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Cholinergic stimulation enhances cytosolic calcium ion accumulation in mouse hippocampal CA1 pyramidal neurones during short action potential trains

Steven M. Beier and Michael E. Barish

MS 0568 Received 6 January 2000; accepted after revision 31 March 2000.

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. Acetylcholine is a regulatory cofactor for numerous activity-dependent processes of central nervous system development and plasticity in which increases in cytosolic calcium ion concentration ([Ca²⁺]_cyto) couple membrane excitation to cellular changes. We examined how cholinergic receptor activation affects temporal and spatial aspects of increases in [Ca²⁺]_cyto during short trains of action potentials in hippocampal CA1 pyramidal neurones. Membrane-impermeant Ca²⁺-sensitive dye was introduced into the cytosol during whole-cell recordings, and Ca²⁺-dependent fluorescence was recorded from somatic, nuclear and proximal dendrite regions with high temporal resolution.

2. In all neuronal compartments, the cholinergic agonist carbachol (5 µM) increased resting [Ca²⁺]_cyto and the maximum [Ca²⁺]_cyto attained during a short action potential train. Carbachol also slowed the recovery of [Ca²⁺]_cyto towards resting levels. The largest increases in peak cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) were seen in the dendrite and apical cell body, while relaxations of the carbachol-induced increase in [Ca²⁺]_cyto showed greater prolongation in the nucleus and basal cell body.

3. Most significantly, the difference between Ca²⁺ signals recorded before and during exposure to carbachol consistently showed a monotonic rise and smooth fall in all cell compartments, suggesting that the increase in [Ca²⁺]_cyto associated with each action potential was not altered by carbachol. Consistent with this view, changes in Ca²⁺ signalling were not accompanied by changes in action potential waveforms.

4. The effects of carbachol were partially reversed by simultaneous exposure to atropine, or partially inhibited by inclusion of heparin in the intracellular solution, indicating the involvement of muscarinic acetylcholine receptors and InsP₃-sensitive Ca²⁺-release channels.

5. Our data indicate that carbachol-induced slowing of [Ca²⁺]_cyto relaxations after each action potential results in enhanced accumulation of Ca²⁺ in the cytosol in the absence of changes in action potential-driven Ca²⁺ entry. By modulating the time course of Ca²⁺ signals, cholinergic stimulation may regulate the activation of Ca²⁺-dependent intracellular processes dependent on patterns of [Ca²⁺]_cyto changes.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

In neurones, increases in the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) are a key mechanism by which action potential generation is coupled to numerous intracellular processes. Recent studies have shown that the cellular actions of [Ca²⁺]_cyto changes in neurones and other cells are determined not only by their amplitude, but also by their origin, frequency, duration and waveform (Gu & Spitzer, 1995; Dolmetsch et al. 1997; De Koninck & Schulman, 1998; Finkbeiner & Greenberg, 1998; Li et al. 1998). These observations implicate both spike frequency patterning and patterns of cytosolic Ca²⁺ signalling in cellular regulation.

The neurotransmitter acetylcholine is an important modulator of central nervous system function in general and hippocampal function in particular. In central neurones, a number of the processes of development and plasticity regulated by activity, including long term potentiation (LTP), synapse maturation and dendritic protein synthesis, employ acetylcholine as a regulatory co-factor (Bear & Singer, 1986; Feig & Lipton, 1993; Segal & Auerbach, 1997). Activation of cholinergic receptors by itself increases [Ca²⁺]_cyto by multiple mechanisms including Ca²⁺ release from intracellular stores (Kudo et al. 1988; Wakamori et al. 1993; Seymour-Laurent & Barish, 1995; Irving & Collingridge, 1998), and their activation also increases the amplitude of intracellular Ca²⁺ transients elicited by depolarization (Müller & Connor, 1991; Tsubokawa & Ross, 1997; present report).

Our goal in this study was to examine, on a per spike basis, how cholinergic receptor activation affects temporal and spatial aspects of the increases in [Ca²⁺]_cyto elicited during short trains of action potentials. By following Ca²⁺ signals after individual action potentials, we were able to distinguish between changes in Ca²⁺ transients linked to Ca²⁺ entry, and changes linked to Ca²⁺ clearance from the cytosol after Ca²⁺ influx. We observed that in the presence of the cholinergic agonist carbachol, activity-dependent increases in [Ca²⁺]_cyto were enhanced and prolonged due to a slowing of Ca²⁺ clearance from the cytosol, rather than by increased Ca²⁺ entry during action potentials. We suggest that by modulation of the temporal integration of Ca²⁺ signalling, cholinergic stimulation may regulate the activation of Ca²⁺-dependent intracellular processes.

Some of these observations have previously been published in abstract form (Beier & Barish, 1997).

	METHODS

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Preparation of hippocampal slices

Acute slices of hippocampus were prepared from postnatal day 11-15 Swiss Webster mice. Neonates were lightly anaesthetized with halothane, decapitated, and their brains rapidly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF; see below) continuously bubbled with carbogen (95 % CO₂-5 % O₂), following procedures consistent with NIH guidelines and approved by our institutional research animal care committee. After cooling for 5-10 min, the brain was trimmed just anterior to the cerebellum, mounted on an already-prepared cooled agar block using ultra-low gelling temperature agarose (Sigma type IX-A) prewarmed to 30°C, and then placed in the refrigerator (4°C) until the agarose gelled. Coronal slices (300 µm thickness) were then cut on a Vibratome (Technical Products International, St Louis, MO, USA) with the brain bathed in an ice-liquid ACSF slush, and individual slices were transferred to a mesh basket submerged in ACSF continuously bubbled with carbogen. Slices were maintained at 35·5°C for 45 min, and thereafter at room temperature until they were used.

Fluorescence imaging

Neurones were filled with Oregon Green BAPTA 488-1 (50 µM; Molecular Probes) from the recording pipette for 20 min before data acquisition commenced. Dye-filled neurones were imaged using a Zeiss × 63 Achromat water-immersion objective (0·9 NA) on an Axioscope FS microscope (Carl Zeiss, Thornwood, NY, USA). Data were acquired at room temperature.

The Ca²⁺-sensitive dye was excited using a 75 W xenon bulb and a 470 nm (40 nm bandpass) filter, and reflected using a 505 nm dichroic mirror (Chroma, Brattleboro, VT, USA). Excitation was controlled by a Lambda-10 filter changer (Sutter Instruments, Novato, CA, USA) and shutter (Uniblitz, Vincent Associates, Rochester, NY, USA). To minimize photodynamic damage, a neutral density filter was used to restrict the intensity of excitation light, and the dye-filled cell was illuminated only during data collection. Preliminary experiments established that photodynamic damage was manifest as increases in action potential duration, leading eventually to spontaneous prolonged (duration, 1-3 s) depolarizations and accompanying massive increases in [Ca²⁺]_cyto.

Emitted fluorescence was filtered at 515 nm (long pass) and passed through a × 4 TV magnifier (Zeiss) to a Hamamatsu intensified CCD camera (Hamamatsu Corporation, Bridgewater, NJ, USA). The output of the camera was digitized at 512 pixels × 512 pixels per frame and 30 frames per second using VideoProbe hardware and software (ETM Systems, Petaluma, CA, USA), before being transferred to a personal computer host for storage and analysis.

Quantitative information was extracted from each image sequence using routines written in the ALI language of Optimas (Media Cybernetics, Silver Spring, MD, USA). Pseudocoloured matrices of extracted data (e.g. Figs 2 and 3) were constructed using Transform (Fortner Software, Sterling, VA, USA).

In our analysis, Ca²⁺-sensitive fluorescence (F) was normalized to initial resting fluorescence (F₀) as (F - F₀)/F₀, or F/F₀ (which was evaluated as (F/F₀ - 1)). F₀ for each pixel was calculated as the average value of F for 20 consecutive frames during a period preceding delivery of the first stimulation pulse. The approximate [Ca²⁺]_cyto corresponding to each value of F/F₀ was calculated as:

	RESULTS

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Overview

Only neurones with resting potentials negative to -60 mV were used in this study; the resting potentials of most neurones were -65 mV or more negative. During recordings the resting potential was adjusted to -65 mV by passing small hyperpolarizing or depolarizing currents through the recording pipette. Action potentials were generated by passing short (duration, 2 ms) depolarizing currents; during each run the standard stimulation regime was four pulses delivered at 4 Hz. This protocol was within the range of action potential frequencies displayed by CA1 pyramidal neurones in situ (Otto et al. 1991), and emphasized the accumulation of Ca²⁺ in the cytosol. Image acquisition commenced 75-125 ms before delivery of pulses, and lasted for a total of 6·7 or 8·3 s (i.e. either 200 or 250 frames were collected during each run).

For each experiment, two control runs were first collected, with 5 min separation, to assess the stability of the preparation. Then, in 'test' experiments, 5 µM carbachol, a non-hydrolysable cholinergic agonist, was perfused over the slice for 10 min before acquisition of the experimental run. In 'sham' control experiments, the two control runs were followed by an additional run after a 10 min interval.

A fluorescence image of a pyramidal neurone filled with Oregon Green BAPTA 488-1 is shown in Fig. 1. In this neurone the nucleus is the bright spherical structure displaced towards the top of the soma, and the apical dendrite exits towards the top right corner. The four cellular areas considered in this study (basal cell body, nucleus, apical cell body, dendrite) are labelled. The pipette can be seen entering from the bottom of the field. Superimposed on the image is the line along which fluorescence intensity information (F) was extracted for computation of F/F₀. This line was positioned to extend from the base of the soma through the nucleus and out along the apical dendrite almost to the point of bifurcation (which is just visible in this image). The line was divided into 300 pixel positions, which are numbered. All of the individual cell data presented in the following figures were taken from this neurone.

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Figure 1. Fluorescence image of a mouse CA1 pyramidal neurone filled using the recording pipette with Oregon Green BAPTA 488-1 (50 µM) Superimposed on the image is a line divided into 300 positions marked off at 50 position intervals (each position corresponds to approximately 1 pixel). Fluorescence values (F) were measured in a 5 pixel-wide band for each position along this line and for each image in a run sequence to construct matrices of F/F0 against time, as in Fig. 2. The four cell regions analysed in subsequent figures are also labelled; in these analyses values of F were extracted from small areas of interest positioned in the centres of the regions indicated. All individual neurone data presented in Figs 2-7 were recorded from this neurone. The soma diameter is approximately 15 µm. The recording pipette can be seen entering from the bottom.

Because the Ca²⁺-sensitive dye will not penetrate internal membrane-delimited organelles, we refer to the cytosolic [Ca²⁺] ([Ca²⁺]_cyto) rather than total intracellular [Ca²⁺] ([Ca²⁺]_i) as might be reported after loading with a membrane-permeant dye.

A pseudocoloured representation of the changes in [Ca²⁺]_cyto along the analysis line during excitation is shown in Fig. 2. On the left is a tracing of the cell, stretched so that each analysis point along the ordinate of the pseudocolour matrices is in registration with the tracing. The approximate positions of the four cell regions are also indicated. The abscissa of each matrix is the frame number from which fluorescence information was extracted, denoted both in frame number and in seconds. Calibration of F/F₀ values was performed as described in Methods.

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Figure 2. Graphical representation of normalized changes in Ca2+-dependent fluorescence A1 and B 1, F/F0 for each position of the analysis line shown in Fig. 1 for each frame of a 200 frame (6·7 s)-long sequence acquired at 30 frames s-1. An outline of the cell with cell regions indicated is shown to the left, and a pseudocolour scale with approximate [Ca2+] calibration is shown to the right. The same values of F0 measured just before delivery of stimuli in A1 were used to calculate the ratio F/F0 in both 'without carbachol' and 'with carbachol' panels, and thus the changes in Ca2+-dependent fluorescence incorporate both resting and dynamic components. Four action potentials were elicited by passing short depolarizing pulses through the recording pipette. A rapid increase in [Ca2+]cyto at the time of each action potential is evident followed by a delayed invasion of the Ca2+ wave into the nucleus, and a large augmentation of the Ca2+ signal in the apical cell body and proximal dendrite during cholinergic stimulation. A2 and B 2, action potentials recorded simultaneously from the same cell and aligned with the F/F0 data above. After application of carbachol (B 2) a small hyperpolarizing current was passed through the recording pipette to return the membrane potential to its original resting value (B 1).

Figure 2A1 (without carbachol) shows F/F₀ for each position along the analysis line for the second control run before application of carbachol. F₀ was calculated from an average F value during the period preceding the first stimulation pulse. The action potentials recorded from this neurone during the run are shown in Fig. 2A2. As expected, each action potential elicited a sharp increase in [Ca²⁺]_cyto, which showed a rapid decrease in cell body and dendritic regions, and a more gradual decline in the portion of the cell body where the signal was dominated by the nucleus.

Figure 2B 1 (with carbachol) was acquired in the presence of 5 µM carbachol. F/F₀ was calculated using the same value for F₀ as used for the control run (Fig. 2A1). This panel thus illustrates both an elevation of resting [Ca²⁺]_cyto and an increased amplitude of Ca²⁺ transients elicited by the train of action potentials (shown in Fig. 2B 2). As before, [Ca²⁺]_cyto changes in the nucleus were delayed and prolonged relative to those in other regions; note the gradual invasion of Ca²⁺ into the nuclear region after its rapid increase in the cytosol (also evident in Fig. 2A1). In continuous recordings of membrane voltage, carbachol routinely elicited a small steady-state 0-5 mV positive shift in resting potential that was removed by passing hyperpolarizing current through the recording pipette. Thus the data in Fig. 2A1 and B 1 were acquired at the same membrane potential.

The difference in F/F₀ between Fig. 2A1 and B 1, calculated for each point in the matrix, is shown in Fig. 3. The green triangles indicate the times at which the four action potentials were elicited. Evident in this figure is the absence of sharp peaks in the difference signal at the times of action potential generation, and a delay between the time of the fourth action potential and the peak of the difference signal.

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Figure 3. Graphical representation of the differences between the F/F0 signals presented in Fig. 2 Data were calculated using pixel-by-pixel subtraction, and are presented using the arbitrary pseudocolour glow scale on the right, with yellow indicating the greatest difference. The times at which the four action potentials were elicited are shown by the green triangles. The lack of a difference in signal time locked to the action potentials, and the delay between the generation of the fourth action potential and the time of maximum difference in Ca2+-dependent fluorescence are evident.

Figures 2 and 3 illustrate four main points that will be described below in more detail. First, carbachol increased the resting [Ca²⁺]_cyto. Second, the maximum [Ca²⁺]_cyto attained during the action potential train was increased, and the recovery of [Ca²⁺]_cyto towards resting levels was slowed. Third, the difference between the Ca²⁺ signals showed a smooth rise, and a maximum that followed the peak of the final action potential. Fourth, changes in the characteristics of Ca²⁺ signalling were not accompanied by large changes in action potential waveform.

Resting [Ca²⁺]_cyto

Chronic exposure to carbachol resulted in an increase in resting [Ca²⁺]_cyto in the absence of changes in resting potential (which, as indicated, were compensated by passing hyperpolarizing current). Changes in resting [Ca²⁺]_cyto for four cell regions are shown in Table 1. The values shown were calculated for the final measurement run following two control runs under two conditions - with or without addition of carbachol to the external solution. In the absence of carbachol, resting [Ca²⁺]_cyto was stable in dendrite and apical cell body, and showed a small increase in nucleus and basal cell body. This small increase could be a reflection of Ca²⁺ accumulation as a result of repetitive stimulation, or unavoidable photodynamic damage. In the presence of carbachol, resting [Ca²⁺]_cyto increased by 10-35 nM in all neuronal compartments, with the largest increases seen in nucleus and basal cell body.

Table 1. Resting calcium ion concentration (nM)

	Without carbachol (n = 6)			With carbachol (n = 6)
	Mean	±S.D.	P	Mean	±S.D.	P
Dendrite	63·1	3·8	n.s.	75·1	10·6	< 0·1
Apical cell body	65·3	17·0	n.s.	86·4	19·8	< 0·05
Nucleus	78·4	10·9	< 0·05	93·4	19·5	< 0·05
Basal cell body	77·6	19·2	n.s.	101·6	22·8	< 0·05

[Ca²⁺]_cyto transients

[Ca²⁺]_cyto transients, the action potential-induced increments in [Ca²⁺]_cyto above resting levels, are presented for the four neuronal compartments in Fig. 4A1-4. The dotted lines mark the times of action potential generation.

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Figure 4. Analysis of the transient components of normalized Ca2+-dependent fluorescence, [Ca2+]cyto (i.e. the action potential-induced increases above resting [Ca2+]cyto) In each case the resting component was eliminated by calculating F/F0 using F0 determined at the beginning of the second control run and again at the beginning of the test run recorded in the presence of carbachol. The dotted lines indicate the times at which action potentials were elicited. A1-4, [Ca2+]cyto for the four cell regions, showing the sharp Ca2+ transients occurring in synchrony with each action potential: , [Ca2+]cyto for the second control run; , [Ca2+]cyto for the subsequent test run. B 1-4, differences in [Ca2+]cyto calculated for each point. Note that the additional cytosolic Ca2+ in the presence of carbachol showed a smooth build-up and decline, and that the peak difference was observed after the last action potential. C 1-4, mean ± S.D. difference traces for five neurones, showing that despite quantitative variation in the behaviour of individual cells, the general properties of the difference signal were consistent. Note that the largest increases in peak [Ca2+]cyto were seen in dendrite and apical cell body, while relaxations of the carbachol-induced increase in [Ca2+]cyto showed greater prolongation in nucleus and basal cell body.

As illustrated, the character of the Ca²⁺ signals varied markedly between the regions. Under control conditions, Ca²⁺ transients were most sharply defined and relaxed most rapidly in the dendrite and apical cell body, while in the nucleus and basal cell body the transient increases in [Ca²⁺]_cyto were smaller and the relaxations were slower. In the presence of carbachol, Ca²⁺ signals relaxed more slowly in all cell regions.

Plots of the difference between the Ca²⁺ signals (Fig. 4B 1-4) showed an essentially monotonic rise and smooth fall in all cell compartments, and this was a consistent finding (Fig. 4C 1-4). Further, the peak of the difference signal was consistently delayed relative to the fourth action potential.

We interpret this smooth rise of the difference signal to indicate that the increment in [Ca²⁺]_cyto (either by Ca²⁺ entry or by Ca²⁺ release) associated with each action potential was not altered during exposure to carbachol. Rather, we suggest that the slower relaxation of the Ca²⁺ transients is responsible for the increase in the [Ca²⁺]_cyto envelope observed in the presence of carbachol. In this scheme, that the Ca²⁺ difference signal shows its maximum with a delay after the last action potential is a reflection of the different relaxation rates of the Ca²⁺ transients.

Increase in maximum [Ca²⁺]_cyto attained during action potential trains

The maximum [Ca²⁺]_cyto attained after the four action potentials was evaluated for measurement runs recorded with or without carbachol, and is shown in Table 2. For the sham trial, peak [Ca²⁺]_cyto remained essentially unchanged between 160 and 190 nM in all regions except the nucleus, in which a small increase was observed. For the test trial, all neuronal compartments showed significant increases in peak [Ca²⁺]_cyto of about 30-40 nM, reaching peak values of 180-210 nM. Characteristically, the increases in resting [Ca²⁺]_cyto and in [Ca²⁺]_cyto transients made different contributions to the total [Ca²⁺]_cyto increase in each cell region. In nucleus and basal cell body, increases in resting [Ca²⁺]_cyto were a larger proportion of the total than in dendrite and apical cell body, where changes in resting [Ca²⁺]_cyto were smaller but differences in [Ca²⁺]_cyto transients were larger.

Table 2. Peak calcium ion concentration (nM)

Changes in relaxation of [Ca²⁺]_cyto transients

Relaxations of the transient [Ca²⁺]_cyto component to resting levels, evaluated by computing the time to decay to half-peak [Ca²⁺]_cyto amplitude (t_½), are shown in Fig. 5. As illustrated by the plots of normalized [Ca²⁺]_cyto decay in Fig. 5A1, and the analysis in Fig. 5A2, mean t _½ was 350-500 ms under control conditions in dendrite and apical and basal cell body, and 1400 ms in nucleus (note the difference in the vertical scale in Fig. 5A2). Carbachol increased these mean t _½ values to 800-1200 and 3000 ms, respectively, 2·2- to 2·4-fold increases in each compartment. In sham control experiments, these values changed by less than 0·1-fold (not shown).

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Figure 5. Slowing of transient [Ca2+]cyto relaxations by exposure to carbachol A1, time course of [Ca2+]cyto relaxations (normalized to peak value) for four cell regions. A2, time for [Ca2+]cyto to relax to half-peak value (t ½). Data are presented as box plots in which the thick horizonal line shows the mean, the thin horizontal line shows the median, the box encloses ±25 % of the data from the median (i.e. 25-75 %), and the bars show ±40 % from the median (i.e. 10 and 90 %); n = 5. In this figure and in Fig. 7, statistical significance is denoted by asterisks: * P < 0·05. B 1, an example of the decomposition of [Ca2+]cyto relaxations of dendrite and apical cell body into multiple exponential components. Normalized pretest and carbachol test F/F0 traces are plotted on semilogarithmic co-ordinates and fitted with double exponential functions. As is evident by inspection of A1, after exposure to carbachol the [Ca2+]cyto relaxations in other cell regions could not be well fitted by exponential functions. B 2, the fitting equation and derived parameters. In both regions, after exposure to carbachol there was a slowing of fast and a decrease in its relative contribution to the overall waveform, while slow was unchanged (fast and slow are shown in s). These changes are representative of those in four neurones that were similarly analysed.

Waveforms of [Ca²⁺]_cyto relaxations in dendrite and apical cell body could often be fitted by the sum of two exponential functions; regional differences in Ca²⁺ signalling elements may have precluded this type of analysis in other cell regions. Figure 5B1 shows [Ca²⁺]_cyto relaxations plotted on semilogarithmic co-ordinates and fitted from the peak of the [Ca²⁺]_cyto transient with the sum of two exponentials according to the relation f = a exp(-t/_fast) + b exp(-t/_slow). Changes in the exponential parameters (Fig. 5B 2) indicate that the actions of carbachol were restricted to the fast component, increasing its time constant and decreasing its contribution to the overall [Ca²⁺]_cyto waveform. In this example carbachol increased _fast from 220-230 ms to 450-590 ms while decreasing a (the relative weight of the fast component) from 80 % to 70 % of the total. At the same time, carbachol had a minimal effect on _slow. Thus at least in dendrite and apical cell body, carbachol selectively reduced the efficacy of the mechanism(s) contributing to the faster component of Ca²⁺ removal from the cytosol.

Action potential waveforms

Figure 6A shows superimposed membrane potential records (from Fig. 2A2 and B 2), and Fig. 6B shows the action potentials at higher temporal resolution. As is evident, action potential waveforms were not changed after exposure to the low concentration of carbachol used. Across populations of neurones, measurements of action potential peak voltage, or time required to decline from 10 to 90 % of peak voltage, did not change significantly between the second control and sham or test measurement runs (Table 3). However, it should be noted that only an afterdepolarization was seen under our recording conditions, and the fast, medium and slow afterhyperpolarizations normally seen in CA1 pyramidal neurones (Storm, 1987, 1989), and subject to muscarinic modulation (Madison et al. 1987), were not evident.

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Figure 6. Comparison of action potential waveforms recorded under control and carbachol-exposed conditions A, superimposed complete voltage records for the cell shown in Figs 1-3. B, the period of action potential generation shown on an expanded time scale, illustrating the lack of a significant change in waveform. It should be noted, however, that action potentials recorded after dialysis with Ca2+-sensitive dye-containing internal solution showed the afterdepolarization but not the afterhyperpolarizations characteristic of CA1 pyramidal neurones.

Table 3. Action potential characteristics

	Without carbachol (n = 6)					With carbachol (n = 7)
	Pretest		Test		P	Pretest		Test		P
	Mean	±S.D.	Mean	±S.D.		Mean	±S.D.	Mean	±S.D.
Peak voltage (mV)	39·3	14·4	41·3	8·6	n.s.	31·4	13·7	37·4	14·7	n.s.
Duration (ms) *	3·6	1·5	3·9	1·2	n.s.	3·3	1·0	3·5	0·8	n.s.
Decay time (ms)	8·2	4·7	6·9	3·6	n.s.	9·4	6·4	7·3	3·1	n.s.

Involvement of muscarinic acetylcholine receptor-linked signalling mechanisms

We explored the potential involvement of the group I muscarinic acetylcholine receptors (mAChRs) signalling through an increase in cytoplasmic inositol 1,4,5-trisphosphate (InsP₃) in experiments using the mAChR antagonist atropine and the InsP₃-sensitive Ca²⁺-release channel blocker heparin.

Simultaneous application of atropine (1 µM) with carbachol partially reversed changes in Ca²⁺ signalling, as illustrated in Fig. 7A1-3. In these experiments, measurements were made in the order: control carbachol carbachol + atropine. Examination of [Ca²⁺]_cyto transients, in this case the average of measurements from the four cell regions (Fig. 7A1), shows substantial but incomplete reversal of the slowing of [Ca²⁺]_cyto relaxations after inclusion of atropine. The population data indicate that atropine incompletely reversed the carbachol-induced increase in peak [Ca²⁺]_cyto (Fig. 7A2), and agonist-induced slowing of [Ca²⁺]_cyto relaxations (Fig. 7A3). In both cases the data acquired in the presence of carbachol + atropine were not significantly different from control values.

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Figure 7. Muscarinic AChR and InsP3R involvement in cholinergic modulation of Ca2+ signalling In these experiments data from the four cell regions (dendrite, apical cell body, nucleus, basal cell body) were averaged because they showed qualitatively similar behaviour illustrating the involvement of mAChRs and InsP3Rs. A, atropine sensitivity of carbachol-induced alterations in [Ca2+]cyto transients. A1, average of [Ca2+]cyto transients measured in the four cell regions for three consecutive runs under different conditions: control, carbachol, carbachol + atropine (1 µM). Addition of atropine attenuated but did not completely reverse slowing of the [Ca2+]cyto relaxation in the presence of carbachol. A2, peak average [Ca2+]cyto, measured at the time of the fourth action potential. Peak average [Ca2+]cyto was increased by carbachol, and the increase was partially reversed by addition of atropine. A3, t ½ for average [Ca2+]cyto relaxations under the same three conditions. Carbachol significantly slowed the return of [Ca2+]cyto to resting levels, and this increase in [Ca2+]cyto was partially reversed by the inclusion of atropine. In the presence of carbachol and atropine, neither peak average [Ca2+]cyto nor t ½ was significantly different from control. * P < 0·05, ** P < 0·01. B, sensitivity of [Ca2+]cyto relaxations to inclusion of heparin (200 µg ml-1) in the pipette internal solution. B 1, an example of average [Ca2+]cyto transients for which heparin substantially inhibited the effects of exposure to carbachol. Peak average [Ca2+]cyto (B 2) and t ½ relaxation (B 3) for these same cells illustrate for both parameters partial block of the action of carbachol by inclusion of heparin (the increases in average [Ca2+]cyto and t ½ were evident but not statistically significant). Data in A2 and 3, and B 2 and 3 are box plots as described in the legend to Fig. 5; n = 5 for each.

Inclusion of heparin (200 µg ml^-1) in the internal solution partially blocked the effects of exposure to carbachol (Fig. 7B 1-3). In the example shown in Fig. 7B 1, carbachol was almost without effect on a heparin-dialysed neurone. In general, increases in [Ca²⁺]_cyto transients (Fig. 7B 2) and their relaxation times (Fig. 7B 3) were attenuated when heparin was included in the internal solution to the point that they were not statistically different from control values.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The major conclusion of this investigation of hippocampal CA1 neurones is that carbachol enhances intracellular Ca²⁺ signalling during short action potential trains both by raising the resting [Ca²⁺]_cyto and by increasing the accumulation of cytosolic Ca²⁺ in the absence of significant alterations of action potential waveforms. In our experiments, chronic exposure to carbachol raised the resting [Ca²⁺]_cyto by 10-20 nM, and delayed the return of [Ca²⁺]_cyto to resting levels (increases in t _½ relaxation times in non-nuclear regions from 400-500 to 800-1000 ms). Thus during individual action potentials, the volume-averaged peak [Ca²⁺]_cyto was raised by 10-30 nM, from 160-190 to 180-210 nM, with the net effect of increasing the time during which [Ca²⁺]_cyto was elevated over resting levels.

Curiously, the actions of carbachol on [Ca²⁺]_cyto were not spatially uniform. For each action potential the increase in [Ca²⁺]_cyto was larger in dendrite and apical cell body, while the decay of [Ca²⁺]_cyto was more prolonged in nucleus and basal cell body. Non-homogeneity in the distributions of calcium channel subtypes (Elliott et al. 1995), calcium-sequestering ATPases and associated organelles (Johnson et al. 1993), Ca²⁺-release channels (Sharp et al. 1993; Seymour-Laurent & Barish, 1995), as well as variations in surface-to-volume ratios and the presence of the nucleus as an initial Ca²⁺ sink and subsequent source (Hernández-Cruz et al. 1990), could all contribute to these regional differences.

Because the enhanced Ca²⁺ accumulation reported here was not associated with changes in the action potential waveform or increases in apparent Ca²⁺ entry during action potentials, we attribute enhanced Ca²⁺ accumulation to alterations in Ca²⁺ clearance from the cytosol. Our data therefore indicate that carbachol-induced changes in intracellular Ca²⁺ dynamics can occur independently of changes in Ca²⁺ entry due to modulations of action potential afterhyperpolarizations (Madison et al. 1987) and/or voltage-gated calcium currents (Gähwiler & Brown, 1987). Müller & Connor (1991) also observed an increase in the [Ca²⁺] attained during periods of repetitive action potential generation in the presence of micromolar concentrations of carbachol. However, in their study, increased [Ca²⁺]_i during epochs of 20-40 Hz firing was accompanied by action potential broadening, and therefore it is difficult to separate the contribution of augmented Ca²⁺ entry (presumed to occur during broader action potentials) from that of altered regulation of intracellular Ca²⁺. The observation of Tsubokawa & Ross (1997) of enhanced dendritic invasion by back-propagating action potentials and increase in peak [Ca²⁺]_i during action potential trains also emphasizes changes in excitability rather than in regulation of internal Ca²⁺.

Quantitative comparisons with other work

Our estimates of the changes in [Ca²⁺]_cyto elicited by single or small groups of action potentials are consistent with other observations. For example, Jacobs & Meyer (1997) estimated that, in cultured pyramidal neurone somata, a single action potential increased average [Ca²⁺]_cyto by 10 nM (at room temperature), and Schiller et al. (1995) observed peak [Ca²⁺]_cyto in neocortical layer V pyramidal neurones in response to single action potentials of 35-111 nM (at 34°C, with a larger peak [Ca²⁺]_cyto observed in dendrites).

The decays of Ca²⁺ transients that we observed were also similar to those reported in other studies of somatic [Ca²⁺]_cyto. For example, Fierro et al. (1998) decomposed Ca²⁺ decay transients for Purkinje cell somata into two exponential components with time constants of 600 and 3000 ms, and Schiller et al. (1995) also reported decay time constants for layer V pyramidal cells of 630-1000 ms (with more rapid relaxations observed in dendrites).

Comparison with other studies suggests that our measurements were minimally distorted by the buffering activity of 50 µM indicator dye. Distortions introduced by the presence of Ca²⁺ indicator dyes were examined explicitly by Helmchen et al. (1996), who analysed the effects of exogenous Ca²⁺ buffering on Ca²⁺ transients in proximal apical dendrites of pyramidal neurones. They observed distortions with fura-2 concentrations as low as 20 µM, and concluded that for rat CA1 pyramidal neurones the estimated peak [Ca²⁺]_cyto after a single action potential at an extrapolated zero fura-2 concentration was 151 nM (a value similar to that reported here). In contrast, Markram et al. (1995) did not observe a significant change in relaxation rate for Calcium Green-1 concentrations between 20 and 100 µM (but did see changes with 500 µM dye).

Possible mechanisms by which cholinergic stimulation may affect intracellular Ca²⁺ dynamics

The sensitivity of [Ca²⁺]_cyto relaxations to atropine and heparin indicates that a major portion of the actions of carbachol involved mAChRs and InsP₃Rs. As an agonist, carbachol does not differentiate between nicotinic and muscarinic mechanisms, and in principle both classes of cholinergic mechanism could make contributions to the changes observed. What types of pathway incorporate these signalling elements? In hippocampal pyramidal neurones, action potential generation results in a rapid accumulation of Ca²⁺ in intracellular organelles including endoplasmic reticulum (Pozzo-Miller et al. 1997), and muscarinic stimulation results in the production of InsP₃, which elicits release from these stores (Kudo et al. 1988; Wakamori et al. 1993; Seymour-Laurent & Barish, 1995; Irving & Collingridge, 1998). Store depletion can then stimulate Ca²⁺ entry in neurones (e.g. Usachev & Thayer, 1999) as in non-neuronal cells (Berridge, 1995; Parekh & Penner, 1997). Thus carbachol acting through mAChRs and InsP₃Rs may in principle enhance accumulation of Ca²⁺ in the cytosol either by compromising extrusion, sequestration or retention mechanisms, or by enhancing Ca²⁺ influx after InsP₃R activation.

We favour a mechanism in which enhanced Ca²⁺ accumulation in the cytosol is driven by reduced Ca²⁺ retention in intracellular stores due to chronic activation of InsP₃-sensitive Ca²⁺-release channels. In the context of the present experiments, open Ca²⁺-release channels could 'short circuit' Ca²⁺ sequestration by sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pumps into the endoplasmic reticulum and thus reduce Ca²⁺ clearance from the cytoplasm. A consequence of elevated resting [Ca²⁺]_cyto could be increased cycling of Ca²⁺ through the stores (Verkhratsky & Petersen, 1998), and chronic Ca²⁺ entry as a consequence of carbachol-induced intracellular store depletion could contribute to an increase in resting [Ca²⁺]_cyto.

Other mechanisms could also act simultaneously to enhance cytosolic Ca²⁺ accumulation. First, while regulation of the SERCA isoform responsible for filling of endoplasmic reticulum-associated Ca²⁺ stores in brain (SERCA2) has not been examined, SERCA inhibition after stimulation of muscarinic receptors could potentially enhance cytoplasmic Ca²⁺ accumulation. Steady cycling of Ca²⁺ through ryanodine-sensitive stores in pyramidal neurones is blocked by the inhibitor cyclopiazonic acid (Garaschuk et al. 1997), and inhibition of SERCA-driven Ca²⁺ sequestration slows the decline of cytosolic Ca²⁺ in other neurones (Friel & Tsien, 1992; Markram et al. 1995; Fierro et al. 1998; Toescu, 1998; Emptage et al. 1999). Additionally, Ca²⁺ extrusion by plasmalemmal Na⁺-Ca²⁺ exchange and Ca²⁺-ATPase contributes to removal of moderate cytosolic Ca²⁺ loads (see Fierro et al. 1998). Regulation of these mechanisms has been examined in non-neuronal cells (see Strehler, 1990), and thus in principle their inhibition after muscarinic activation could enhance accumulation of cytosolic Ca²⁺.

Finally, part of the atropine-insensitive portion of carbachol-induced augmentation could be mediated by activation of neuronal nicotinic acetylcholine receptors (nAChRs), since neuronal nAChRs are permeable to Ca²⁺ (Sands & Barish, 1991; Vernino et al. 1992; Zhou & Neher, 1993). While there are conflicting reports on whether application of cholinergic agonists to somata of CA1 pyramidal neurones does (Albuquerque et al. 1996) or does not (Frazier et al. 1998; Irving & Collingridge, 1998) elicit inward current or increases in [Ca²⁺]_cyto, synaptic stimulation of CA1 pyramidal neurones does elicit a small current sensitive to nAChR blockers (Hefft et al. 1999), and hippocampal pyramidal neurones express mRNAs for multiple nAChR subunits (Alkondon et al. 1994; Rubboli et al. 1994; Didier et al. 1995). However, while Ca²⁺ entry could contribute to changes in resting [Ca²⁺]_cyto, it seems less likely that nAChR activation alone would result in a slowing of Ca²⁺ removal from the cytosol, since the increase in resting [Ca²⁺]_cyto and the [Ca²⁺] changes resulting from action potentials are small and unlikely to saturate Ca²⁺ sequestration or extrusion mechanisms.

In these experiments, carbachol-induced changes in action potential waveforms, which should affect Ca²⁺ entry, were minimal. We did not observe changes in action potential duration or repolarization time course, and the increased [Ca²⁺]_cyto attributable to carbachol did not show a sawtooth rise indicative of increased [Ca²⁺]_cyto linked to individual action potentials. Further, we did not observe the appearance of plateau potentials after carbachol stimulation (Fraser & MacVicar, 1996). However, other studies have noted carbachol-induced modulation of potassium currents and action potential waveforms (Madison et al. 1987; Müller & Connor, 1991), and some of these changes affected action potential components not present in our recordings, possibly because of the intracellular dialysis associated with prolonged whole-cell loading of Ca²⁺-sensitive dye. These afterhyperpolarizations are also absent from other recordings made under similar conditions (e.g. Sandler & Barbara, 1999). Therefore our data indicate that changes in the temporal integration of cytosolic Ca²⁺ signals can occur in the absence of changes in excitability, but leave open the additional possibility of modulation of action potential-driven Ca²⁺ entry.

Potential contributions of Ca²⁺-induced Ca²⁺ release to action potential-driven increases in [Ca²⁺]_cyto

In none of the cell regions did enhancement of intracellular Ca²⁺ signals appear to involve amplification of Ca²⁺ entry by Ca²⁺-induced intracellular Ca²⁺ release. This process would have been evident as a rapidly rising increase in [Ca²⁺]_cyto correlated with the generation of each action potential. The general issue of contributions of Ca²⁺ release to action potential-induced increases in [Ca²⁺]_cyto in neurones is not clear. Some results indicate that amplification due to Ca²⁺-induced Ca²⁺ release is a factor in regulating [Ca²⁺]_cyto after imposed depolarizations or action potentials (Friel & Tsien, 1992; Jacobs & Meyer, 1997; Sandler & Barbara, 1999), while other results suggest that Ca²⁺ release does not contribute to action potential-linked Ca²⁺ signalling (Nohmi et al. 1992; Markram et al. 1995; Garaschuk et al. 1997). Whether these differences reflect variations in Ca²⁺ store loading (Verkhratsky & Petersen, 1998) or other aspects of cell physiology, heterogeneity of cell type, or differences in experimental conditions, remains to be resolved. This situation is in contradistinction to synaptically elicited Ca²⁺ signals, which do appear to be amplified by Ca²⁺ release from intracellular stores (Alford et al. 1993; Pozzo-Miller et al. 1997; Emptage et al. 1999).

Significance of regulated enhanced cytosolic Ca²⁺ accumulation

These results indicate that a major action of cholinergic stimulation will be an alteration in the dynamics of the rise and fall of [Ca²⁺]_cyto during fluctuating epochs of action potential generation (see Otto et al. 1991). It should be noted that, depending on the mechanism of action of carbachol, these results could also imply a complementary pattern of decreased Ca²⁺ accumulation in intracellular stores. Regardless, as indicated in the introductory remarks, recent evidence suggests that Ca²⁺-dependent effector pathway stimulation is sensitive to the frequency and duration of [Ca²⁺]_cyto increases (for reviews see Barish, 1998; Berridge, 1998; Meldolesi, 1998; Putney, 1998). In many systems, activation and deactivation rates are balanced to favour accumulation of Ca²⁺-dependent activation during oscillations of varying periods, with variation in these rates leading to frequency specificity. Thus the amplitude and frequency of Ca²⁺ oscillations affects accumulation of Ca²⁺-dependent phosphorylation on calcium-calmodulin-dependent protein kinase II (CaMKII) subunits, and translocation of transcription factors from cytoplasm to nucleus and their accumulation in the nucleus. In our experiments cholinergic stimulation varied the relaxation rate of Ca²⁺ transients in the absence of any change in action potential waveform, thereby increasing the integrated Ca²⁺ exposure of Ca²⁺-sensitive effector systems. Possible sites where modulation of Ca²⁺ dynamics could be significant include induction of LTP and protein synthesis in pyramidal neurone dendrites, both of which are promoted by the association of carbachol application with afferent stimulation (Bear & Singer, 1986; Feig & Lipton, 1993; Segal & Auerbach, 1997).

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Acknowledgements

We thank Dr Kerry Delaney for helpful discussions of the experiments and the manuscript, and Ms M. Jill Flanagan for assistance with manuscript preparation. Supported by NIH NINDS R01 grants NS34581 and NS23857.

Corresponding author

M. E. Barish: Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.

Email: mbarish{at}coh.org

Author's present address

S. M. Beier: Program in Neuroscience, Stanford University, Stanford, CA 94305, USA.

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