Muscarinic Modulation of the Oscillatory and Repetitive Firing Properties of 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
Abstract
Introduction
Methods
Results
Discussion
References

Klink, Ruby and Angel Alonso. Muscarinic modulation of the oscillatory and repetitive firing properties of entorhinal cortex layer II neurons. J. Neurophysiol. 77: 1813-1828, 1997. Neurons in layer II of the entorhinal cortex (EC) are key elements in the temporal lobe memory system because they integrate and transfer into the hippocampal formation convergent sensory input from the entire cortical mantle. EC layer II also receives a profuse cholinergic innervation from the basal forebrain that promotes oscillatory dynamics in the EC network and may also implement memory function. To understand the cellular basis of cholinergic actions in EC, we investigated by intracellular recording in an in vitro rat brain slice preparation the muscarinic modulation of the electroresponsive properties of the two distinct classes of medial EC layer II projection neurons, the stellate cells (SCs) and non-SCs. In both SCs and non-SCs, muscarinic receptor activation with carbachol (CCh, 10-50 µM) caused atropine-sensitive (300 nM) membrane depolarization. In SCs, the CCh-induced membrane depolarization was associated with subthreshold membrane potential oscillations and "spike cluster" discharge, which are typically expressed by these cells on depolarization. CCh, however, caused a decrease of the dominant frequency of the membrane potential oscillations from 9.2 ± 1.1 (SD) Hz to 6.3 ± 1.1 Hz, as well as a decrease of the intracluster firing frequency from 18.1 ± 1.7 Hz to 13.6 ± 1.3 Hz. In addition, spike cluster discharge was less robust, and the cells tended to shift into tonic firing during CCh. In contrast to SCs, in non-SCs, CCh drastically affected firing behavior by promoting the development of voltage-dependent, long-duration (1-5 s) slow bursts of action potentials that could repeat rhythmically at slow frequencies (0.2-0.5 Hz). Concomitantly, the slow afterhyperpolarization (sAHP) was replaced by long-lasting plateau postdepolarizations. In both SCs and non-SCs, CCh also produced conspicuous changes on the action potential waveform and its afterpotentials. Notably, CCh significantly decreased spike amplitude and rate of rise, which suggests muscarinic modulation of a voltage-dependent Na+ conductance. Finally, we also observed that whereas CCh abolished the sAHP in both SCs and non-SCs, the membrane-permeant analogues of adenosine 3',5'-cyclic monophosphate, 8-(4-chlorophenylthio)-adenosine-cyclic monophosphate and 8-bromo-adenosine-cyclic-monophosphate, abolished the sAHP in SCs but not in non-SCs. The data demonstrate that cholinergic modulation further differentiates the intrinsic electroresponsiveness of SCs and non-SCs, and add support to the presence of two parallel processing systems in medial EC layer II that could thereby differentially influence their hippocampal targets. The results also indicate an important role for the cholinergic system in tuning the oscillatory dynamics of entorhinal neurons.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The entorhinal cortex (EC) is crucially located within the parahippocampal region, providing bidirectional connections between the neocortex and the hippocampal formation (Witter et al. 1989). Studies of cortical connectivity have shown that the superficial layers (II and III) of the EC receive convergent inputs from a variety of associational cortices and that this convergent information is then funneled to the hippocampal formation via the perforant path (Insausti et al. 1987; Ramon y Cajal 1902). In turn, the hippocampal formation feeds back onto the deep layers of the EC, which give rise to projections that remarkably reciprocate the input channels. Neuropsychological studies in rats, monkeys, and humans (Alvarez and Squire 1994; Squire 1992) have demonstrated a role of the neocortical-hippocampal-neocortical circuitry in high-order cognitive processes, particularly in learning and memory. The parahippocampal region and hippocampal formation together form the medial temporal lobe memory system, which is primarily involved in the early stages of declarative memory formation. The central involvement of the EC in human cognition is underscored by the pathological changes targeting the superficial EC layers in conditions such as Alzheimer's disease (Braak and Braak 1991; Van Hoesen et al. 1991) and schizophrenia (Arnold et al. 1991).

Acetylcholine is critically involved in promoting cortical activation and plasticity (Dunnett and Fibiger 1993; Winkler et al. 1995). Cholinergic innervation of the cerebral cortex originates almost exclusively from a continuum of cells located in the basal forebrain (Amaral and Kurz 1985; Rye et al. 1984; Shute and Lewis 1967), and the discovery that these cholinergic neurons degenerate in Alzheimer's disease (Geula and Mesulam 1994; Price 1986) has suggested that they play an important role in mechanisms of learning and memory (Dunnett and Fibiger 1993). In the EC, basal forebrain cholinergic afferent fibers densely innervate layer II in the rat (Alonso and Köhler 1984), primate (Alonso and Amaral 1995), and human (De Lacalle et al. 1994). It is the layer II EC cells that give rise to the most prominent component of the perforant path, which activates the intrinsic hippocampal circuitry (Steward 1976; Witter and Groenewegen 1984). In the EC layer II and several hippocampal subfields, the activation of the cholinergic system during various wake states and rapid-eye-movement sleep promotes the development of a very prominent rhythmic population activity known as the "theta rhythm" (Alonso and García-Austt 1987; Bland 1986; Dickson et al. 1994; Mitchell and Ranck 1980; Mitchell et al. 1982). The theta rhythm has been suggested to be instrumental in implementing synaptic plasticity (Huerta and Lisman 1993, 1996; Larson and Lynch 1986), a basic mechanism for learning.

It has been proposed that the EC might achieve its memory functions through synchronizing mechanisms (Damasio 1990) by which the activity patterns of the multiple cortical inputs that converge on EC neurons may be temporally coordinated for the production of a memory representation (Alonso and Klink 1993). Synchronization and temporal coordination of neuronal population activity can best be produced via intrinsic oscillatory mechanisms in some of the network elements (König et al. 1995; Lampl and Yarom 1993; Llinàs et al. 1991). Interestingly, the electrophysiological study of medial EC (MEC) layer II neurons (Alonso and Klink 1993; Alonso and Llinàs 1989; Klink and Alonso 1993) demonstrated that the most abundant MEC layer II cell type, the stellate cells (SCs), displays robust intrinsic rhythmicity.

The present study focuses on the analysis of cholinergic actions on oscillatory and repetitive firing properties in MEC layer II neurons as a step in understanding the cellular basis of cholinergic involvement in rhythmic population activities and learning and memory in the EC. It has been shown, in many cortical regions, that muscarinic receptor activation produces a long-lasting depolarization and enhancement of the intrinsic excitability of principal neurons (Krnjevic 1993) and also regulates synaptic transmission (Cox et al. 1994; Hasselmo and Bower 1992). The present study demonstrates profound and differential neuromodulatory actions of muscarinic receptor activation on SCs and pyramidal-like cells of EC layer II that collectively may facilitate network oscillations and bursts of activity. The results also support the view that two parallel channels of information processing with different integrative properties exist in layer II of the MEC (Alonso and Klink 1993). Part of this work has already been published in abstract form (Klink and Alonso 1992).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Brain slices (350 µM) were derived from male Long-Evans rats (150-250 g) following standard procedures. Dissection methods, recording procedures, and intracellular staining methods have been described in detail elsewhere (Alonso and Klink 1993). Briefly, after Nembutal (30 mg/kg) anesthesia animals were decapitated, the brain was rapidly removed, and a block of tissue containing the retrohippocampal region was placed in a cold (6-10°C) oxygenated Ringer solution (see below). Horizontal slices (350 µm) were cut with the use of a vibratome and then allowed to recover at room temperature for >= 2 h in oxygenated incubation chambers. For recording, a single slice was transferred to a recording chamber, submerged at 35 ± 1°C (mean ± SD), and superfused with a solution containing (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. Intracellular glass electrodes were filled with 2-3 M potassium acetate (tip resistance 40-120 MOmega ), 2-3 M potassium chloride (40-80 MOmega ), or 1-2% biocytin in 2-3 M potassium acetate (100-200 MOmega ). Histochemical procedures followed for biocytin revealing were as described elsewhere (Alonso and Klink 1993). To block Na+ conductances, tetrodotoxin (TTX, 1 µM) was used. Carbachol (CCh, 10-50 µM) was added to control or TTX Ringer solution and bath applied for variable periods of time ranging from 30 s to the entire duration of the experiment. In some experiments, 5-hydroxytryptamine (30 µM), 8-(4-chlorophenylthio)-adenosine-cyclic monophosphate (8-CPT-cAMP, 100-300 µM), or 8-bromo-adenosine-cyclic-monophosphate (8-Bromo-cAMP, 1 mM) were bath applied to compare with CCh actions. Atropine (300 nM, to block muscarinic responses) was superfused for 20 min before test applications of CCh. All drugs were purchased from Sigma except for TTX (Calbiochem).

Electrophysiological parameters were measured as follows: action potential waveform was quantified in normal Ringer solution and during CCh by eliciting a single spike from the control resting membrane potential in response to a threshold current pulse; spike amplitude and fast afterhyperpolarizing potential (AHP) were measured relative to threshold; spike rate of rise was taken as spike amplitude divided by duration from threshold to peak. The dominant frequency of subthreshold membrane potential oscillations was estimated by computing power spectra of 3-s traces digitized at 3 kHz. The area under input-output relation plots was calculated by integrating the corresponding curves with the use of the graphics and data analysis package Origin. Data are given as means ± SD. Statistical significance was tested according to the one- or two-tailed Student's t-test.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

The present study is based on a database of 60 neurons from layer II of the MEC, recorded for >= 30 min in stable conditions and tested with bath applications of the cholinergic agonist CCh. Of these neurons, 10 were also intracellularly injected with biocytin for morphological characterization. In control Ringer solution, all neurons had a resting membrane potential negative to -55 mV, an input resistance >15 MOmega , and a spike amplitude >60 mV. As previously found (Alonso and Klink 1993), neurons fell into two distinct electrophysiological and morphological categories, the SCs (n = 34) and the non-SCs (n = 26), whose main characteristics are summarized in Fig. 1. In brief, SCs have multiple primary dendrites and a widely branching upper dendritic tree (Fig. 1A, top). Their subthreshold voltage responses to current pulses are markedly nonlinear, exhibiting pronounced time-dependent rectification in both the depolarizing and hyperpolarizing direction (Fig. 1B, top). The most remarkable electrophysiological characteristic of SCs is their ability to generate subthreshold, sinusoidal-like, membrane potential oscillations that reach their maximal amplitude and have a frequency of ~8 Hz at about -54 mV (Figs. 1C, top, and 3A). On DC depolarization, tonic firing is not readily induced in SCs. Instead, a 1- to 3-Hz repetitive bursting pattern, constituted by nonadapting spike clusters, emerges (Fig. 4A). The majority of non-SCs has a pyramidal-like morphology, with one or two major apical dendrites and a profuse basal dendritic tree (Fig. 1A, bottom). Their responses to current pulses are easily distinguished from those of SCs by a less pronounced time-dependent rectification and delayed firing on threshold depolarization (Fig. 1B, bottom). Moreover, non-SCs never exhibit persistent rhythmic subthreshold oscillations or spike clusters and, when DC depolarized, readily go into tonic firing (Fig. 1C, bottom).


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FIG. 1. Basic morphological and electroresponsive properties of medial entorhinal cortex (MEC) layer II neurons. A: camera lucida reconstruction of a stellate cell (SC; top) and a non-SC (bottom). B: voltage-current relationships in SCs (top) and non-SCs (bottom). Note the pronounced time-dependent rectification in response to inward and outward current pulses in the SC and delayed firing in the non-SC. C: membrane voltage behavior on close to rheobase DC depolarization in an SC (top) and a non-SC (bottom). Note the robust subthreshold sinusoidal-like membrane potential oscillations inthe SC.


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FIG. 3. CCh decreases the frequency of the SC subtreshold oscillations. A: subthreshold membrane oscillations at different levels of DC polarization in control Ringer solution (left) and during CCh (right). B: power spectral plots of top traces in A; CCh reduced the dominant frequency of subthreshold membrane potential oscillations from 10 to 6 Hz.


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FIG. 4. CCh modulates spike clusters. A: in control Ringer solution, an SC exhibits the typical spike clustering phenomenon on DC depolarization. Note that even with injection of 0.54 nA (top), tonic firing was not induced and clustering was still prominent. B: 30 µM CCh depolarized the cell to a level at which spike clusters develop (0.00 nA, middle), and further minor manual DC depolarization induces tonic discharge (0.07 nA, top). C: plots of instantaneous intracluster firing frequency vs. interspike interval (ISI) number, for a series of clusters of 3-5 spikes in control (left) and CCh (right). Control plot corresponds to A, top (0.54 nA); CCh plot corresponds to B, middle (0.00 nA).

CCh (10-50 µM) applied at rest (SCs: -61.6 ± 2.2 mV; non-SCs: -63.2 ± 3.6 mV) in normal Ringer solution (n = 52) caused membrane depolarization in most SCs (27 of 29) and in all non-SCs (23). This depolarization was large enough to reach firing threshold in 7 of 29 SCs and in 5 of 23 non-SCs. CCh applied during Na+ conductance block with TTX (1 µM) depolarized all SCs and non-SCs (n = 46). No statistically significant difference (P > 0.1) was found in the magnitude of a 30 µM CCh-induced depolarization between SCs (4.3 ± 2.7 mV; n = 24) and non-SCs (3.7 ± 1.8 mV; n = 18). In a large majority of neurons (50 of 60), the CCh-induced depolarization was not accompanied by a measurable change in apparent input resistance at the control resting membrane potential (e.g., Fig. 2). In the remaining neurons, the apparent input resistance increased by 19.7 ± 6.4%. Again, there was no significant difference between SCs and non-SCs with regard to the magnitude of the input resistance increase (SCs: 18.5 ± 5.3%, n = 6; non-SCs: 21.2 ± 9.2%, n = 4). The CCh-induced depolarization as well as all other CCh neuromodulatory actions on EC layer II neurons did not develop in the presence of atropine (300 nM; n = 5, not shown), therefore indicating their mediation through muscarinic receptors.


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FIG. 2. Depolarizing response of SCs to cholinergic receptor activation. A: 30 µM carbachol (CCh), applied at rest for the duration indicated by bar, depolarized the membrane potential to the subthreshold oscillatory voltage level, without measurable change in apparent input resistance at the control resting level. B: traces at an expanded time scale taken at the time points indicated by small arrows in A. Before CCh application (left), the cell fired at the anodal break from a resting membrane potential of -59 mV.

SCs: oscillatory properties

As illustrated in Figs. 2 and 3, the depolarizing action of CCh brought the membrane potential of the SCs to within the voltage range in which subthreshold oscillations develop. It was a consistent observation that during CCh the voltage threshold for the ocurrence of subthreshold oscillations was shifted by ~1-3 mV in the negative direction (Fig. 3A). More notably, in all SCs CCh substantially decreased the frequency of the subthreshold oscillations by ~30%. Similarly to the case illustrated in Fig. 3, A and B, the mean frequency of the subthreshold oscillations in control Ringer solution was 9.2 ± 1.0 Hz (measured at -53.5 ± 0.8 mV; n = 26), and it decreased to 6.3 ± 1.1 Hz (measured at -55.2 ± 1.3 mV) during CCh application.

The discharge in repetitive spike clusters typical of the SCs was also affected by CCh. Essentially, as in the typical case illustrated in Fig. 4, A and B, the phenomenon of spike clusters was less robust during CCh in the sense that the cells switched more readily from cluster discharge into tonic firing on membrane depolarization. Concomitant with the CCh-induced decrease in the frequency of the subthreshold oscillations described above, there was a parallel decrease in the instantaneous intracluster firing frequency (Fig. 4C). Although the instantaneous intracluster firing frequency for clusters of 3-5 spikes averaged 18.1 ± 1.7 Hz in control, it decreased to 13.6 ± 1.3 Hz in CCh (n = 6).

SCs: spike train and action potential properties

In SCs, the trains of spikes evoked by depolarizing current steps exhibit pronounced adaptation that, for large current pulses (>0.5 nA), leads to cessation of firing 100-200 ms after pulse onset (Alonso and Klink 1993). This adaptation is primarily caused by the buildup of a Ca2+-dependent slow AHP (sAHP) (Klink and Alonso 1993). We consistently observed that CCh application markedly reduced the spike train adaptation evoked by 250- to 350-ms depolarizing current pulses, which caused repetitive firing to be present for the entire duration of the pulse (Fig. 5A). Concomitant with the adaptation block, CCh also abolished the sAHP following the current-pulse-triggered spike trains (Fig. 5B) without affecting a medium-duration AHP (*).


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FIG. 5. CCh action on SC spike train adaptation and slow afterhyperpolarization (sAHP). A: spike trains evoked by a 1.0-nA current pulse in control (left) and during CCh (right). Note the cessation of firing toward the end of the pulse in control compared with the regular dischargue during CCh. B: control (left) and CCh (middle) traces from A at a condensed time scale, and superimposition (right); CCh selectively blocked the sAHP without affecting the medium afterhyperpolarizing potential (AHP) (*).

The actions of CCh on the SC input-output relationships were investigated in detail by analyzing plots of instantaneous firing frequency versus injected current (f-I relationship; Fig. 6A), instantaneous firing frequency as a function of time after onset of the current pulse (f-t relationship; Fig. 6B), and number of spikes as a function of injected current (Sn-I relationship; Fig. 6C), in control and CCh-containing Ringer solution (n = 8). In control, all SCs displayed a bilinear f-I relationship for the first interspike interval (ISI), and a simply linear relationship for subsequent ISIs (Fig. 6A). During CCh, in most SCs (7 of 8), the first ISI f-I relationship became linear, but its average slope (340.3 ± 202.7 Hz/nA) was not significantly different from that of the primary segment of the control f-I relationship (362.4 ± 97.1 Hz/nA). This means that CCh did not significantly increase firing frequency at the begining of the current pulse. In fact, on average, the slope of the f-I relationship for the second to the fourth ISIs was not significantly affected by CCh; it could slightly decrease in some cells (Fig. 6A) or increase in others. CCh-induced increases in firing frequency (reflecting block of adaptation) were, however, always present after the fourth ISI and were particularly manifest for large current amplitudes. This CCh action was especially apparent when comparing the f-t plots in control and CCh. Figure 6B depicts a typical f-t plot for a 1.0-nA current pulse in control (black-square) and CCh (black-triangle), demonstrating that CCh only had an obvious effect on adaptation after the fourth ISI. Note in fact that during CCh (black-triangle) the cell fired at a constant rate for the second half of the currrent pulse, whereas in control (black-square), firing frequency monotonically decreased and the cell ceased firing after ~125 ms of pulse onset.


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FIG. 6. CCh actions on SC input-output relations. A: instantaneous firing frequency vs. injected current (f-I) in control (left) and CCh (right). For the 1st ISI, the bilinear f-I relation in control became simply linear in CCh without a significant change in mean slope; for the 2nd and 3rd ISIs, f-I slopes were slightly smaller in CCh. B: instantaneous firing rate vs. time (f-t) for a 240-ms pulse of 1.0 nA in control (black-square) and during CCh (black-triangle). CCh had only a clear effect on adaptation after 50 ms of pulse onset. Dotted lines: double-exponential fits to data points. C: spike number vs. injected current (Sn-I). CCh (black-triangle) increased spike numbers at all injected current intensities, with a more pronounced effect at higher amplitudes. All plots taken from the same cell.

It has been shown that changes in the area under the Sn-I curve represent a good quantitative estimate of changes in adaptation (Barkai and Hasselmo 1994). Figure 6C illustrates the Sn-I plot for the same cell as in Fig. 6, A and B, and demonstrates that, for each current step, the cell fired more spikes in CCh (black-triangle) than in control (black-square), especially at high stimulus intensities. The same was observed in all neurons tested (n = 8). Integration of the Sn-I curves gave corresponding Sn-I areas for control and CCh. On average, CCh largely increased the Sn-I value by 88.8%, from 5.4 ± 1.9 to 9.7 ± 2.8 spikes per pulse × nanoamperes.

CCh also produced clear modulatory effects on the single spike afterpotentials and the action potential waveform itself (Fig. 7). In SCs, the action potential is followed by both a fast AHP (Fig. 7A, black-triangle) and a medium AHP (Fig. 7A, *) separated by a depolarizing afterpotential (Alonso and Klink 1993). As in the case shown in Fig. 7A, in all SCs, CCh reduced the fast AHP (by 37 ± 12%; n = 22) without affecting the medium AHP, and in 56% of SCs, the depolarizing afterpotential was no longer manifest in CCh. Figure 7B shows in detail the alteration in action potential waveform that was typically produced by CCh. It can be observed that CCh produced a small positive shift in spike threshold, and decreased spike amplitude, rate of rise, and rate of repolarization. Although reduction in fast AHP and rate of repolarization may be explained by CCh block of repolarizing potassium conductances (Hille 1992), the other changes cannot be ascribed to this mechanism. We have thus quantified these changes. In 88% of neurons (n = 22), CCh increased the spike threshold by ~1 mV, decreased spike amplitude by 8.9 ± 2.8%, and decreased spike rate of rise by 24.4 ± 7.7%. In the remaining 12% of neurons, only the above mentioned decrease in fast AHP and a concomittant decrease in rate of repolarization were observed.


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FIG. 7. CCh actions on SCs action potential and afterpotentials. A: CCh reduced the fast AHP (black-triangle) without affecting the medium AHP (*). Insets: action potential at an expanded time scale. B: in another SC, details on action potential waveform. CCh raised firing threshold by ~1 mV, reduced amplitude by ~10%, and reduced spike rate of rise by ~30%. Superimposition labeled Control + CCh shows CCh trace delimited by arrows.

SCs: subthreshold Na+-dependent potentials

SCs exhibit pronouced "sags" in membrane potential in response to low-amplitude hyperpolarizing and, particularly, depolarizing current pulses applied from rest [(Alonso and Klink 1993); Fig. 8 in this paper]. We demonstrated in a previous study that although the hyperpolarization-induced sag response is primarily generated by a time dependent inward vectifier (Ih-like current), the depolarization-induced sag and anodal break potentials are largely due to Na+ conductance activation (Klink and Alonso 1993). Given the above-described modulatory actions of CCh on the Na+-dependent action potential and subthreshold oscillations, we also investigated whether CCh modulated the subthreshold time-dependent voltage responses to current pulses. Figure 8A (Control) illustrates a prominent slow depolarizing potential followed by a sag to a lower level in response to an 0.1-nA current pulse (top), and a hyperpolarization-induced sag and associated rebound potential in response to a -0.4-nA current pulse (bottom). Remarkably, during perfusion with CCh (30 µM) the slow depolarization-induced potential was largely reduced whereas the hyperpolarization-induced sag was not significantly affected (Fig. 8A, CCh 30 µM and Control + CCh). The CCh block of the slow depolarizing potential completely recovered after washout (Fig. 8A, Washout). This result, which was observed in all neurons tested (n = 18), may be explained by a CCh block of a voltage-dependent Na+ current. Consistent with this idea, CCh produced no detectable action on the subthreshold time-dependent responses to depolarizing (Fig. 8B) or small hyperpolarizing (not shown) current pulses during Na+ conductance block with TTX (n = 6). Interestingly, in most cases we observed that the CCh reduction of the Na+-dependent subthreshold responses developed before any measurable steady membrane potential depolarization took place.


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FIG. 8. CCh blocks a tetrodotoxin (TTX)-sensitive subthreshold potential in SCs. A: voltage responses to low-amplitude subthreshold depolarizing (top) and hyperpolarizing (bottom) current pulses in control, during CCh (30 µM), and in washout. Note (superimposition at right, labeled Control + CCh) that CCh selectively blocked a voltage "sag" response triggered by depolarization and the rebound potential at the break of the hyperpolarizing current pulse. B: in another SC, the depolarization-induced sag response suppressed by CCh (top) is also fully blocked by TTX (bottom left); addition of CCh to TTX (bottom middle) did not affect the voltage responses to the current pulses.

Non-SCs: oscillatory properties

In contrast to SCs, non-SCs do not display rhythmic subthreshold membrane potential oscillations and, when DC depolarized, readily go into tonic firing (Figs. 1C and 10). This typical regular spiking was drastically affected by CCh. Remarkably, in most non-SCs (16 of 26), bath application of CCh induced a voltage-dependent repetitive bursting discharge that could emerge either spontaneously, by the direct depolarizing action of CCh, or on additional current injection. The bursts typically consisted of long-duration clusters of spikes with an accelerando-decelerando pattern separated by a silent interval with little hyperpolarization. We considered that a cell displayed a repetitive bursting discharge when, every time it was depolarized to about -55 mV, a sequence of at least three slow bursts emerged. The CCh concentrations required to evoke this bursting response were relatively low, usually in the range of 10-30 µM. A typical example is illustrated in Fig. 9. In this neuron, bath application of CCh caused a small (~4 mV) membrane depolarization to about -60 mV (Fig. 9A). From this initial CCh resting level, application of an 0.1-nA short current pulse fired the cell and triggered a regenerative slow bursting pattern sustained by a very long-lasting (~1 min) depolarizing afterpotential (Fig. 9A; traces within the rectangle are expanded in Fig. 9B). Individual bursts lasted for 2-5 s and repeated about every 6 s. Once the membrane potential returned to the resting CCh level, further minimal DC depolarization (Fig. 9A, right-arrow) resumed the slow bursting activity which then became self sustained. Figure 9C illustrates the initial burst from Fig. 9B (enclosed in rectangle) at an expanded time base and demonstrates that the burst considerably outlasted the duration of the pulse with firing frequency peaking toward the end of the pulse (at ~114 Hz). Subsequent spontaneous bursts, although not stereotyped, were rather consistent in pattern in terms of firing frequency and burst duration. As in this case, in all other non-SCs, CCh spontaneous bursts were always initiated by a ramplike depolarization that triggered spiking at a gradually accelarating frequency that peaked toward the end of the burst, then decreased rapidly with an abrupt membrane repolarization. For any given cell and membrane potential, individual burst durations and interburst intervals could vary considerably (up to ~100%), however. In those non-SCs that did not display a repetitive bursting sequence during CCh (10 of 26), low-amplitude current pulses that reached threshold nonetheless triggered a long-lasting burst riding on a plateau depolarization. CCh-triggered postdepolarizations and plateau potentials were also observed in all non-SCs tested in the presence of TTX (1 µM, n = 18; Fig. 9D), thus indicating that this phenomenon does not depend on the activation of the persistent Na+ current that these neurons possess (Klink and Alonso 1993).


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FIG. 10. Voltage dependence of CCh-induced bursting pattern. A: in a non-SC, in normal Ringer solution, increasing levels of DC depolarization (from bottom to top) elicited tonic firing at corresponding increasing frequencies. B: in the same neuron, CCh (30 µM) caused bursting discharge in a voltage-dependent manner. Bursting can be turned off by DC hyperpolarization (not shown) or by substantial DC depolarization (top; 0.11 nA).


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FIG. 9. CCh promotes bursting in non-SCs. A: CCh (30 µM) depolarized the cell by ~4 mV without measurable change in apparent input resistance at the control resting level. Application of a 230-ms, 0.1-nA depolarizing pulse (up-arrow  under current trace) triggered a transient slow repetitive bursting behavior that could be switched on permanently by further DC depolarization. B: enlargement of boxed area in A. Triggered bursts were sustained by a long-lasting (50 s) plateau potential; individual bursts lasted for 2-5 s and occurred about every 6 s. C: enlargement of boxed area in B. The initial burst considerably outlasted the duration of the current pulse. D: afterpotentials to current pulse depolarization in normal Ringer solution (left), in the presence of CCh only (30 µM, middle), and in the presence of CCh (30 µM) and TTX (1 µM) to block Na+ conductances (right).

The ability of CCh to switch the firing pattern of the non-SCs from regular spiking to rhythmic bursting, and the voltage dependence of the bursting mode, are further demonstrated in Fig. 10. Figure 10A illustrates the typical regular discharge of a non-SC in control Ringer solution at different levels of membrane potential depolarization and Fig. 10B illustrates the actions of DC membrane depolarization on the CCh-induced bursting discharge in the same neuron. Note that membrane depolarization first decreased the bursting frequency and increased the duration of the bursts (0.09 nA), and then gradually switched the firing pattern into rhythmic single spiking (0.10 and 0.11 nA). For comparison, note that the 0.30-nA level in Fig. 10A (Control) and the 0.11-nA level in Fig. 10B (CCh) correspond to approximately the same mean firing frequency (~15 Hz; note different time scale).

Non-SCs: action potential properties

As in the SCs, the action potential in non-SCs is followed by a fast AHP and a medium AHP (Alonso and Klink 1993). Similarly to the SCs, in all non-SCs examined (n = 15), CCh reduced the fast AHP by 26 ± 15% without affecting the medium AHP. Also, as in the SCs, in non-SCs CCh-induced changes in action potential waveform were manifest. CCh increased threshold by ~1 mV, and decreased amplitude by 18.6 ± 9.5% and rate of rise by 40.0 ± 12.6%. The CCh-induced reduction in action potential amplitude and rate of rise was larger than that observed in SCs, but the difference did not reach statistical significance (P > 0.001).

In non-SCs the sAHP is not modulated via adenosine 3',5'-cyclic monophosphate

The finding that CCh promotes long-lasting postdepolarizations raises the question of whether muscarinic activation unmasks an afterdepolarizing potential or induces it. If, in addition to a slow Ca2+-dependent K+ current, a slow inward current is also significantly activated during the sAHP, the reversal potential for the sAHP should be different from the K+ equilibrium potential. We estimated sAHP reversal potential in both SCs and non-SCs by evoking trains of spikes at different membrane potentials. The estimated sAHP reversal potential was approximately the same in SCs and nonSCs (-95 mV, n = 2, and -94 mV, n = 3, respectively), and this approximates the expected K+ equilibrium potential for the extracellular K+ concentration used in our experiments. This observation suggest that an inward curent does not significantly contribute to the sAHP.

We also considered that if CCh unmasks an afterdepolarizing potential by blocking the sAHP, then other agents that block the sAHP should, in principle, also promote postdepolarization and bursting activity in non-SCs. In addition to acetylcholine, monoamines are also known to efficiently block the sAHP in hippocampal pyramidal cells and other brain neurons (McCormick and Williamson 1989; Nicoll 1988). However, although in hippocampal pyramidal cells modulation of the sAHP by acetylcholine involves the Ca2+-calmodulin kinase II pathway (Müller et al. 1992), modulation of the sAHP by monoamines (including serotonin, norepinephrine, histamine, and dopamine) is mediated via adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (Pedarzani and Storm 1993, 1995). For this reason, we examined whether membrane-permeant analogues of cAMP could block the sAHP without unmasking any afterdepolarizing potential. To our surprise, bath application of 8-CPT-cAMP (n = 4; Fig. 11A, left 2 panels) and 8-Bromo-cAMP (n = 2; not shown) did not affect the sAHP or spike train adaptation (insets) in non-SCs. Consistent with this observation, we also found that serotonin had no effect on the non-SC sAHP (Fig. 11A, right 2 panels; n = 6), while still causing membrane hyperpolarization via a direct action (not shown). On the other hand, similar to what was observed in other neurons, 8-CPT-cAMP (n = 4; Fig. 11B, left 2 panels), 5-hydroxytryptamine (n = 4; Fig. 11B, right 2 panels), and 8-Bromo-cAMP (n = 1; not shown) always greatly reduced spike adaptation (insets) and totally abolished the sAHP in SCs. This result demonstrates that SCs and non-SCs are not only different with regard to their basic electrophysiology and morphology (Alonso and Klink 1993), but also with respect to their pharmacology and metabolism.


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FIG. 11. sAHP can be modulated via the adenosine 3',5'-cyclic monophosphate (cAMP) 2nd-messenger pathway in SCs, but not in non-SCs. A: in non-SCs, neither 300 µM 8-(4-chlorophenylthio)-adenosine-cyclic monophosphate (8-CPT-cAMP) (left 2 panels) nor the monoamine 5-hydroxytryptamine (5HT, 30 µM; right 2 panels) affect the sAHP or spike train adaptation (insets). B: in SCs, 200 µM 8-CPT-cAMP (left 2 panels) and 5HT (right 2 panels) block the sAHP and adaptation (insets). In all cases, the sAHP was evoked by generating a train of 27 spikes at 70 Hz with 2-ms suprathreshold current pulses. The spike trains in the insets were generated by depolarizing constant current pulses. In both SCs and non-SCs, 5HT caused a ~2-mV hyperpolarization (not shown).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The intent of the present study was to evaluate the possible cholinergic modulation of the intrinsic excitable properties of neurons from layer II of the MEC. Our findings demonstrate that the firing characteristics of the two types of projection neurons present in this crucial cortical layer, the SCs and the non-SCs, are prominently yet differentially influenced by CCh. Whereas muscarinic receptor activation modulated in a rather subtle manner the subthreshold membrane voltage oscillations and spike clusters typical of the SCs, muscarinic actions were very robust on the non-SCs and could transform their regular tonic firing into a slow repetitive bursting discharge. In addition, our data also demonstrate that SCs and non-SCs differ not only with regard to their electrophysiology and morphology, as previously reported (Alonso and Klink 1993; Klink and Alonso 1993), but also with respect to their metabolism, because, for example, the sAHP can be modulated by cAMP-dependent protein kinase in SCs, but not in non-SCs.

Despite the differential effects on firing pattern, application of CCh (30 µM) produced, via direct muscarinic receptor activation, a modest membrane depolarization of similar amplitude (~4 mV) in both SCs and non-SCs. This issue is discussed in detail in an accompanying manuscript (Klink and Alonso 1997), in which we provide evidence that the mechanism underlying the CCh-induced depolarization largely relies on the activation of a voltage- and Ca2+-dependent nonspecific cationic conductance in both SCs andnon-SCs.

Muscarinic actions on SCs

Muscarinic receptor activation facilitated the development of oscillatory activity in SCs by depolarizing the membrane potential to within the voltage range at which the characteristic Na+-dependent subthreshold membrane potential oscillations of these neurons develop. That subthreshold oscillations persisted during relatively high concentrations of CCh (30-50 µM) clearly indicates that a classical M current is not essential for their generation (Gutfreund et al. 1995; Klink and Alonso 1993). Membrane potential depolarization and development of subthreshold 5- to 10-Hz (theta-like; Alonso and Llinàs 1989) oscillations in the SCs by muscarinic receptor activation is consistent with the well-established role of the basal forebrain cholinergic system in promoting theta rhythmicity in hippocampal cortex and EC (Alonso and García-Austt 1987; Green and Arduini 1954). We observed that although the subthreshold oscillatory frequency of the SCs is ~9 Hz when DC depolarized in control conditions, the frequency of the spontaneous subthreshold oscillations decreased to ~6 Hz during CCh. This frequency decrease agrees with the low-frequency range of the cholinergic-dependent theta rhythm (Bland 1986). In neocortical neurons, however, muscarinic receptor activation appears to facilitate the frequency of Na+-dependent intrinsic subthreshold oscillations (Metherate et al. 1992). It may be that in the neocortex the cholinergic system is biased to promote high-frequency (gamma range) oscillatory activity that constitutes the so called low-voltage fast activity typical of cortical activation (Alonso et al. 1996; Buzsàki et al. 1988; Vanderwolf 1988). In a manner similar to that in which different ionic conductances in different neurons may determine the same pattern of activity (Alonso and Llinàs 1989, 1992), the same modulatory system (in this case cholinergic) in different neurons and networks (archicortex and periallocortex vs. neocortex) may well produce distinct patterns of activity.

The phenomenon of spike clustering in SCs persisted, although it diminished, during CCh superfusion. This result is somewhat paradoxical because we observed that, as described in many other cortical neurons (Constanti and Sim 1987; Cox et al. 1994; Hasselmo and Bower 1992; Lancaster and Nicoll 1987; Madison and Nicoll 1984; McCormick and Prince 1986; Schwindt et al. 1988; Tseng and Haberly 1989), muscarinic receptor activation blocked adaptation and the Ca2+-dependent sAHP following current-pulse-triggered spike trains. The observation is consistent, however, with our previous finding that spike clusters also remain during Ca2+ conductance block (Klink and Alonso 1993) and suggests that although the sAHP contributes to cluster generation, it is not an essential factor.

Our analysis of the SC input/output relationship as assessed by 250- to 350-ms current pulse injection produced results similar to those previously reported in other cortical neurons (Barkai and Hasselmo 1994). CCh had no or a small effect, on the initial firing rates (up to about the 4th ISI) but the block of adaptation at later times resulted in a substantial increase in the total number of spikes per current pulse. In no case did we observe, however, an aceleration in firing frequency or the production of an afterdepolarization, as occurred in the non-SCs (discussed below). In a recent biophysical model of rat piriform cortex, simulated cholinergic block of adaptation has been shown to implement an appropriate dynamic for learning within the network (Barkai et al. 1994; Liljenström and Hasselmo 1995). In the case of the EC, this mechanism may also work and operate in conjunction with the cholinergic facilitation of oscillations.

Muscarinic receptor activation produced manifest modulatory actions on the action potential waveform of the SCs, and also equivalent effects on the action potential of the non-SCs. The most obvious effect of CCh was the block of the fast AHP with a concomitant broadening of the spike at the base and decrease in the action potential rate of repolarization. In addition, CCh also decreased the rate of rise and amplitude of the action potential. Such conspicuous cholinergic modulatory actions on the action potential do not appear to occur in neocortical neurons (Cox et al. 1994; McCormick and Prince 1986). In hippocampal pyramidal cells, one early study (Segal 1982) reported a decrease in a fast AHP by CCh, but this observation was not repeated (Benardo and Prince 1982; Lancaster and Nicoll 1987). However, in a recent study reevaluating cholinergic modulation of the action potential in hippocampal neurons (Figenschou et al. 1996), results identical to ours have been reported, namely positive shift in threshold, increase in spike duration, and reduction in spike amplitude and rate of rise.

We have previously demonstrated that the fast AHP in EC layer II neurons is generated by a Ca2+-dependent K+ conductance (Klink and Alonso 1993). Because it is well known that in many neurons muscarinic agonists can depress Ca2+ currents (Allen and Brown 1993; Berhein et al. 1991; Gähwiler and Brown 1987; Howe and Surmeier 1995), it seems likely that the fast AHP block may be an indirect result of Ca2+ current reduction by CCh. Alternatively, it may also be that muscarinic receptor activation can directly modulate a fast Ca2+-dependent K+ conductance (Figenschou et al. 1996).

Of particular interest was the observed consistent decrease in the spike's amplitude and rate of rise by CCh. This result may be explained by a reduction in the Na+ conductance(s) underlying action potential generation, because the slope of the rising phase of the action potential is directly related to the instantaneous depolarizing current density. Consistent with this interpretation was also the observed decrease by CCh of the slow Na+-dependent subthreshold potentials to current pulses (slow depolaring potential and anodal break potential; Fig. 8). It has been shown that, like voltage-gated Ca2+ and K+ channels, Na+ channels can be targets for neuromodulation (Cohen-Armon et al. 1989; Gershon et al. 1992; Li et al. 1992, 1993). In fact, recent studies described a decrease in peak Na+ current by CCh in acutely isolated neostriatal neurons (Howe and Surmeier 1994) and hippocampal neurons (Cantrell et al. 1996). Regarding the possible mechanism of action of CCh on the Na+ current, it is well known that in many cells muscarinic receptor stimulation can increase intracellular Ca2+ concentration via inositol triphosphate-mediated mobilization of Ca2+ from intracellular stores (Berridge 1993), and it has recently been shown that an increase in intracellular Ca2+ concentration can decrease total Na+ current by reducing the fraction of Na+ channels available for activation (Bulatko and Greeff 1995). Muscarinic modulation of the voltage-gated Na+ conductances in SCs may also be related to the observed changes in the frequency of the Na+-dependent subthreshold oscillations by CCh.

Induction of repetitive bursting in non-SCs

Muscarinic receptor activation had a profound action on the firing pattern of the non-SCs. Although in control conditions current-pulse-triggered spike trains displayed moderate adaptation and were followed by a sAHP, during muscarinic activation firing frequency accelerated during the train and the sAHP was replaced by a prolonged postdepolarization that could sustain robust burst firing. In addition, during muscarinic activation, DC depolarization could induce a repetitive bursting sequence in most cases. Because the slow bursting oscillations produced by CCh were voltage dependent, being blocked by hyperpolarization and changing in frequency to become inactivated with depolarization, the underlying mechanism must be intrinsic to the cells and not synaptically mediated. We did not test whether muscarinic activation could trigger slow afterpotentials and bursting activity during Ca2+ conductance block, because in non-SCs spike repolarization is for the most Ca2+ dependent and these neurons possess a robust noninactivating Na+ conductance. In consequence, in non-SCs, Ca2+ conductance block results by itself in the production of robust Na+-dependent postdepolarizations (Klink and Alonso 1993). However, we observed that during TTX-sensitive Na+ conductance block, CCh always produced the development of prolonged postdepolarizations that could sustain Ca2+ spiking. In an accompanying paper (Klink and Alonso 1997) we provide further evidence that the CCh-induced depolarization and slow postdepolarizations are, in fact, largely mediated by a Ca2+-dependent nonspecific cationic current. We thus propose that CCh induces bursting by blocking the slow Ca2+-dependent K+ current and activating a Ca2+-dependent nonspecific cationic current that sustains a regenerative process once the intracellular Ca2+ concentration rises over a certain threshold. Consistent with this interpretation is the fact that slow bursts during CCh could be triggered by the firing of one or a few Na+ spikes that allow Ca2+ entry (Klink and Alonso 1993). Cholinergic induction of a slow postdepolarization has also been described in other cortical neurons and similarly attributed, in most cases, to the cholinergic activation of an inward nonspecific cationic current (Andrade 1991; Caeser et al. 1993; Hasuo and Gallagher 1990; Schwindt et al. 1988). In EC layer II non-SCs, however, the phenomenon appears to be more robust than in other brain neurons, giving rise to a very prominent slow repetitive bursting activity.

In hippocampal pyramidal cells in slices, CCh application (>= 50 µM) can induce synchronized oscillatory bursting at 4-10 Hz (MacVicar and Tse 1989), or, during block of ionotropic glutamatergic and GABAergic transmission, very slow (0.002-0.05 Hz) long-lasting (8-30 s) repetitive bursts (Bianchi and Wong 1994). In contrast to the results presented in the present study, CCh-induced hippocampal bursts are synaptically mediated. In a few experiments (<10%), however, we did observe a transient synaptically mediated rhythmic population activity of EC neurons at the begining of the CCh perfusion. We recently found that this epileptic-like activity develops with nearly every CCh application and has a persistent character when an interface slice chamber is used. The properties and basis of this network oscillation are currently under investigation (Dickson and Alonso 1995).

cAMP pathway modulates the sAHP in SCs but not in non-SCs

We found that in the SCs the sAHP could efficiently be blocked by bath application of the cAMP membrane-permeant analogues 8-CPT-cAMP and 8-Bromo-cAMP or by the classical monoamine serotonin, as typically observed in other neurons (Pedarzani and Storm 1993 and references therein). However, interestingly and to our surprise, in the non-SCs none of these agents had any effect on the spike frequency adaptation or the sAHP. It thus appears that in the non-SCs the signaling cAMP pathway is not operative for sAHP ion channel modulation. To our knowledge, this apparent inefficiency of the cAMP pathway has not been reported in any other neuronal type. For the moment, we can only speculate that the EC layer II network appears to be specifically designed so that activation of the cholinergic and not of any monoaminergic system can induce bursting activity. Considering the nodal location of EC layer II within the temporal lobe, and given that non-SCs posses robust voltage-gated slow inward currents and lack, in contrast to SCs, pronounced Ca2+-independent repolarizing K+ currents (Klink and Alonso 1993), it does make sense that few neurotransmitters may block the Ca2+-dependent sAHP in non-SCs to prevent the production of paroxysmal-like depolarizations and epileptogenic activity. The ascending cholinergic system thus appears specifically suited to switch the EC network into an oscillatory bursting mode, and at the cost of risking the production of epileptic activity (Dickson and Alonso 1995).

Functional implications

The present study demonstrates that muscarinic receptor activation exerts a profound and differential modulatory action on the two morphologically and electrophysiologically distinct projection cell types in EC layer II, the SCs and the non-SCs. We have further demonstrated that the non-SCs, unlike any other CNS neuron studied so far to our knowledge, do not have their sAHPs modulated via the cAMP messanger pathway. These data add further support to the existence of two parallel output systems of information in EC layer II that could act very differently on their hippocampal targets.

The function of the EC has to be understood from its position as an interface between the hippocampus and the neocortex and considering the established role of the neocortical-hippocampal-neocortical circuit in memory function. Neurons in EC layer II are particularly important in this (declarative) memory system because polysensory information from multiple cortical association areas converges on them and their output represents the almost exclusive source of sensory information to the hippocampus (Insausti et al. 1987; Ramon y Cajal 1902). The basal forebrain cholinergic system, which plays a role in memory (Dunnett and Fibiger 1993; Winkler et al. 1995), densely innervates EC layer II neurons. Electrophysiologically, at the macroscopic level, activation of the cholinergic system produces theta rhythm in the EC and hippocampus, and rhythmic theta-related events appear to influence memory mechanisms and function (Huerta and Lisman 1993, 1996; Winson 1978). In the EC layer II, the genesis of the theta rhythm appears also to be directly related to the rhythmic subthreshold oscillations of the SCs (Alonso and García-Austt 1987; Alonso and Llinàs 1989).

Lampl and Yarom (1993) recently showed that subthreshold membrane potential oscillations can operate as powerful synchronizing devices in a frequency-dependent manner. Hopfield (1995) has also recently shown that a network of neurons having subthreshold membrane potential oscillations can exquisitely encode information by the use of action potential timing with respect to an ongoing collective oscillatory pattern. The muscarinic induction of intrinsic oscillations and modulation of oscillatory frequency in the EC SCs might thus be of crucial importance in setting the proper temporal dynamics for the coordination of the multiple cortical inputs that converge on these neurons and for the ultimate generation of sensory representations within the hippocampal region.

    ACKNOWLEDGEMENTS

  We thank Drs. C. Dickson and B. Jones for comments on the manuscript.

  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.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society