Apical Dendritic Location of Slow Afterhyperpolarization Current in Hippocampal Pyramidal Neurons: Implications for the Integration of Long-Term Potentiation

RESULTS

We have used an indirect route to address the question of the location of active AHP channels. Consider a perfectly space-clamped cell that has a voltage-independent conductance. If the cell is voltage-clamped at a potential different from the reversal potential, a steady current will be measured by the voltage clamp. If the clamp potential is suddenly stepped to a different value, the membrane potential and membrane current will relax to their new values, with a time course determined by the speed of the voltage-clamp circuit, the series resistance of the electrode, and the capacitance of the cell (Jackson, 1992). In an extended structure such as a pyramidal neuron, however, distant dendritic membrane will settle more slowly to the new membrane potential after a voltage step imposed at the soma, because of the cable properties of the dendrites (Jack et al., 1983). As a consequence, relaxation of the membrane current arising in distant membrane will also be slowed, and current of increasingly distant origin will be slowed to an increasing extent (for review, see Spruston et al., 1994).

To test this prediction we took advantage of the anatomy of hippocampal pyramidal neurons, which receive fast inhibitory synaptic inputs from two distinct sets of GABAergic interneurons. One set forms baskets of synapses around the somas of pyramidal neurons (Andersen et al., 1964). The other set forms inhibitory synapses on the apical dendritic tree (Alger and Nicoll, 1982a). These two sets of interneurons can be stimulated selectively in vitro in a transverse hippocampal slice (Alger and Nicoll, 1982a; Pearce, 1993).

CA1 pyramidal neurons were voltage-clamped near −50 mV, and inhibitory postsynaptic currents (IPSCs) were evoked either by stimulation in the cell body region (somatic IPSC) or in s. radiatum (dendritic IPSC; Fig.1A). Both IPSCs were blocked completely by picrotoxin (100 μm;n = 3), confirming that they were mediated by GABA_A receptors. Near the peak of the synaptic current, the membrane potential was stepped to the reversal potential of the IPSC (near −65 mV). After the voltage step, the synaptic current relaxed to its new value with a time course that was faster for the somatic IPSC (τ = 3.5 ± 0.5 msec, mean ± SEM;n = 5) than for the dendritic IPSC (τ = 8.8 ± 0.3 msec; n = 4), consistent with its electrotonically closer location to the recording electrode at the soma (p < 0.001, paired t test; Fig.1B). Note that the somatic relaxation is not instantaneous because the voltage-clamp step at the soma is slowed by the resistance of the electrode in series with the cell capacitance (Jackson, 1992). To determine the relaxation time course for the AHP current, sI_AHP was evoked by a 50–200 msec step to 0 mV from a holding potential of −50 mV. Near the peak of the outward current, the membrane potential was stepped to −65 mV. The current relaxed to its new value with a time constant of 7.4 ± 0.4 msec (n = 8), slower than that for the somatic GABA_A IPSC (p < 0.001), but not significantly different from that for the dendritic IPSC (p > 0.05; Fig. 1C). These results suggest that active AHP channels are not concentrated on the soma.

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Fig. 1.

Current generating the AHP has a more distal distribution than current underlying the somatic GABAergic IPSC.A, Schematic diagram illustrating the experimental setup. The cell was voltage-clamped at the soma at a holding potential of −50 mV. Somatic and dendritic inhibitory postsynaptic currents (IPSCs) were independently stimulated in s. pyramidale and s. radiatum, respectively. Excitatory postsynaptic currents were blocked with CNQX (10 μm) and d-APV (50 μm). B, a andc show superimposed traces with and without a voltage step to the IPSC reversal potential (−65 mV). The stimulus protocol is shown in the inset; arrow indicates synaptic stimulation. The relaxations after the voltage step are shown expanded in b and d and have been fitted with a single exponential for comparison. As expected for an electrotonically distant input, the dendritic IPSC (a, b) relaxed at a slower rate (τ = 10 msec) than the somatic IPSC (c,d; τ = 3.7 msec) recorded in the same cell. C, The AHP current in the same neuron, with and without a voltage step to −65 mV (a). The current relaxation is shown expanded inb and has been fitted with a single exponential. The AHP current relaxed with a time course (τ = 8.3 msec) slower than that of the somatic IPSC, indicating that it must have a large contribution from extra-somatic channels. Calibration: 50 pA, 20 msec (B,a and c); 50 pA, 200 msec (Ca).

To estimate the electrotonic locations on the neuron that would be compatible with these relaxation times, we constructed an equivalent cable model of a CA1 pyramidal cell (Fig. 2C; for details, see Materials and Methods). Varying the distribution of the synaptic conductance over the model cell, we found that the somatic GABA_A relaxation was simulated best when the conductance was placed on the soma of the model (Fig.2A,B, left panels, and 2Ca,filled region). In contrast, the AHP current relaxation was fitted best when the conductance change was either distributed over the equivalent proximal dendrite within ∼200 μm of the soma (Fig.2Cb, filled region) or injected at a point 50–100 μm from the soma (Fig. 2Cb, arrow) in different cells (n = 5; Fig. 2A,B,right panels). The AHP relaxation was clearly not fitted by a conductance concentrated at the soma. For the dendritic GABA_A transient, the conductance had to be distributed further out in the dendrite, or injected at a point 100–125 μm along the equivalent dendrite from the soma, to predict correctly the transient (not shown). In two cells, we were also able to measure a GABA_B IPSC that is thought to have a distant dendritic location (Alger and Nicoll, 1982b). In both cases, the time course of the relaxation was very slow (time constants 16.9 and 16.5 msec), consistent with a distant location for this conductance.

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Fig. 2.

Electrotonic locations of the AHP and somatic GABA_A IPSC channels, estimated using an equivalent cable model of a CA1 pyramidal neuron. A, Somatic IPSC (left) and AHP current (right) recorded in the same cell, when interrupted by a step from the holding potential (−50 mV) to −65 mV. B, The same currents shown expanded, with predictions from the model superimposed (dashed lines). Labels a–c correspond to different channel distributions in the model shown in C. Trace a (soma) best fits the IPSC switchoff, and trace b (proximal apical dendrite) best fits the AHP. C, Schematic diagram showing the equivalent cylinder model used to fit the data. The dendritic trees (apical at right) are shown to scale, using a different scale for the lengths and diameters, but the soma (circle) is not to scale. Filled regions represent the membrane surface that was given a constant conductance density: a, soma only; b, initial 166 μm of the apical dendrite;c, all of the apical dendrite. Adding the same conductance density to the soma made little difference to the predictions of modelsb and c, because the soma has a much smaller surface area than the dendrites. The arrow in bis 75 μm from the soma; injecting the AHP conductance at this point gave an identical switchoff to the distributed conductance inb.

Hippocampal pyramidal neurons are polarized cells with a basal and an apical dendritic tree. We next asked whether the AHP conductance was located preferentially in either or both trees. To address this question, we took advantage of the laminar nature of the excitatory synaptic inputs to pyramidal neurons (Andersen et al., 1980). Because the decay time constant of the excitatory synaptic currents (3–5 msec;Hestrin et al., 1990) is faster than the membrane time constant (20–30 msec; Spruston and Johnston, 1992), the peak amplitude of a subthreshold EPSP reflects the local input impedance rather than the impedance of the whole cell (Carlen and Durand, 1981). EPSPs were evoked in either s. radiatum or s. oriens, alone or during the AHP (Fig. 3A). The peak amplitude of the EPSP in s. radiatum was reduced during the AHP (to 0.81 ± 0.04 of control, mean ± SEM; n = 8), whereas the EPSP evoked in s. oriens was affected marginally (0.98 ± 0.07; n = 8; Fig. 3B; s. radiatum and s. oriens are significantly different: p < 0.05). When the AHP was blocked by application of isoproterenol (10 μm;n = 3) or carbachol (1 μm;n = 2), the shunting of the EPSP after a depolarizing voltage step was blocked completely (Fig. 3C). This result indicates that AHP channels are located preferentially in the apical dendritic tree. These observations were also reproduced by the model (not shown; see Materials and Methods).

What are the physiological consequences of a dendritic location of the AHP? The action of the AHP in shunting the amplitude of local EPSPs is reminiscent of shunting inhibition (Carlen and Durand, 1981). Inhibitory inputs to pyramidal neurons have a significant effect on processes requiring synaptic integration. In particular, blockade of GABA_A inhibition reduces the threshold for the induction of long-term potentiation (LTP) (Wigstrom and Gustafsson, 1983). We thus tested whether activation of the AHP has a similar role. Field EPSPs in area CA1 were evoked at a frequency of 0.1 Hz. After a stable baseline was obtained, a weak tetanus (50 Hz, 0.5 sec) was delivered, which caused a potentiation that returned to baseline over the next 30 min (Fig. 4A). The AHP was then reduced by application of the selective β-adrenergic agonist isoprenaline (Madison and Nicoll, 1986a). This concentration of isoprenaline had no effect on the control EPSP (Fig. 4A) or on the inhibitory postsynaptic potential (n = 2; not shown); however, the same weak tetanus delivered in the presence of isoprenaline produced a sustained potentiation (Fig. 4A;n = 5 slices). When the actions of isoprenaline at β receptors were blocked by co-application of the selective antagonist propranolol, the second weak tetanus also led to decremental potentiation, whereas a subsequent strong tetanus (100 Hz, 1 sec) produced a sustained potentiation (Fig. 4B).

DISCUSSION

In this article we have studied both the electrotonic location of the slow AHP current in hippocampal pyramidal neurons and its possible physiological role. We conclude that this current is localized to the proximal apical dendritic tree (within ∼200 μm from the soma in the equivalent cable model), but relatively little of the current originates on the soma itself. This is based on two lines of evidence. (1) The switchoff of the AHP current was slower than that for a conductance located at the soma (Fig. 2), and (2) the amplitudes of EPSPs evoked in s. radiatum were shunted to a larger extent than those of EPSPs evoked in s. oriens (Fig. 3). If AHP channels were largely somatic, both EPSPs would be equally shunted (Carlen and Durand, 1981; our unpublished simulations). Note that our measurements can localize only active AHP channels; this distribution may reflect the inhomogeneous influx of calcium rather than the distribution of AHP channels themselves.

Although these experiments show clearly that the AHP conductance is not restricted to the somas of CA1 pyramidal cells, our conclusion about its exact dendritic location is obviously model-dependent. We have made three assumptions in our simulations. (1) The neuron is purely passive, apart from the GABA, AHP, or EPSP conductances, (2) its passive properties are uniform, and (3) the conductances are either distributed uniformly over the surface of the model cell or injected at a single point on the equivalent dendrite. Assumption 1 seems to hold for the small (15 mV) hyperpolarizing voltage steps used here, because no time-varying currents, apart from the capacity transients, were apparent during the step without the AHP or IPSC. Even if such currents were present, they are unlikely to have been affected by the presence or absence of a small synaptic or AHP conductance and would have been subtracted out in our protocol. Assumption 2 is commonly accepted (Spruston and Johnston, 1992). Assumption 3 is the most parsimonious one that yields good fits to the data, although other more heterogeneous distributions may fit the switchoffs equally well. Nevertheless, it is qualitatively clear from our simulations that the active AHP conductance cannot be concentrated at the soma, and neither can it be significant in the distal dendritic tree (Fig.2B,C). Note that distance along the equivalent dendrite is not a simple function of dendritic distance in the real cell, because it depends on the branching pattern. The first 100 μm or so of the apical tree of CA1 pyramidal cells, however, is dominated by a single thick process emanating from the soma (Major et al., 1993), and so for this region, dendritic distance will be nearly the same for both real and equivalent cells. It is attractive to suppose that AHP channels are concentrated here so they can act to gate incoming signals from more distal parts of the apical dendritic tree.

Tetanic stimulation of afferent fibers in area CA1 can lead to the generation of a form of synaptic plasticity called LTP. LTP is triggered by depolarization of the postsynaptic cell attributable to summation of EPSPs and unblock of NMDA channels, which allows the influx of extracellular calcium (Bliss and Collingridge, 1993). Tetani delivered at lower frequencies, below the threshold for induction of LTP, give rise to a decremental form of potentiation called short-term potentiation (STP) (Malenka, 1991). During trains of afferent stimuli, summation of EPSPs and generation of action potentials also activate the AHP (Nicoll, 1988; Sah, 1996). This would reduce calcium influx through NMDA channels both directly, via the AHP hyperpolarization, and indirectly, by shunting EPSPs and reducing their depolarizing drive (Fig. 3). Blockade of the AHP during a given tetanus would therefore result in a larger depolarization, a larger calcium influx, and a lower threshold for the induction of LTP (Fig. 4; also see Hopkins and Johnston, 1981). Because activation of the AHP is relatively slow, these experiments lend support to the idea that some minimum duration of calcium influx is necessary to convert STP to LTP (Malenka, 1991;Malenka et al., 1992). It is notable that the threshold for LTP induction is lower in basal than in apical dendrites, a difference that is largely abolished by application of forskolin (Arai and Lynch, 1992). Forskolin increases cyclic AMP levels and blocks the AHP (Madison and Nicoll, 1986b). Thus, this finding is consistent with an apical location for AHP channels.

A number of neurotransmitter systems, including acetylcholine and norepinephrine, act via distinct second messenger systems to block the slow AHP. It is interesting that both cholinergic and monoaminergic transmitter systems have been implicated in the consolidation of memory (Squire, 1987). Activation of these systems and the subsequent blockade of the AHP would facilitate the induction of LTP. Although it remains possible that these second messenger systems interact in other ways with the generation of LTP (Chetkovich and Sweatt, 1993), blockade of the AHP could be one mechanism whereby these transmitters modulate the induction of LTP.

In conclusion, these studies show that the conductance generating the slow AHP in hippocampal pyramidal neurons is located predominantly in the proximal apical dendritic tree. Activation of this conductance during trains of action potentials acts as an adjustable gain control, short-circuiting the apical tree and preferentially shunting synapses impinging on the apical dendritic membrane.