Ionic Mechanisms of Muscarinic Depolarization in Entorhinal Cortex Layer II Neurons
Ruby Klink and
Angel Alonso
Department of Neurology and Neurosurgery, Montreal Neurological Institute and McGill University, Montreal, Quebec H3A 2B4, Canada
 |
ABSTRACT |
Klink, Ruby and Angel Alonso. Ionic mechanisms of muscarinic depolarization in entorhinal cortex layer II neurons. J. Neurophysiol. 77: 1829-1843, 1997. The mechanisms underlying direct muscarinic depolarizing responses in the stellate cells (SCs) and non-SCs of medial entorhinal cortex layer II were investigated in tissue slices by intracellular recording and pressure-pulse applications of carbachol (CCh). Subthreshold CCh depolarizations were largely potentiated in amplitude and duration when paired with a short DC depolarization that triggered cell firing. During Na+ conductance block, CCh depolarizations were also potentiated by a brief DC depolarization that allowed Ca2+ influx and the potentiation was more robust in non-SCs than in SCs. Also, in non-SCs, CCh depolarizations could be accompanied by spikelike voltage oscillations at a slow frequency. In both SCs and non-SCs, the voltage-current (V-I) relations were similarly affected by CCh, which caused a shift to the left of the steady-state V-I relations over the entire voltage range and an increase in apparent slope input resistance at potentials positive to about
70 mV. CCh responses potentiated by Ca2+ influx demonstrated a selective increase in slope input resistance at potentials positive to about
75 mV in relation to the nonpotentiated responses. K+ conductance block with intracellular injection of Cs+ (3 M) and extracellular Ba2+ (1 mM) neither abolished CCh depolarizations nor resulted in any qualitatively distinct effect of CCh on the V-I relations. CCh depolarizations were also undiminished by block of the time-dependent inward rectifier Ih with extracellular Cs+. However, CCh depolarizations were abolished during Ca2+ conductance block with low-Ca2+ (0.5 mM) solutions containing Cd2+, Co2+, or Mn2+, as well asby intracellular Ca2+ chelation with bis-(o-aminophenoxy)-N,N,N
,N
-tetraacetic acid. Inhibition of the Na+-K+ ATPase with strophanthidin resulted in larger CCh depolarizations. On the other hand, when NaCl was replaced by N-methyl-D-glucamine, CCh depolarizations were largely diminished. CCh responses were blocked by 0.8 µM pirenzepine, whereas hexahydro-sila-difenidolhydrochloride,p-fluoroanalog (p-F-HHSiD) and himbacine were only effective antagonists at 5- to 10-fold larger concentrations. Our data are consistent with CCh depolarizations being mediated in both SCs and non-SCs by m1 receptor activation of a Ca2+-dependent cationic conductance largely permeable to Na+. Activation of this conductance is potentiated in a voltage-dependent manner by activity triggering Ca2+ influx. This property implements a Hebbian-like mechanism whereby muscarinic receptor activation may only be translated into substantial membrane depolarization if coupled to postsynaptic cell activity. Such a mechanism could be highly significant in light of the role of the entorhinal cortex in learning and memory as well as in pathologies such as temporal lobe epilepsy.
 |
INTRODUCTION |
Acetylcholine, via muscarinic receptors, is known to exert profound control over the excitability of many CNS neurons by modulating multiple ionic channels. In cortical neurons, excitatory actions of muscarinic agonists have been attributed to suppression of several K+ currents (see Krnjevic 1993
for recent review). These include the voltage- and time-dependent M current, the slow Ca2+-activated K+ current, the fast transient outward current, and a resting "leak" conductance. However, it has been frequently recognized that block of K+ conductances cannot account for all depolarizing actions of muscarinic receptor activation in CNS neurons. Membrane conductance changes observed in hippocampal pyramidal cells during muscarinic depolarization led to the postulate of cholinergic activation of a cationic conductance (Benson et al. 1988
; Segal 1982
). Also, the muscarinic induction of a slow afterdepolarizing potential in hippocampal (Benardo and Prince 1982
; Caeser et al. 1993
; Gähwiler and Dreifuss 1982
; Fraser and MacVicar 1996
) and neocortical (Andrade 1991
; Schwindt et al. 1988
) pyramidal cells has been attributed to the cholinergic potentiation of a Ca2+-activated nonspecific cationic conductance. Moreover, it is well known that in mammalian smooth muscle cells, muscarinic receptor activation causes membrane depolarization by acting primarily on a nonselective cationic conductance that is potentiated by a rise in intracellular Ca2+ concentration ([Ca2+]in) (Benham et al. 1985
; Inoue and Isenberg 1990b
; Pacaud and Bolton 1991
).
In the accompanying paper (Klink and Alonso 1997
) we describe the modulatory action of muscarinic receptor activation on the intrinsic excitability of the two electrophysiologically and morphologically distinct types of projection neurons in layer II of the medial entorhinal cortex (MEC), the stellate cells (SCs) and non-SCs (Alonso and Klink 1993
). Bath application of the cholinergic agonist carbachol (CCh), acting via atropine-sensitive receptors, was found to depolarize both SCs and non-SCs while differentially modulating their oscillatory and firing behavior. In non-SCs, in particular, firing was drastically modified with induction of a slow rhythmic bursting pattern ostensibly driven by a tetrodotoxin (TTX)-insensitive plateau potential.
The present work aims at elucidating the principal ionic mechanism generating the CCh-induced depolarization in entorhinal cortex (EC) layer II neurons. In particular, the induction of slow TTX-insensitive plateau potentials in non-SCs led us to examine whether activation of a Ca2+-dependent cationic conductance plays a role in cholinergic depolarization, and if so, through which pharmacological subtype of muscarinic receptor. Part of this work has already been published in abstract form (Klink and Alonso 1994
).
 |
METHODS |
The materials and methods used for the preparation of EC slices and recording are the same as those described in the accompanying and previous papers (Alonso and Klink 1993
; Klink and Alonso 1997
), with the exception that in the present experiments an interface recording chamber was used. The normal Ringer solution contained (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 2.6 MgSO4, 26 NaHCO3, and 10 glucose, pH adjusted to 7.4 by saturation with 95% O2-5% CO2. In experiments in which the effects of Cd2+, Co2+, Mn2+, or Ba2+ were tested, phosphate and sulfate were omitted to avoid precipitation and divalent cations were maintained at a normal concentration by adjusting the Mg2+ concentration. To block Na+ conductances, TTX (1 µm) was routinely used. N-methyl-D-glucamine (NMDG) Ringer solution consisted of normal Ringer solution or TTX Ringer solution in which NaCl (124 mM) was replaced with NMDG (124 mM). CCh (10 mM dissolved in bath solution) was delivered by pressure-pulse (15-20 psi) applications of variable duration (5-1,000 ms) through a patch pipette (tip diameter 2 µM) positioned in close proximitiy to the entry point of the recording electrode. The muscarinic antagonists atropine, pirenzepine, himbacine, and hexahydro-sila-difenidolhydrochloride,p-fluoroanalog (p-F-HHSiD) were bath applied at the specified concentrations for exactly 20 min at a rate of 2 ml/min before CCh test applications. Antagonists were tested at the following concentations (µM) in the following order: atropine, 0.2, 0.3; pirenzepine, 0.3, 1.3, 0.6, 1.0, 0.8; himbacine, 1.5, 13.0, 10.0; p-F-HHSiD, 2.5, 7.5, 5.0, 4.0. We estimated the "minimal" antagonist concentration necessary to inhibit the CCh response as that concentration that reduced the response to <10% of control. 2-Amino-5-phosphonovaleric acid (AP-5; 100 µM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM),
-methyl-4-carboxyphenylglycine (MCPG; 500 µM), and strophanthidin (20-60 µM) were bath applied for
20 min before test applications of CCh. The apparent slope input resistance of the cells was estimated from the first derivative (with the use of the Origin analysis package) of voltage-current (V-I) relationship plots constructed from the membrane voltage responses to square current pulses.
Strophanthidin, atropine, and AP-5 were purchased from Sigma; pirenzepine and p-F-HHSiD from RBI; and CNQX and MCPG from Tocris Cookson.
Data are given as means ± SD. Significance was tested according to the one- or two-tailed Student's t-test.
 |
RESULTS |
The present study is based on intracellular recordings from 79 neurons in layer II of the MEC to which pressure-pulse applications of the cholinergic agonist CCh were delivered. At the CCh application intervals used in this study (
5 min), multiple test pulses could be delivered to the same neuron without any observable desensitization of the CCh response. Recording selection criteria were as described in the preceeding paper (Klink and Alonso 1997
). Neurons fell into the two previously described morphological and electrophysiological classes (Alonso and Klink 1993
), the SCs (resting potential =
62.8 ± 2.2 mV, mean ± SD; n = 37, 3 of which were morphologically identified) and non-SCs (resting potential =
63.6 ± 2.0 mV; n = 42, 4 of which were morphologically identified).
Characteristics of the depolarization
In control Ringer solution, pulse applications of CCh (5-600 ms; n = 48) resulted in membrane potential depolarizing responses of similar characteristics in SCs and non-SCs (Fig. 1A). For any given neuron, the amplitude and duration of the depolarization increased with increasing CCh pulse durations and typically consisted of a fast rising and a slow decaying phase (Fig. 1B).

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| FIG. 1.
Depolarizing responses of entorhinal cortex (EC) layer II neurons to pressure-pulse applications of carbachol (CCh) in normal Ringer solution. A: at the arrow, CCh was pressure-ejected for the specified duration (in ms). In a stellate cell (SC; top) and a non-SC (bottom), CCh depolarized the membrane potential to firing level. B: pressure-pulse CCh applications of increasing duration produced responses of larger amplitude and longer duration. Depolarizations consisted of a fast rising phase and a slow decaying phase.
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In TTX containing Ringer solution, however, CCh applications (5-1,000 ms; n = 49) resulted in membrane potential depolarizations of apparent larger amplitude and duration in non-SCs than in SCs (Fig. 2A). Also, in non-SCs exclusively, CCh frequently triggered (n = 11 of 30) an initial spikelike response followed by a plateau potential (Fig. 2A, right). In addition, in some non-SCs (n = 6 of 30) the initial spikelike response repeated for a few cycles (2-5) at a slow frequency (0.02-0.2 Hz; Fig. 2B). This oscillatory phenomenon appeared to be cell specific and relatively dose independent. For any given non-SC that displayed the oscillatory response to CCh, increased doses of CCh increased the frequency of the oscillation but not the number of cycles (Fig. 2C). With regard to the amplitude of the CCh depolarizations, in those non-SCs that did not display an initial spikelike response to CCh depolarizations averaged 10.5 ± 11.3 mV (n = 13; pulse duration of 372 ± 309 ms), and in those non-SCs that displayed spikelike potentials the plateau phase averaged 10.2 ± 4.2 mV (n = 11; pulse duration of 194 ± 184 ms). In SCs, CCh depolarizations were smaller, averaging 4.3 ± 2.1 mV (pulse duration of 222 ± 216 ms; n = 19).

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| FIG. 2.
In tetrodotoxin (TTX) Ringer solution, depolarizing responses to CCh are more robust in non-SCs than in SCs. A, left: typical depolarizing response of an SC to 300-ms pulse application of CCh. Note that the membrane depolarized fast by ~6 mV and then repolarized exponentially. A, right: typical depolarizing response in a non-SC. Note the initial spikelike response followed by a plateau phase. B: in another non-SC, CCh triggered large-amplitude spikelike membrane potential oscillations at a slow frequency (0.06-0.02 Hz). C: in another non-SC exhibiting an oscillatory response, increasing CCh doses gradually increases the frequency of the oscillation (from 0.07 Hz for 5 ms CCh to 0.22 Hz for 100 ms CCh) without affecting the number of cycles.
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In the accompanying paper we show that bath applications of CCh block, in both SCs and non-SCs, the slow afterhyperpolarization that follows a train of spikes, and, in non-SCs particularly, promote the development of robust depolarizing afterpotentials. Similarly, when CCh pulse-triggered subthreshold depolarizations were coupled to an additional brief (1-10 s) manual DC depolarization that elicited spiking, the magnitude of the CCh depolarizing response was largely enhanced in non-SCs and also, although to a lesser degree (see next paragraph), in SCs. This potentiation phenomenon was observed in all neurons tested (n = 16; 9 SCs and 5 non-SCs) and is illustrated for a typical non-SC in Fig. 3. Figure 3A shows a 4-mV depolarization that lasted for ~4 min in response to a 500-ms CCh pulse. Figure 3C shows, in a subsequent CCh application of equal duration, that a manual DC depolarization that triggered cell firing applied at the peak of the initial response induced a bistable state in which the cell continued to fire for a very prolonged period of time. This additional plateau depolarization could not be cut short by hyperpolarizing the membrane potential to the initial resting potential (Fig. 3C, *). Also, note that neitherduring the initial subthreshold depolarization (Fig. 3A) nor the potentiated suprathreshold depolarization (Fig 3C) did the apparent input resistance significantly change (see below).

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| FIG. 3.
Cell firing potentiates CCh-triggered depolarization. A: in a non-SC, a pressure-pulse CCh application resulted in a subthreshold, 4-mV depolarization. B: traces at an expanded time scale, taken at arrows marked 1 and 2 in A, showing no significant change in apparent input resistance at the resting membrane potential. C: in the same cell, in a subsequent CCh application of equal magnitude, a manual DC depolarization (horizontal bar) that triggers cell firing is applied at the peak of the intial depolarizing response. This manipulation caused a postdepolarization plateau that sutained firing, and this activity could not be turned off by a short (~10 s) DC hyperpolarization (*). Note that, as in A, membrane depolarization was not associated with a significant change in apparent input resistance at the resting membrane potential.
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During Na+ conductance block with TTX, the magnitude of CCh pulse depolarizations was also greatly potentiated by superimposition of a brief manual DC depolarization to about
40 mV (n = 17) (Fig. 4). It became apparent that during TTX the phenomenon was far more prominent in non-SCs than in SCs, and this was related to the ease by which Ca2+ spiking (Fig. 4B, *) was triggered in non-SCs versus SCs. In SCs, initial CCh depolarizations of 2.6 ± 1.2 mV increased by 2.2 ± 1.8 mV (~100%; n = 8) after ~10-s manual DC depolarizations positive to
50 mV. In non-SCs, initial CCh depolarizations of 2.1 ± 1.4 mV increased by 7.0 ± 3.9 mV (~350%; n = 9) after similar manual DC depolarizations.

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| FIG. 4.
In TTX Ringer solution, the CCh depolarizing response is potentiated in amplitude and duration by DC depolarization that elicits Ca2+ spiking. A: in a non-SC, a pressure-pulse CCh application resulted in a 4-mV depolarization. B: in the same cell, a subsequent application elicited the same depolarization, which was potentiated by 250% (additional 10 mV) after a 10-s manual DC depolarization (horizontal bar) that triggered multiple Ca2+ spikes (*). A series of depolarizing and hyperpolarizing current pulses was also applied to estimate voltage-current (V-I) relationships during the CCh depolarization (see Figs. 5 and 6).
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V-I relationships
As in the case illustrated in Fig. 3, in most neurons tested (17 of 22) the CCh-triggered depolarization was not associated with a measurable change in apparent input resistance at resting membrane potential, and in the other cells only a minor increase in apparent input resistance (8.4 ± 3.6%) was observed. To assess conductance changes more fully, V-I relationships estimated before and during CCh depolarizations were compared. Because layer II neurons possess the time-dependent hyperpolarization-activated inward rectifier Ih (Klink and Alonso 1993
), both peak and steady-state V-I plots were constructed from the voltage responses to inward and outward current steps (Fig. 4).
Figure 5 depicts, for an SC (A) and a non-SC (B), typical peak (left) and steady-state (right) V-I plots constructed before (
,
) and during CCh-triggered depolarization (
,
). The V-I relations of SCs and non-SCs, although differing with regard to the range and magnitude of inward and outward rectification (Klink and Alonso 1993
), were similarly affected by CCh. In all neurons tested (n = 12), CCh induced a shift to the left of the steady-state V-I relations over the entire voltage range explored (
40 to
95 mV; Fig. 5, right). This shift was, however, larger at potentials positive to about
70 mV. With respect to the peak V-I relations (Fig. 5, left), CCh induced an overall increase in their slope with convergence of the V-I relations at
80 to
90 mV. To rule out that these observations were caused by spurious effects due to the mode of application of CCh, we constructed V-I relations after bath applications of CCh (n = 5), and exactly the same pattern was observed.

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| FIG. 5.
V-I relationships of both SCs and non-SCs were similarly affected by CCh. A and B: in an SC (top) and a non-SC (bottom), peak (left) and steady-state (right) V-I relations in control ( , ) and toward the maximum of CCh-triggered depolarizations ( , ). Insets: slope resistance vs. membrane potential (Rin-Vm) plots estimated (as described in detail in METHODS) from the corresponding V-I plots. Note that the most striking CCh action is a parallel shift in the negative direction of the steady-state V-I relation and that the slope resistance was increased by CCh only at potentials positive to about 70 mV. V-I plots were constructed from the initial peak and steady-state voltage responses to ~300-ms current pulses applied from the resting level. CCh was applied under continuous superfusion with TTX.
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To better detect the changes in the slope of the V-I relations induced by CCh, which reflect CCh's actions on the membrane apparent input resistance, we estimated the first derivative of the control and CCh V-I plots (see METHODS) and constructed plots of slope input resistance versus membrane potential (Rin-Vm plots; n = 12; Fig. 5, A and B, insets). In all cases, comparison of control and CCh peak Rin-Vm plots demonstrated an overall increase in the slope input resistance at potentials positive to about
75 mV and no significant change at more negative voltages. With respect to the steady-state Rin-Vm plots, in all cases these also demonstrated an increase in the slope input resistance at potentials positive to about
70 mV, but, in most cases (8 of 12 neurons), a decrease at more negative voltages.
Given the potentiation of the CCh responses by membrane depolarization (and thus Ca2+ influx; Fig. 4), it appeared of interest to look for possible differences in the V-I relationships of nonpotentiated (e.g., Fig. 4A) versus potentiated (e.g., Fig. 4B) CCh depolarizations. As in the case illustrated in Fig. 6, A and B (same neuron as in Fig. 4), in all neurons tested (n = 4), the potentiated steady-state V-I relations (
) demonstrated a selective increase in slope with respect to the CCh nonpotentiated V-I relations (
) at potentials positive to about
75 mV (Fig. 6, left). This voltage-dependent enhancement was more clearly manifested when comparing the CCh nonpotentiated and potentiated Rin-Vm plots (Fig. 6B).

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| FIG. 6.
Potentiation of the CCh response by DC depolarization produced voltage-dependent changes in the CCh V-I relation. A: steady-state V-I relations for the cell and protocols shown in Fig. 4. The CCh V-I relation ( ) exhibited the typical leftward shift relative to the TTX V-I relation ( ). After potentiation of the response by DC depolarization, the CCh V-I relation ( ) exhibited a voltage-dependent increase in slope relative to the nonpotentiated V-I relation. B: steady-state Rin-Vm plots show that the increase in slope input resistance in the potentiated V-I relation is above about 75 mV.
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Several mechanisms could be responsible for the parallel shifts in the V-I relationships induced by CCh, such as 1) stimulation of an electrogenic pump or ion exchanger; 2) changes in a distant dendritic conductance; 3) compound actions of opposite direction in different conductances; or 4) changes in a conductance with a positive reversal potential well out of the range explored. In the following sections we describe experiments aimed to distinguish between these various possibilities.
Effect of various ion channel blockers
In hippocampal pyramidal cells, inhibition of a leak K+ conductance seems to be the main mechanism underlying the cholinergic depolarization observed at rest (Krnjevic 1993
), and the K+ channel blocker Ba2+ has been shown to abolish the CCh-induced depolarization (Benson et al. 1988
). In MEC layer II neurons, Ba2+ (1 mM; n = 3) (not shown) did not diminish the CCh responses, thus suggesting that a mechanism other than K+ conductance block may be primarily responsible for muscarinic-dependent depolarization in EC layer II neurons.
To give stronger support to the above hypothesis, we also tested whether a more powerful block of K+ conductances by intracellular injection of Cs+ ions would block or affect the V-I characteristics of the CCh depolarizing response. In an initial set of experiments we performed intracellular injections of Cs+ alone (3 M; n = 4) or in combination with extracellular Ba2+ perfusion (1 mM; n = 3) to maximize leak K+ conductance block. As in the case illustrated in Fig. 7A, under either of the above experimental conditions, short CCh pulse applications (100 ms, top; 40 ms, bottom) always resulted in very robust membrane depolarizations in all SCs and non-SCs tested (n = 7). Moreover, the changes in the V-I relations induced by CCh in Cs+-injected neurons (Fig. 7B) were qualitatively the same as those observed in neurons recorded with K+-containing electrodes and described in detail above. It thus appears that in MEC layer II neurons (SCs and non-SCs) K+ conductances do not play an important role in the CCh depolarizing response.

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| FIG. 7.
CCh applications during K+ channel block with intracellular Cs+ resulted in robust depolarizations and actions on the V-I relations equivalent to those obtained in normal recording conditions. A: in a non-SC injected with Cs+ (3 M), a 100-ms (top) pressure-pulse CCh application resulted in a robust, long-lasting depolarization; the trace has been interrupted for 4 min, 42 s. Another shorter application (40 ms, bottom) resulted in a slower depolarizing potential of much shorter duration. In both cases no change in apparent input resistance was observed at the control membrane potential. Holding current: 0.15 nA; Ringer solution contained TTX and Ba+ 1 mM. Inset: before CCh application, removal of holding current results in a resting potential of 8 mV. B: in the same non-SC, peak and steady-state V-I relations were constructed for hyperpolarizing pulses from a membrane potential of 62 mV. The peak (top, ) and steady-state (bottom, ) CCh V-I relations exhibited a leftward shift relative to the control TTX V-I relations ( , ).
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As mentioned above, CCh frequently, although not always, induced a small decrease in the steady-state slope resistance at potentials negative to about
70 mV (Figs. 5 and 8,A and B, left). This could be caused by a CCh enhancement of the time-dependent inward rectifier Ih that EC layer II neurons posses (Klink and Alonso 1993
). Indeed, CCh application during exclusive Ih block with extracellular Cs+ (2 mM; n = 3) did not cause any decrease in slope resistance at negative voltages (Fig. 8B, right), although CCh still produced membrane depolarizations (6.3 ± 2.4 mV) undiminished from those in control (6.0 ± 2.8 mV), and a parallel shift in the V-I relation (Fig. 8A, right; n = 3). CCh was also effective in causing robust membrane depolarization and a parallel shift in the V-I relation during combined K+ channel block with intracellular Cs+ (3 M) and extracellular Ba2+ (1 mM), and Ih block with extracellular Cs+ (3 mM, n = 6; Fig. 8, C and D). Under these pharmacological conditions the CCh-induced membrane depolarization cannot be ascribed to a combined Ih enhancement and K+ conductance block by CCh.

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| FIG. 8.
Extracellular Cs+ blocks inward rectification and the CCh-induced decrease in slope resistance below 70 mV, but not the CCh depolarization and leftward shift in V-I relation. A, left: steady-state V-I relation (control TTX; ) exhibits the usual leftward shift with CCh ( ). A, right: during perfusion with Cs+ (2 mM), inward rectification is blocked, and CCh still causes the typical parallel shift on the V-I relation. B: Rin-Vm plots show that the CCh-induced decrease in slope input resistance seen below 70 mV in control Ringer solution (left) is not produced during Cs+ superfusion (right). All plots taken from the same SC. C: in a non-SC, combined potassium conductance and inward rectifier (Ih) block was evidenced by a 168% increase in input resistance and a disappearance of the time-dependent Ih sag (right; holding current 0.13 nA). D: under the same conditions, the steady-state V-I relation (control; ) exhibits the usual leftward shift with CCh ( ; right; same cell as as in C).
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Ca2+ dependence
The data presented above indicate that in MEC layer II SCs and non-SCs, CCh-induced depolarizations and main associated changes on V-I relations do not result from major CCh actions on K+ conductances or Ih. We then tested for the possible Ca2+ dependence of the CCh depolarizations by pulse applying the drug during Ca2+ conductance block with inorganic Ca2+ channel blockers. As in the case illustrated in Fig. 9A, superfusion with low-Ca2+ solutions (0.5 mM) containing either Cd2+ (n = 4), Co2+ (n = 2), or Mn2+ (n = 3) largely abolished (from 8.0 ± 4.5 mV to 1.1 ± 1.2 mV) the depolarizing response to CCh in both SCs (n = 4) and non-SCs (n = 5). This was true for both the direct depolarizing effect and the postdepolarization (potentiated response) following DC membrane depolarization (note the current trace in Fig. 9A). This result suggests that Ca2+ entry and a possible rise in [ca2+]in may be responsible for the CCh-induced depolarization. To test this possibility we performed CCh applicationsin neurons recorded with electrodes containing the Ca2+ chelator bis-(o-aminophenoxy)-N,N,N
,N
-tetraacetic acid (BAPTA)(200 mM). As illustrated in Fig. 9B, 40-50 min after impalement, the depolarizing responses to CCh were abolished in all cells (n = 2 SCs and 3 non-SCs; Fig. 9B, 42
), with the exception of minimal effects triggered when the CCh dose was highly increased (Fig. 9B, 48
). However, this residual CCh action could not be potentiated by DC depolarization. These data suggests that muscarinic receptor activation in MEC layer II neurons causes membrane depolarization by activating a depolarizing conductance and that this activation is dependent on [Ca2+]in. To further eliminate the unlikely possibility that blockade of the CCh responses by Ca2+ channel blockers results from block of TTX-insensitive presynaptic cholinergic receptor-mediated release of a depolarizing neurotransmitter, we applied CCh during block of ionotropic and metabotropic glutamate receptors. A mixture of AP-5 (100 µM), CNQX (10 µM), and MCPG (500 µM) had no effect on the CCh-induced depolarizing responses (n = 3; not shown).

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| FIG. 9.
CCh-induced depolarization and potentiated plateau potentials are dependent on Ca2+ entry and a rise in intracellular Ca2+. A: in a non-SC, a pressure-pulse CCh application produced a 5-mV depolarization that was potentiated to 8 mV after DC depolarization (left). After perfusion with a low-Ca2+ (0.5 mM) + Co2+ solution the initial and postdepolarization response to CCh were totally blocked (middle). The CCh response recovered after washout of the low-Ca2+ + Co2+ solution (right). B: in a non-SC injected with bis-(o-aminophenoxy)-N,N,N ,N -tetraacetic acid (BAPTA; 200 mM), a pressure-pulse CCh application 10 min after impalement produced a robust depolarization of 26 mV (left). Forty-two min after impalement, the same pressure-pulse CCh application did not cause any direct response (middle). Forty-eight min after impalement, a pressure-pulse CCh application of double duration was required to observe a minimal CCh response that could not be potentiated by DC depolarization (right).
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Investigation of the electrogenic Na+-K+ ATPase pump mechanism
The parallel shift in V-I relationships caused by CCh could be due to an activated ionic flux through an electrogenic pump or exchange mechanism, thereby affecting membrane battery without producing changes in membrane resistance. Depolarization induced by cholinergic depression of the sodium pump was investigated, because receptor-mediated, Ca2+-dependent modulation of this ATPase by neurotransmitters has been described in central neurons (Phillis and Wu 1981
). As in the case illustrated in Fig. 10, in all neurons tested (n = 2 SCs and 2 non-SCs), CCh pulse application during superfusion with TTX and strophanthidin (20-60 µM), a reversible inhibitor of the Na+-K+ ATPase (Thompson and Prince 1986
), resulted in an enhanced membrane potential depolarization that persisted until strophanthidin washout. This result indicates that the CCh-induced depolarization is not due to Na+-K+ ATPase block.

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| FIG. 10.
CCh-induced depolarization is enhanced after Na+-K+ ATPase inhibition. A: in a non-SC, a pressure-pulse CCh application resulted in a spikelike depolarization followed by a slowly decaying plateau potential. B: during superfusion with strophanthidin (35 µM), the same pressure-pulse application resulted in in an enhanced plateau that sustained a 2nd spikelike depolarization. Membrane potential depolarization persisted at a steady level until washout of strophanthidin was completed.
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CCh depolarization consists mostly of a Na+-sensitive component
Because in mammalian smooth muscle cells activation of muscarinic receptors induces a cationic current that is largely facilitated by a rise in [Ca2+]in (Benham et al. 1985
; Inoue and Isenberg 1990a
,b
; Sims 1992
), we next considered the possibility that the CCh depolarization results from activation of a nonspecific cation channel with a high permeability to Na+. Na+ dependence was tested for by replacing extracellular Na+ with equimolar concentrations of NMDG+, which does not permeate nonspecific cation channels (n = 3 SCs; n = 5 non-SCs). As illustrated in Fig. 11, in control Ringer solution (A) or TTX Ringer solution (C) CCh applications that evoked robust depolarizations in control were largely reduced but still potentiated by manual depolarization during Na+ substitution (NMDG+ 124 mM; Na+ 26 mM). The blockage was entirely reversible after washout of NMDG+ (Fig. 11C, bottom). It is to be noted that perfusion with NMDG+ did not compromise cell integrity (Fig. 11B).

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| FIG. 11.
CCh-induced depolarization is largely Na+ dependent. A: in control Ringer solution, a pressure-pulse CCh application in a non-SC resulted in a large suprathreshold depolarization (top). During equimolar Na+ (124 mM) substitution with N-methyl-D-glucamine+ (NMDG+), the CCh response was reduced to an initial 1-mV depolarization and a 4-mV plateau potentialfollowing DC depolarization (bottom). B: perfusion with NMDG+ per se largely abolished Na+ spiking and caused a 2-mV hyperpolarization, but did not significantly affect other membrane properties. C: in TTX Ringer solution, a pressure-pulse CCh application in the same cell as in A caused a 20-mV depolarization (top). After perfusion with NMDG+ the same pressure-pulse application produced no initial depolarization and a 4-mV plateau potential following DC depolarization (middle). The response to CCh totally recovered after washout of NMDG+ (bottom).
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Pharmacology of the CCh depolarization
As described in the accompanying paper (Klink and Alonso 1997
), CCh effects on EC layer II neurons were entirely blocked by atropine (300 nM) (also shown here in Fig. 12A) and thus dependent on the activation of muscarinic receptors. To further clarify the muscarinic receptor subtype responsible for the depolarizing action of CCh, we made use of pirenzepine, himbacine, and p-F-HHSiD, reported "selective" antagonists for the pharmacologically defined M1, M2, and M3 receptor subtypes, respectively (Hulme et al. 1990
). Although binding studies have shown that each of the above antagonists has similar affinities for more than one molecularly defined receptor subtype (m1-m5), each of these subtypes shows a unique binding profile, as shown by Dörje et al. (1991)
. Thus, by exploring the CCh depolarization blocking profile for the three selected antagonists, one may narrow down pharmacologically the identification of the receptor subtype involved in the CCh depolarization. To address this issue, we estimated the minimal bath concentration of antagonist (see METHODS) necessary to inhibit the CCh depolarization. For pirenzepine, tested at concentrations from 0.3 to 1.3 µM (n = 6), the minimal effective blocking concentration was found to be 0.8 µM (Fig. 12B); for himbacine (1.5-13 µM; n = 3), 13 µM (Fig. 12C illustrates a residual CCh depolarization for a 10 µM antagonist concentration); and for p-F-HHSiD (1-5 µM; n = 4), 5 µM. We then used these concentrations to estimate the p-F-HHSiD/pirenzepine, himbacine/pirenzepine, and p-F-HHSiD/himbacine selectivity ratios, which were 6.25, 16.25, and 0.38, repectively. Comparison of these ratios with those of the same antagonists estimated from their published affinities at the five cloned muscarinic receptors (Dörje et al. 1991
) indicated a close match with the m1 receptor subtype (p-F-HHSiD/pirenzepine = 3.55; himbacine/pirenzepine = 16.99; p-F-HHSiD/himbacine = 0.21), thus suggesting that the CCh depolarization is largely mediated by the m1 receptor subtype.

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| FIG. 12.
Pharmacolgy of the CCh-induced response. A-C: in 3 different cells, pressure-pulse applications of CCh that elicit large suprathreshold depolarizations in normal Ringer solution (left), and pharmacological block of the responses (right) by 300 nM atropine, 800 nM pirenzipine, and 10 µM himbacine, respectively. Note that the depolarizing response was not fully blocked by a dose of himbacine ~10 times larger than that required for a complete block with pirenzipine.
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DISCUSSION |
The results of the present study indicate that the slow membrane depolarization caused by CCh in EC layer II neurons is primarily generated by the activation of a Ca2+-dependent nonspecific cationic conductance. This observation is in contrast to what is found in hippocampal (Benson et al. 1988
; Madison et al. 1987
) or neocortical principal cells (McCormick and Prince 1986
), where muscarinic receptor activation causes membrane depolarization primarily via block of K+ conductance, although other depolarizing mechanisms, including the one reported here, probably also participate (Blitzer et al. 1991
; Colino and Halliwell 1993
; Guérineau et al. 1995
). The robust Ca2+ dependence of the cholinergic actions in EC neurons may have important functional implications given the role of this structure in learning and memory (Alvarez and Squire 1994
; Squire 1992
) and the role of Ca2+ in neural plasticity (Gosh and Greenberg 1995
; Henzi and MacDermott 1992
).
Despite the differential modulatory actions of CCh on the oscillatory and firing pattern of SCs and non-SCs from EC layer II (Klink and Alonso 1997
), the ionic mechanism underlying the basic CCh depolarizing response appeared to be the same in both cell types. In essence, in both SCs and non-SCs, CCh produced qualitatively equivalent changes on the V-I relations and the response displayed an equivalent ionic dependence and pharmacological profile. However, the CCh depolarizations were of larger amplitude and duration and were more strongly potentiated by Ca2+ entry in non-SCs than in SCs. This must be related to differences in the intrinsic membrane properties between both types of neurons such as, for example, the presence of a very robust subthreshold outward rectifier in SCs but not in non-SCs (Klink and Alonso 1993
). This K+ conductance is not blocked by CCh (Klink and Alonso 1993
) and may effectively counteract the CCh-triggered inward current.
After cloning of the five muscarinic receptors (m1-m5), it was recognized that although the antagonist binding properties of the m2 and m3 receptors correlated well with those of the pharmacologically defined M2 and M3 subtypes, the antagonist binding properties of both m1 and m4 receptors were similar to those of the M1 receptors (Dörje et al. 1991
). The fact that in EC layer II neurons CCh-induced depolarizations were blocked by low concentrations (<1 µM) of pirenzepine (M1 selective) and only by ~10-fold higher concentrations of himbazine [m2 "specific," but with an affinity for m4 similar to that of pirenzipine for m1 (Dörje et al. 1991
)] is a clear indication that the depolarizing responses were primarily (if not exclusively) mediated via the m1 receptor subtype. In fact, m1 receptors are known to have a preferential postsynaptic localization in cortical tissue (Levey et al. 1995
), and it has been recently described that EC layer II projection neurons do express this receptor subtype (Rouse and Levey 1995
). These data are consistent with a direct postsynaptic action of CCh on layer II neurons, as further indicated by our results demonstrating the persistence of the CCh response during synaptic transmission block with TTX, as well as during block of both ionotropic and metabotropic glutaminergic transmission.
CNS neurons possess several K+ conductances that may be subject to modulation by acetylcholine and other neurotransmitters (see Storm 1990
for review). Decrease of several K+ conductances has been shown to be the mechanism mainly responsible for the depolarization caused by muscarinic receptor activation in hippocampal and other cortical pyramidal cells (Benson et al. 1988
; Halliwell and Adams 1982
; Madison et al. 1987
; McCormick and Prince 1986
; Wang and McCormick 1993
). However, in MEC layer II principal cells, K+ conductance block does not seem to play a significant role in the CCh-triggered depolarization, as suggested by the absence of membrane conductance decrease with CCh and the enhanced depolarizing responses during K+ conductance block with with extracellular Ba2+ and/or intracellular application of Cs+.
EC layer II neurons display time-dependent inward rectification mediated by a hyperpolarization-activated cation conductance Ih, and this conductance has been shown to be subject to neuromodulation by neurotransmitters in other neurons (Bobker and Williams 1989
; Colino and Halliwell 1993
; Pape and McCormick 1989
; Schwindt et al. 1992
). Indeed, the small decrease in the steady-state slope resistance below
70 mV that we observed with CCh suggests a minor enhancement of Ih by muscarinic receptor activation. This enhancement could have some contribution to the CCh depolarization. However, we found undiminished CCh responses during Ih block with extracellular Cs+, indicating that the potential Ih contribution to the CCh depolarization, if present, must be minimal. In addition, during K+ conductance block, CCh depolarizations were not accompanied by a membrane conductance increase at the resting level. This result is not compatible with Ih having a significant contribution to the CCh depolarization in EC layer II neurons.
On the other hand, CCh depolarizations were blocked by 1) lowering extracellular Ca2+ concentration and bath applying the Ca2+ channel blockers Cd2+, Co2+, or Mn2+; 2) intracellular Ca2+ chelation with BAPTA; and 3) substitution of extracellular Na+ by NMDG. Taken together, these results suggest that in EC layer II neurons, CCh induces membrane depolarization by activating a cationic conductance, other than Ih, largely permeable to Na+ ions, and that a rise in [Ca2+]in largely due to Ca2+ influx is required for activating or controlling muscarinic receptor stimulation effects. It may be argued that simultaneous Na+ and Ca2+ dependence could denote activation of an electrogenic Na+-Ca2+ exchange mechanism (Eisner and Lederer 1989
). However, because the effectiveness of the Na+-Ca2+ exchange process decreases with increased intracellular Na+ concentration (Fontana et al. 1995
; Harrison and Boyett 1995
), the robust CCh-triggered depolarization during inhibition of the Na+ pump with strophanditin is not compatible with a significant contribution of a Na+-Ca2+ exchanger to the depolarization.
A recent study in insect motoneurons has also described the muscarinic induction of a Na+ current that requires external Ca2+ (Trimmer 1994
), and it is also well known that in mammalian smooth muscle cells, activation of muscarinic receptors induces a cationic current that is largely facilitated by a rise in [Ca2+]in (Benham et al. 1985
; Inoue and Isenberg 1990a
,b
; Sims 1992
). With respect to the mammalian CNS, early studies by Segal (1982)
and Benson et al. (1988)
on CCh actions on hippocampal pyramidal cells already suggested that in addition to block of K+ conductances, muscarinic receptor activation could promote activation of a cationic current. More recently Caesar et al. (1993) and Fraser and MacVicar (1996)
have provided more direct evidence that in hippocampal pyramidal cells, muscarinic agonists activate a Ca2+-dependent cation current that is mainly carried by Na+ and that gives rise to an afterdepolarizing potential and long-lasting plateau potentials. Also, the activation of muscarinic receptors in prefrontal cortex neurons elicits a slow, possibly Ca2+-dependent, cationic current (Andrade 1991
; Haj-Dahmane and Andrade 1995
) that appears to be the primary drive underlying muscarinic depolarization (Haj-Dahmane and Andrade 1996
). In locus coeruleus neurons, muscarine also causes membrane depolarization, in part, by activating a cation conductance that is not Ca2+ dependent (Shen and North 1992
). A similar voltage- and Ca2+-insensitive cationic current activated by muscarinic agonists has also been recently described in the hippocampus (Guérineau et al. 1995
).
It is known that in several cell types the m1 receptor subtype couples efficiently to the activation of phospholipase C (McKinney 1993
), which leads to the production of inositol triphosphate and subsequent mobilization of Ca2+ from intracellular stores (Berridge and Irvine 1989
). This intracellular Ca2+ signal is likely to play an important role in mediating the CCh depolarization. The fact that the CCh responses were largely abolished by Ca2+ conductance block suggests that a cooperativity between calcium influx and calcium store release may be required to produce a substantial depolarizing response (Lo and Thayer 1995
; Shmigol et al. 1995
; further discussed below). It may also be that Co2+, Cd2+, and Mn2+ directly diminished the activated cationic conductance (Inoue 1991
).
Depending on the cell type, the reversal potentials for muscarinic-activated Ca2+-dependent cationic currents have been estimated in the range of
25-20 mV (Colino and Halliwell 1993
; Guérineau et al. 1995
; Shen and North 1992
), which is well positive to the maximum voltage reached in our V-I relations (
40 mV). A positive reversal potential could explain a V-I parallel shift induced by CCh, provided that the current activated in EC neurons is voltage independent. However, CCh did not induce a "strictly" V-I parallel shift in EC neurons, because it also caused an increase in slope resistance at potentials positive to
70 mV. This nonlinearity suggests the presence of an additional voltage-dependent component in the muscarinic-activated current. It appeared that this component was selectively enhanced when the CCh response was potentiated by depolarization (Ca2+ entry; Fig. 6).
CCh response in non-SCs may reflect complex Ca2+ dynamics
In non-SCs, the CCh-triggered depolarization often displayed an oscillatory phenomenon (Fig. 2, B and C). Agonists like acetylcholine that lead to Ca2+ mobilization are known to induce repetitive patterns of [Ca2+]in spikes with a distinct spatial organization (reviewed in Berridge 1993
). Also, in several preparations, periodic increases in [Ca2+]in closely coupled to oscillatory Ca2+-dependent currents have been imaged in response to muscarinic receptor activation (Komori et al. 1993
; Lechleiter et al. 1991
). We suggest that the slow membrane voltage oscillations triggered by CCh in non-SCs may thus reflect complex interactions between spatially and temporally organized Ca2+ release of intracellular stores, the Ca2+-dependent cationic current, and influx of extracellular Ca2+, resulting from activation of a muscarinic receptor subtype coupled to inositol triphosphate production. In fact, the pattern of the CCh-triggered membrane oscillations that we observed followed closely described patterns of [Ca2+]in oscillations in many tissues (reviewed in Berridge and Irvine 1989
). The dynamic and spatial properties of these oscillations vary in form and frequency from cell to cell; however, they are so remarkably constant for individual cells that they have been referred to as "fingerprints" (Berridge and Irvine 1989
). In non-SCs, we have observed a similar fingerprinting phenomenon because repeated applications of CCh in a given cell always produced an identical oscillatory response but this response could be different in different cells.
[Ca2+]in oscillations have been modeled in several cell types (Friel 1995
and references therein). In neurons, the general scheme involves extracellular Ca2+ entry to replenish the oscillator (the actual stores) and a positive feedback mechanism whereby Ca2+ amplifies its own release. The importance of a threshold level of voltage-gated Ca2+ entry for reestablishing Ca2+ mobilization has been demonstrated experimentally (Jaffe and Brown 1994
; Komori et al. 1993
). The preceeding explains the dependence of the CCh depolarization on operative Ca2+ channels that we have observed in layer II neurons, even in the absence of oscillatory responses.
Functional implications
We have provided evidence that a major excitatory effect of muscarinic receptor activation on MEC layer II neurons occurs by induction of a Ca2+-activated cationic conductance perhaps mediated through the m1 receptor subtype. The activation of this conductance is largely potentiated by activity that causes Ca2+ influx. This property introduces a Hebbian-like mechanism whereby muscarinic receptor activation may only be translated into substantial membrane depolarization and repetitive firing if coupled to postsynaptic cell activity. As cells of origin of the perforant path (Ramon y Cajal 1902
), EC layer II neurons occupy a pivotal position in the hippocampal system known to play a crucial role in the making of long-term memories (Alvarez and Squire 1994
; Squire 1992
). Because the cholinergic system is also known to participate in learning and memory, the presently described cholinergic mechanism may be most instrumental in these tasks (Lisman and Idiart 1995
). On the other hand, activity-dependent potentiation of the muscarinic depolarization introduces a positive feedback mechanism both at the cellular and at the network level that may lead to hyperexcitability and epileptogenesis (Dickson and Alonso 1995
). Also, the apparent Ca2+ mobilizing properties of the m1 transduction process and the Ca2+-dependent properties of the activated conductance may cooperate to sustain an elevated [Ca2+]in signal. This signal might be important in mediating plastic changes but may also lead to metabolic compromises and neurodegeneration in the long term, such as that observed in Alzheimer's disease, where EC layer II is most affected (Dunnett 1991
).
 |
ACKNOWLEDGEMENTS |
We thank Drs. C. Dickson, B. Jones, and E. Hamel for comments on the manuscript. Himbacine was a generous gift from W. C. Taylor.
This work was supported by the Canadian Medical Research Council.
 |
FOOTNOTES |
Address for reprint requests: A. Alonso, Dept. of Neurology and Neurosurgery, Montreal Neurological Institute, 3801 University St., Montreal, Quebec H3A 2B4, Canada.
Received 22 March 1996; accepted in final form 10 December 1996.
 |
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Control of firing mode of corticotectal and corticopontine layer V burst-generating neurons by norepinephrine, acetylcholine, and 1S,3R-ACPD.
J. Neurosci.
13: 2199-2216, 1993.[Abstract]