Cerebral Cortex Advance Access originally published online on April 20, 2005
Cerebral Cortex 2006 16(2):200-211; doi:10.1093/cercor/bhi098
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Layer 6 Cortico-thalamic Pyramidal Cells Preferentially Innervate Interneurons and Generate Facilitating EPSPs
Department of Pharmacology, The School of Pharmacy, University of London 29–39 Brunswick Square, London WC1N 1AX, UK
Address correspondence to Prof. A.M. Thomson, Department of Pharmacology, The School of Pharmacy, 29–39 Brunswick Square, London WC1N 1AX, UK. Email: alex.thomson{at}ulsop.ac.uk.
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Abstract |
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The properties of the connections made by the axons of pyramidal cells with cortico-thalamic (CT)-like morphology with a range of postsynaptic layer 6 targets were studied with dual intracellular recordings in slices of adult rat and cat neocortex. The cells were filled with biocytin and identified morphologically and, where appropriate, immunofluorescently. CT-like pyramids contacted interneurons with a very high probability (up to 1:2) but contacted other layer 6 pyramidal cells only rarely (
1:80). The excitatory postsynaptic potentials (EPSPs) that they elicited both in pyramidal cells and in a variety of types of interneurons (including those immunopositive for parvalbumin and for somatostatin) facilitated, the second EPSP being larger than the first over a range of interspike intervals. Facilitation was not, however, maximal at the shortest intervals; in fact, depression was apparent at some connections at short interspike intervals. Facilitation in the majority of connections peaked at intervals of 25–35 ms and then declined slowly. Nor did these connections display the augmentation typical of many other strongly facilitating connections. Third EPSPs were smaller on average than second EPSPs, and fourth and subsequent EPSPs could be depressed (relative to first EPSPs). The properties of the outputs of these CT-like pyramidal cells are therefore quite distinct from those of other pyramidal cells, both within layer 6 and in other layers, possibly reflecting their unique role as both first order thalamo-cortical recipient and cortico-thalamic output neurons.
Key Words: cortico-thalamic • EPSP (excitatory postsynaptic potential) • layer 6 pyramidal cell • facilitation • interneuron • microcircuitry
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Introduction |
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Layer 6 of the neocortex contains several quite distinct classes of pyramidal cells. Cortico-thalamic (CT) cells that project to the primary sensory, or ‘specific’ sensory, thalamic nuclei as well as to the nucleus reticularis thalami (nRT) are upright pyramidal cells with an apical dendritic tuft in layer 4. Their local axonal arbour is narrow, each branch turning up towards the superficial layers, forming drumstick side branches and a terminal axonal arbour in layer 4 (Zhang and Deschenes, 1998
). Typically, cells with this morphology fire with an accommodating, tonic pattern (Mercer et al., 2005). In contrast, phasically firing cortico-cortical (CC) pyramidal cells display a wide range of dendritic morphologies, including short upright pyramidal cells whose apical dendrites do not extend beyond upper layer 5, bipolar cells and inverted pyramids. These CC pyramidal cells have long, horizontally oriented axonal arbours that are confined to the deeper layers.
It is becoming increasingly apparent that different subclasses of pyramidal cells in each of the layers make and receive different connections, not only in terms of their interactions with distant cortical or subcortical structures, but also within the local circuit. For example, it is the large, tufted layer 5 pyramids that receive the powerful descending input from layer 3 pyramids (Thomson and Bannister, 1998), while the smaller, short layer 5 pyramids lacking a tuft in layers 2/1 provide the majority of the intra-laminar excitatory input to neighbouring layer 5 pyramids. In a recent paired recording study in adult neocortical slices, selectivity in intra-laminar target selection was also suggested for layer 6 pyramidal cells. CC-like pyramidal axons were found to be up to four times more likely to make synaptic connections with other layer 6 pyramidal cells (of all classes) than were the axons of CT-like cells (Mercer et al., 2005).
The excitatory postsynaptic potentials (EPSPs) activated by CC-like cells displayed paired pulse and brief train depression at all interspike intervals studied (Mercer et al., 2005). This confirmed a suggestion made by Beierlein and Connors (2002) that the ‘depressing’ EPSPs activated by other layer 6 pyramidal cells in paired recording experiments may have involved presynaptic CC cells, while the facilitating EPSPs elicited by electrical stimulation of the thalamus in thalamo-cortical slices might result from the antidromic activation of CT cell axons. It was therefore of interest to determine whether CT cells do indeed generate facilitating EPSPs in other pyramidal cells, since strongly facilitating EPSPs have only previously been widely reported in postsynaptic dendrite targeting (Thomson et al., 1995) and particularly in somatostatin immunopositive interneurons (Reyes et al, 1998). Although one study reported facilitating pyramid–pyramid EPSPs in rat layers 3 and 5 at 28 days of age (Reyes and Sakmann, 1999), the vast majority of EPSPs recorded with paired intracellular recordings in both adult and juvenile pyramidal cells exhibited paired pulse and brief train depression (Thomson and West, 1993, 2003; Thomson et al., 1993, 2002a,b; Markram et al., 1998; Thomson and Bannister, 2003).
Apart from pyramidal cells, the postsynaptic targets most consistently reported to receive ‘depressing’ EPSPs from neighbouring pyramidal cells are the parvalbumin-containing interneurons (Reyes et al., 1998; Thomson and West, 2003). It was of interest therefore to explore the possibility that CT-like pyramids might also generate facilitating EPSPs in these interneurons, i.e. whether the output characteristics of CT cells differ fundamentally from those of many other classes of pyramidal cells.
To answer these questions, paired intracellular recordings were made in layer 6 of rat and cat visual cortex and rat somatosensory cortex. Synaptically connected neurons were filled with biocytin and processed for immunofluorescent identification of interneuronal markers and histochemically for morphological identification of connected pairs of neurons and reconstruction. This paper focuses on the excitatory connections made in layer 6 by the axons of pyramidal cells with CT-like morphology. Connections made by CC-like pyramidal cells are reported elsewhere (Mercer et al., 2005).
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Materials and Methods |
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Dual intracellular recordings were made from synaptically connected neurons in rat somatosensory and visual cortex and in cat visual cortex as described previously (Thomson and West, 2003
; Mercer et al., 2005).
Male Sprague–Dawley (n = 20) or Wistar (n = 8) rats (120–200 g) were anaesthetized with inhaled Fluothane and intraperitoneal pentobarbitone sodium (Sagatal, 60 mg/kg). Male cats (n = 7, 2.5–3.4 kg) were anaesthetized intravenously with a mixture of 
-chloralose (70 mg/kg) and pentobarbitone sodium (6 mg/kg) for a different series of experiments (procedures similar to Wang and Ramage, 2001). Rats were perfused transcardially, and cats (following an overdose of barbiturate) via the carotid arteries, with ice-cold modified artificial cerebrospinal fluid (ACSF) with added pentobarbitone (60 mg/l) in which 248 mM sucrose replaced NaCl. Rats were decapitated and the brain removed. Visual cortex was removed from cats via a hole in the skull. Slices of neocortex, 450–500 µm thick, were cut (Vibroslice, Camden Instruments, UK) and transferred to an interface recording chamber where they were maintained for 1 h in sucrose-containing medium, before switching to standard ACSF containing (in mM) 124 NaCl ,25.5 NaHCO3, 3.3 KCl, 1.2 KH2PO4, 1.0 MgSO4, 2.5 CaCl2, 15 D-glucose equilibrated with 95% O2/5% CO2 at 35–36°C. All procedures complied with UK Home Office regulations for animal use.
Paired intracellular recordings were made with conventional sharp micro-electrodes, containing 2 M KMeSO4 and 2% w/v biocytin, tip resistance 90–150 M
. Presynaptic neurons were depolarized with combinations of square-wave and ramped currents, typically delivered at 1 pulse per 3 s to elicit trains of action potentials (APs) with different firing patterns and instantaneous frequencies and postsynaptic responses were recorded (Spike-2, Cambridge Electronic Designs, Cambridge, UK). Cells were filled with biocytin and slices fixed and processed histologically for identification of recorded neurons. Slices in which interneurons were recorded and filled were processed first for immunofluorescent identification of the interneuronal markers parvalbumin, calbindin and/or somatostatin, then for avidin–horseradish peroxidase with 2,4-diamino butyrate as described previously (Hughes et al., 2000; Thomson et al., 2002a).
During off-line analysis (in house software), data sets in which the first EPSP shape and amplitude and the postsynaptic membrane potential were stable, were selected. Single sweeps were checked by hand to ensure that every presynaptic AP was recognized by the software and that the trigger points used for subsequent analysis were accurately aligned with the rising phase of each AP. Sweeps including artifacts or large spontaneous events were excluded from averaged records. All sweeps in which the second AP followed the first AP within a given time window were then selected. The second EPSPs within each window were then averaged, using the rising phase of the second AP as the trigger. This second EPSP average was then superimposed on an average of all responses to single APs. The amplitude of the averaged second EPSP was then measured from its peak to the appropriate point on the falling phase of the averaged first EPSP. Averaged responses to later APs in trains were analyzed similarly. These points were then plotted against the interval between the first and second spike. Where postsynaptic responses exhibited an adequate signal-to-noise ratio, single sweep events were also measured (by hand with cursors) using an average of all single spike EPSPs, scaled to match the amplitude of the first EPSP in each sweep, to measure second EPSPs.
Single sweep data were plotted against interspike interval and/or the time interval from the first AP (for third and subsequent EPSPs in trains) and smoothed (running average of 10–30 points) to reveal trends (Figs 1A, 3B, 4F and 6F,G). Where enough single sweep measurements with a suitable range of interspike intervals for both second and third EPSPs had been collected, the combined effects of the interval between the first and second AP (first interspike interval) and the interval between the second and third AP (second interspike interval) on the amplitude of the third EPSP was tested by plotting the third EPSP amplitude against these two interspike intervals. The resulting 3 dimensional plots were then rendered as contour maps with third EPSP amplitude indicated by colour (PSI Plot; see Figs 1E, 3E and 6D).
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Properties of EPSPs Activated in Layer 6 Pyramidal Cells by Presynaptic CT-like Pyramidal Cells
The properties of the EPSPs generated in layer 6 pyramidal cells by presynaptic CC-like cells and the relative probabilities of CC- and CT-like pyramids innervating other neurons are reported in detail elsewhere (Mercer et al., 2005). In brief, in 28 experiments in rat neocortex and seven in cat neocortex some 1500 pairs of layer 6 pyramidal cells were tested. These yielded 56 pyramid–pyramid connections. Despite approximately equal numbers of recordings from CC-like and CT-like pyramids, however, only five of the rat pyramid–pyramid connections (one pair recorded only briefly) and none of the cat pairs tested involved a presynaptic CT-like cell.
One CT to CT-like cell connection is illustrated in Figure 1 and the properties of four CT to pyramid connections are summarized in Table 1, where they are compared with those elicited by presynaptic CC-like cells (data from Mercer et al., 2005). The EPSPs activated by CT-like cells were smaller (average first EPSP amplitude) than EPSPs activated by CC-like pyramids. This smaller amplitude correlated with a higher proportion of apparent failures of transmission and a higher coefficient of variation (CV). They also displayed, on average, a briefer time course, although this did not reach significance with these small samples.
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CT-like Pyramidal Cells Target Layer 6 Interneurons
Of 21 pairs tested in rat and 38 in cat in which one of the cells was a layer 6 pyramid and the other a layer 6 interneuron, a pyramid to interneuron connection was identified in five pairs in rat (1:4) and eight pairs in cat (1:5, one interneuron receiving two CT inputs). Of these, nine pairs involved a morphologically identified presynaptic CT-like cell, while only two involved a CC-like presynaptic pyramidal cell (two presynaptic pyramids were not identified). It is therefore reasonable to conclude that CT-like pyramids are approximately four times more likely to innervate interneurons in layer 6 than are CC-like cells and that for CT-like pyramid to interneuron connections the connectivity ratio is very high (approaching 1:2), a finding that contrasts dramatically with the low innervation rate of other pyramidal cells by CT-cells. These pairs and the properties of the EPSPs are summarized in Table 2. The relatively small average first EPSP amplitudes, large proportions of failures of transmission and high CVs resemble those for CT-like inputs onto pyramidal cells (Table 1).
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Layer 6 Interneurons Synaptically Connected to Layer 6 Pyramidal Cells
This section describes the interneurons that were synaptically connected to CT-like pyramidal cells, their morphology, immunocytochemistry and firing patterns (see Table 2 for summary). The dynamic properties of the excitatory connections received by layer 6 interneurons are described in subsequent sections. Of the 12 interneurons receiving excitatory inputs from layer 6 pyramids (eight from CT-like cells, one receiving two such inputs, two from CC-like and two from unidentified pyramidal cells), four were too faintly stained for immunofluorescence or full reconstruction. Four were immunopositive for parvalbumin (Figs 2C, 3C and 4A). One of these was reciprocally connected to a CC-like pyramid (not illustrated), one was postsynaptic to a CT-like cell and presynaptic to two others (Fig. 3) and one was reciprocally connected to one CT-like pyramid and excited by another (Fig. 4). All four were fast spiking, multipolar cells with smooth dendrites and a dense axonal arbour in layer 6. The parvalbumin interneuron innervated by the CC-like pyramid was one of the smaller interneurons recovered (dendritic height 190 µm, width 500 µm), and both its dendrites and axon were almost entirely confined within layer 6. One parvalbumin immunopositive interneuron innervated by a CT-like pyramid had five major dendrites extending into layer 5, but its axon was confined to layer 6 (Fig. 3C). The remaining parvalbumin immunopositive interneurons had axons and dendrites that spanned layers 5 and 6, extending a few branches into deep layer 4 (Figs 2C and 4A). A fifth parvalbumin immunopositive cat layer 6 interneuron is illustrated in Figure 5 for comparison with Figure 4. Although no presynaptic partner was found for this interneuron, it is interesting to note the striking similarity between these two interneurons.
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One somatostatin immunopositive interneuron innervated by a CT-like cell had fast spikes, but with an accommodating firing pattern. It had a bitufted dendritic arbour that extended into lower layer 5 and a sparse axonal arbour spanning upper layer 6 and lower layer 5 (Fig. 6B). Another bitufted interneuron targeted by a CT-like pyramid that was immunonegative for parvalbumin (somatostatin not tested) also had dendrites and a sparse axonal arbour that spanned layers 5 and 6, but had, in addition, seven major dendrites extending into lower layer 4. The final CT-like pyramidal target was a smaller (dendritic width 300 µm), low threshold spiking, sparsely spiny, multipolar, somatostatin immunonegative interneuron with a sparse axonal arbour in layer 6. Finally a large bitufted interneuron innervated by an unidentified pyramid had dendrites spanning layers 5 and 6 with two major branches entering layer 4 and a sparse axonal arbour spanning the deep layers.
The common morphological features of these interneurons were therefore that many were relatively large cells (vertical dendritic length 190–740 µm, horizontal 300–820 µm) with dendrites that spanned layers 5 and 6, some with branches extending into lower layer 4. The parvalbumin immunopositive cells were fast spiking and multipolar. The low threshold spiking (or burst-firing) and accommodating cells were often bitufted and had less dense axonal arbours. Axonal arbours did not extend much beyond dendritic arbours, and were either confined to layer 6 or spanned the deep layers. Overall they innervated layer 6 more densely than layer 5 with only a few minor axonal branches entering lower layer 4.
EPSPs Activated by CT-like Pyramidal Cells Display Paired Pulse Facilitation
In this and subsequent sections the properties of the EPSPs elicited by action potentials in CT-like pyramidal cells are described. The figures are presented on a cell pair by cell pair basis so that the data relating to any one connection are presented together. This does result in the need to reference several figures in discussion of each of the properties studied. We have attempted to provide consistency in the style of presentation, however, to aid comparisons across pairs.
The most surprising finding of this study was perhaps the facilitation displayed by EPSPs activated by CT-like pyramidal cells in all cell types studied, compared with the depression uniformly displayed by connections involving presynaptic CC-like cells. The dynamic properties of ten connections were studied in some detail; four involving postsynaptic pyramidal cells (e.g. Fig. 1A–C,E), four involving parvalbumin immunopositive interneurons (one in rat, three in cat, Figs 2A,B, 3A,B,D–F and 4B–F), one involving a somatostatin immunopositive interneuron (in rat, Fig. 6A,C–G) and one a parvalbumin immunonegative interneuron (Table 2).
In all pairs, the paired pulse ratio was >100% at an interspike interval of 18 ms (average second EPSP amplitude as a percentage of average first EPSP amplitude, range 115–311%). That this facilitation was at least partially of presynaptic origin was demonstrated by the smaller proportion of apparent failures of transmission in response to the second AP than in response to the first (for examples of EPSP amplitude distributions see Figs 3F and 6E) and by a slope >1 in plots of normalized CV–2 against normalized mean EPSP amplitude (M) (Fig. 7). Since, in a binomial distribution, CV–2 is independent of quantal amplitude [CV–2 = np/(1 – p), where n is the number of release sites and p is the probability of release from a given release site], linearly related to the number of release sites and supralinearly related to the probability of release, while M is linearly related to all three parameters (M = npq, where q is the quantal amplitude), a slope >1 indicates a change in probability of release.
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Complex Relationships between EPSP Amplitude and Interspike Interval
The facilitation and its decay could not be described by a single component that was maximum at short interspike intervals and that decayed with increasing intervals as seen at many strongly facilitating connections (Thomson et al., 1993, 1995; Ali and Thomson, 1998; Markram et al., 1998). The range of paired pulse ratios at short intervals was very broad with one CT to CT and two CT to parvalbumin immunopositive interneuron EPSPs displaying paired pulse depression (second EPSP amplitude 40–88% of first) at intervals between 8 and 12 ms. Moreover, even in those connections where facilitation was apparent at all intervals tested, it was rarely maximum at the shortest intervals. It first increased, reaching a population maximum of 213 ± 54% (mean ± SD, 10 pairs: average second EPSP amplitude at a given interval for each pair normalized to the average amplitude of the first EPSP and expressed as a mean for the population) at intervals between 25 and 35 ms, then decreased with increasing intervals.
In addition to this level of complexity, many of the relationships between EPSP amplitude and either the immediately preceding interspike interval or the interval from the first spike were not smooth. These plots included multiple peaks and troughs occurring at intervals between 10 and 15 ms. Similarly, no simple relationship between third EPSP amplitude and the two preceding interspike intervals was apparent. This is illustrated in Figures 1E, 3E and 6D, where third EPSP amplitude is plotted against first and second interspike interval and the three-dimensional plots are rendered as contours. There appears to be no single second interspike interval that results in the largest third EPSPs; rather, there appear to be certain combinations of the preceding two intervals that result in larger third EPSPs and combinations that result in smaller third EPSPs.
Release Site Refractoriness Does Not Explain the Delayed Peak in the Facilitation
A delayed peak in the facilitation could be due either to a slowly developing form of facilitation or to a combination of a rapidly decaying depressing mechanism and a more slowly decaying facilitation, as discussed previously for some facilitating hippocampal synapses (Thomson and Bannister, 1999). To test for the expression of release site refractoriness, second EPSPs that followed failures of transmission (when no release-dependent refractoriness should be present), were compared with second EPSPs that followed release in response to the first spike. In the somatostatin immunopositive interneuron (Fig. 6G) and in two CT to CT pairs, EPSPs that followed first spike failures were on average larger than those that followed first spike EPSPs, indicating that release site refractoriness did indeed contribute to the control of EPSP amplitude in these connections. However, even when only those second EPSPs that followed first spike failures were analyzed, the facilitation was not maximum at the shortest intervals. Therefore, although release site refractoriness was apparent in these connections and clearly reduced second and subsequent EPSP amplitudes at shorter interspike intervals, it could not explain the delayed peak in facilitation. Moreover, release site refractoriness could not be demonstrated at all connections and was therefore either absent or, more probably, only weakly expressed because of the low probability of release. In one CT to CT pair and three CT to parvalbumin immunopositive interneuron pairs, no clear relationship between the amplitudes of the first and second EPSPs was apparent at any interspike interval tested. These connections also failed to demonstrate a clear relationship between second and third EPSP amplitudes indicating that the delayed peak in third EPSP amplitude was also independent of release-dependent depression.
The expression of release independent depression can only be unambiguously demonstrated where depression of second EPSPs is apparent after first spike failures (Thomson and Bannister, 1999). In only three connections was there depression at the short intervals at which this form of depression is expressed and in these, the EPSPs that followed first spike failures were on average larger than average first EPSPs. Thus, while release-independent depression cannot be excluded, it was not sufficiently powerful to explain the delayed peak in facilitation reported here.
Little or No Augmentation of Later EPSPs in Trains
Many of the strongly facilitating EPSPs reported previously (Thomson et al., 1993, 1995; Ali and Thomson, 1998; Markram et al., 1998; Reyes et al., 1998; Beierlein et al., 2003) increased in amplitude with successive spikes in trains, the third EPSP being on average larger than the second, the fourth larger than the third and so on until a plateau was reached. Moreover the augmentation of these later EPSPs decayed very slowly once initiated by a short interval pair or brief burst of spikes. This was not, however, the case for the facilitating EPSPs recorded in this study in layer 6 (see Figs 1C, 2A, 3B,D, 4E,F and 6C,F). In Figure 8 the average amplitudes of EPSPs in short trains are plotted as a percentage of the second EPSP amplitude and the EPSPs elicited by CT-like cells compared with augmenting EPSPs elicited in other interneurons [the grey area representing the mean ± 1 SD for a population of 21 augmenting connections that includes both previously published (Ali and Thomson, 1998; Thomson et al., 1993) and unpublished examples]. Despite the wide range of second EPSP facilitation at the outputs of CT-like cells, third and subsequent EPSPs in trains were consistently less facilitated than second EPSPs. Indeed, in all but the two most strongly facilitating connections, third and subsequent EPSPs were either the same size as, or smaller than first EPSPs.
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Discussion |
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This paper describes the properties of EPSPs elicited in pyramidal cells and interneurons in layer 6 by APs in layer 6 CT-like cells (see Table 3 for comparison with connections in other layers). The most striking findings were the selection by these pyramidal cells of interneurons as postsynaptic targets and the facilitation seen in all types of connections made by CT-like cell axons, particularly when compared with the depression uniformly expressed at the connections made by layer 6 CC-like pyramidal cells (Mercer et al., 2005
). The connections made by CC-like and CT-like pyramids were often recorded in the same experiments and therefore under precisely the same experimental conditions. That layer 6 provides a facilitating excitatory input to layer 4 spiny cells has been demonstrated with paired intracellular recordings in cat visual cortex (Tarczy-Hornoch et al., 1999), and that this may be due to inputs from CT-cells was demonstrated by electrical stimulation of the thalamus in rat somatosensory thalamo-cortical slices (Beierlein et al., 2003). Here we document the dynamic properties of the facilitating inputs from CT-like cells to other layer 6 neurons and demonstrate the striking difference between the outputs of these cells and those of most other pyramidal outputs studied to date. CT-like Cells Preferentially Innervate Interneurons in Layer 6
The numbers of CT-like pyramid to layer 6 pyramid connections described here is small, but this reflects the very low incidence of such connections. Extrapolating from an average probability of any two layer 6 pyramids being connected of 1:27 (Mercer et al., 2005) and the approximately fourfold higher probability of a CC-like cell rather than a CT-like cell being presynaptic, these connections are rare for intralaminar pyramid–pyramid connections (1:80). In striking contrast, CT-like cells were found to be four times more likely to innervate an interneuron than were CC-like cells. Extrapolating again, the connectivity ratio for CT-like cell to layer 6 interneuron could be as high as 1:2 and appeared to involve a range of interneuron classes. While a striking preference for interneurons over excitatory cells as targets in another layer, e.g. layer 3 pyramidal axon innervation of layer 4 interneurons (Thomson et al., 2002a) and layer 6 pyramidal preference for aspiny cells in layer 4 (McGuire et al., 1984), have been described, this degree of target selectivity within the layer of origin has not previously been described.
Slowly Developing Facilitation without Augmentation at CT-like Axon Synapses
All these synapses exhibited facilitation at some interspike intervals, some profound, but the most powerful facilitation was not typically seen at the shortest intervals, as has more commonly been reported for other facilitating connections (Thomson et al., 1993, 1995; Ali and Thomson, 1998; Markram et al., 1998). The possibility that the delayed peak of facilitation resulted from a more rapidly decaying depressing mechanism competing with facilitation at short interspike intervals was explored. However, neither release site refractoriness nor release independent depression, the two mechanisms that account for the earliest post-spike depression, was found to be sufficiently powerfully expressed to account for the delay. It is possible, therefore, that in these synapses a different, more slowly developing form of facilitation is expressed. Despite the unusual time course of facilitation at these synapses, there was a striking difference between the outputs of CT-like cells and of pyramidal cells in other layers onto a range of apparently similar targets (see Fig. 9).
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In addition, although these synapses could facilitate strongly, they did not display augmentation (see Fig. 8). Third EPSPs were smaller than second EPSPs, and later EPSPs in trains could exhibit depression (cf. first EPSPs) following a facilitated second EPSP. Whether there is a causal relationship between the expression of augmentation and of maximal facilitation at the shortest intervals remains to be determined. It is interesting to note, however, that at strongly augmenting connections, the amplitudes of third and later EPSPs in trains were more strongly correlated with the duration of the very first interspike interval in the train than with the interval that immediately preceded them (for discussion, see Thomson, 2003). This suggests that something happens during that brief interval including the first two spikes that primes augmentation and that this does not occur in synapses in which peak facilitation is delayed.
Complex Relationships between Interspike Interval and EPSP Amplitude
In a previous study of pyramid–pyramid and pyramid-parvalbumin interneuron connections, the ‘notch’ filter was apparent as a trough that occurred at 20 ms in plots of EPSP amplitude against interspike interval (Thomson and West, 2003). Plots illustrated here from connections formed by CT-like cell axons also exhibited a trough at intervals close to 20 ms. However, in the present study these plots often included additional troughs resulting in their appearing to include multiple peaks and troughs with repeat intervals at 10–15 ms. We cannot be certain whether or not these apparently ‘oscillatory’ changes in synaptic efficacy also occurred in the connections included in the previous study, but were less apparent there because intervals <10 ms and >30 ms were not studied in as much detail.
To date, none of the many mechanisms that control and fine tune the release of transmitter has been fully elucidated, and we can only speculate as to the possible mechanism underlying what appears to be an oscillatory modulation of transmitter release probability. The most thoroughly documented neuronal oscillatory activity with this sort of frequency is that underlying subthreshold membrane potential oscillations at gamma frequencies (Llinas et al., 1991; Traub et al., 2004), although subthreshold membrane potential oscillations as fast as 100 Hz have largely been reported only in fast spiking interneurons (e.g. Thomson et al., 2002b). Whether pyramidal axon terminal membranes can support membrane potential oscillatory frequencies that their somata typically cannot follow and whether such a subthreshold oscillation would modulate transmitter release are, of course, unknown. The complex relationships seen between third EPSP amplitudes and the first two interspike intervals might, however, suggest the sort of inherent mechanism that, like subthreshold membrane potential oscillations, can be triggered and reset by a preceding AP and its afterhyperpolarization. Whether these apparent oscillations in synaptic efficacy, like subthreshold membrane potential oscillations, dampen with longer intervals was not determined. These cells, with their moderate spike frequency adaptation (cf. e.g. CC-like layer 6 pyramidal cells), will continue to fire at a lower frequency during prolonged depolarizations. However, the relatively transient nature and non-stationary instantaneous frequencies of responses reported for layer 6 cells in vivo suggest that the properties of their outputs during brief, irregular spike trains may be more functionally relevant than their steady state characteristics.
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Conclusions |
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The CT-like cells described here resemble the first order simple cells recorded intracellularly in cat visual cortex and labelled with biocytin (Hirsch et al., 1998
). They form therefore a very distinct cell class, receiving direct input from specific thalamic nuclei and innervating cortical layer 4 and both the GABAergic nRT and ‘specific’ thalamic regions. In contrast to layer 6 CC-cells, their firing patterns and the properties of their outputs will favour more maintained information transfer, possibly with some preference given to the firing patterns that include certain combinations of interspike intervals. |
Acknowledgments |
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This work was supported by the Medical Research Council (UK) and Novartis Pharma (Basel). Our grateful thanks to Dr Andrew Ramage, Department of Pharmacology, Royal Free and UCL Medical School, London for providing the cat tissue.
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