basket and 3 bistratified cells) were classified as R-AHP interneurones by the rounded shape of their spike after-hyperpolarizations, which were longer lasting than the spike AHPs of CFS cells near spike threshold. R-AHP cells had the highest input resistances, but with their brief time constants, deep spike AHPs and relative lack of spike accommodation or frequency adaptation (during large depolarizing pulses, not illustrated) they resembled CFS cells. Two of the interneurones defined as CFS were basket cells. The other two were not recovered sufficiently for identification. However, one interneurone (not included in Table 1 because its AP did not overshoot 0 mV) resembled other CFS cells and was identified as a bistratified cell (cell 960719A, shown in Fig. 2A). The APs of CFS cells had simple, fast spike AHPs and trains of spikes showed little accommodation or adaptation. CFS cells had relatively low input resistances, brief time constants and a linear current-voltage response negative to resting potential (Table 1, Fig. 1).

Morphology of postsynaptic interneurones

The recorded, filled and recovered postsynaptic interneurones could be divided into two broad classes, basket and bistratified cells, based on their axonal and dendritic morphology. These classifications were based on those described previously (Buhl et al. 1994a;Sík et al. 1995; Buhl et al. 1996; Halasy et al. 1996).

Properties of pyramidal cell to bistratified cell EPSPs

The properties of spontaneous EPSPs recorded in postsynaptic bistratified cells are summarized for each cell in Table 2. The rate of spontaneous EPSPs in these cells was relatively high and ranged from 2.8 to 22 s⁻¹ and their average amplitude from 1.40 to 4.45 mV (mean, 2 ± 1.1 mV). There appeared to be no relationship between frequency and size of spontaneous EPSPs. The properties of EPSPs in response to single APs, or the first of a train of APs, are also summarized in Table 2. The average amplitude of these EPSPs recorded in seven pairs varied widely and could be as large as 9 mV in average amplitude (Fig. 2Aa and 2Ba). These EPSPs were relatively brief with rise times < 2 ms and widths at half-amplitude between 4 and 11 ms (Figs 2Aa, 2Ba, 3Aa, 3Ba and 5A). The apparent failure rate in response to the first presynaptic AP ranged from 0 (no failures) to 36 % (mean, 6.4 ± 12.2 %). The EPSPs displayed non-conventional voltage relations with an average increase in amplitude of 23.7 ± 14.9 % with a 10 mV depolarization of the postsynaptic interneurone from rest (n= 4; Fig. 3Aa and Ba).

The properties of the EPSPs recorded in bistratified cells in response to second and third APs delivered in brief trains are shown in Table 3. Second EPSPs were the same shape as first EPSPs, but generally smaller and third and fourth EPSPs were smaller than second EPSPs (n= 5; Fig. 5Aa and Ba). With an interspike interval of < 15 ms the second EPSP amplitude was on average 72 ± 27 % of the first EPSP (n= 3). Failures were observed in response to second APs where no failures had been apparent in responses to first APs (11 of 800 and 30 of 695, respectively). Failure rates in response to third APs were higher than following second APs in the two pairs so tested.

Properties of EPSPs recorded in pyramidal cell to basket cell connections

The properties of spontaneous and AP-elicited EPSPs recorded in basket cells are shown in Table 4. Spontaneous EPSPs occurred at a similar frequency to those observed in bistratified neurones, but were typically of smaller amplitude. EPSPs elicited by the first AP in a train were smaller and a little briefer than those recorded in bistratified cells, although with small samples these differences do not reach significance. Typical failure rates were higher than for bistratified cell EPSPs, possibly reflecting the lower average amplitudes. However, the depression observed during responses to trains of presynaptic APs was similar (Table 5 and Figs 4, 6 and 7). Basket cell EPSPs did not exhibit a consistent response to changes in membrane potential. Two EPSPs so tested increased in amplitude and two decreased with postsynaptic depolarization (Fig. 4).

The third group of interneurones were recorded with the electrode placed in the stratum pyramidale but were not sufficiently well recovered histologically for morphological classification. However, their properties and those of their EPSPs are similar to those of the other two groups and are therefore included in Tables 4 and 5 (n= 2).

EPSPs elicited in interneurones by action potentials in pyramidal cells

Depression at pyramidal cell to basket and bistratified cell connections

In this study of the paired pulse and brief train effects at pyramidal cell-interneurone synapses in CA1, depression was found to be prevalent at synapses onto stratum pyramidale interneurones. This is in marked contrast to the profound facilitation observed using the same protocols at certain classes of pyramidal cell-interneurone connection in the neocortex (Thomson et al. 1993a, 1995; Deuchars & Thomson, 1995; Thomson, 1997). However, pyramidal cell to horizontally orientated oriens/alveus interneurone connections in the hippocampus also display marked facilitation (Ali & Thomson, 1997) and some, as yet incompletely characterized, neocortical pyramidal cell- interneurone connections also exhibit paired pulse depression (Thomson & Deuchars, 1997). Since the paired pulse effects that have been observed to date are consistent across morphological subclasses within any one region, the effect seems to be dependent on the identity of the postsynaptic cell. However, the factor(s) that determine(s) how the postsynaptic target influences the behaviour of the presynaptic terminal have yet to be established.

The mechanisms involved in presynaptically mediated synaptic depression have been studied more extensively at pyramidal cell-pyramidal cell connections in the neocortex of adult (Thomson, 1997 for discussion of the time course of depression) and juvenile rats (Markram & Tsodyks, 1996; Markram, 1997). There are several putative mechanisms (see Markram, 1997) but a current hypothesis is that such synaptic depression is due to refractoriness at release sites, i.e. once a release site has released, it is refractory for tens of milliseconds (Thies, 1965; Betz, 1970; Koerber & Mendel, 1991; Thomson et al. 1993b;Stevens & Wang, 1995; Thomson, 1997). The larger the proportion of available release sites that has released, the smaller will be the proportion available for the next release.

Functional importance of synaptic depression in hippocampal local circuits

The functional consequences of synapses exhibiting paired pulse and brief train depression have been studied and modelled (Markram & Tsodyks, 1996; Abbott, Varela, Sen & Nelson, 1997). These models propose that small changes in presynaptic firing rate (especially changes from low to high rates) will generate a phasic response, but that these connections convey little information about static firing rates. Once a population of pyramidal cells has been active for even a few tens of milliseconds and their synapses onto basket and bistratified cells have depressed, little additional activation of these interneurones will result from an increase in firing in these pyramidal cells. Only recruitment of additional pyramidal cells will enhance their activation. This is in marked contrast to the patterns of activity that most effectively excite horizontally oriented oriens/alveus interneurones (Ali & Thomson, 1997, 1998). These interneurones will be more effectively recruited by a small population of pyramidal cells firing at high frequency than by a large population firing slowly and tonically.

The dynamic behaviour of CA1 pyramidal inputs onto three types of interneurones and onto other pyramidal cells in the CA1 region have therefore been studied in some detail. Relatively few, rather sparse axonal branches of pyramidal cells have been seen to cross stratum pyramidale and to enter stratum radiatum in these studies, the majority of their axonal arbour being confined to stratum oriens and the alveus. The innervation that basket and bistratified cells can receive from CA1 pyramidal cells, therefore, may largely be confined to the oriens. It is perhaps surprising that basket and particularly bistratified cells with dendrites that also span stratum radiatum receive such a strong input from a relatively discrete portion of their dendritic tree. The properties of the inputs that bistratified cells receive in stratum radiatum and that basket cells receive in radiatum and lacunosum-moleculare (where entorhinal cortex input to CA1 occurs), and the interactions amongst these several inputs, remain to be determined.