Muscarinic activation of a non-selective cationic conductance in pyramidal neurons in rat basolateral amygdala

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Neuroscience Vol. 88, No. 1, pp. 159–167, 1999 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00210-3

MUSCARINIC ACTIVATION OF A NON-SELECTIVE CATIONIC CONDUCTANCE IN PYRAMIDAL NEURONS IN RAT BASOLATERAL AMYGDALA J. YAJEYA,*‡ A. DE LA FUENTE JUAN,* V. M. BAJO,† A. S. RIOLOBOS,* M. HEREDIA* and J. M. CRIADO* Departments of *Physiology and Pharmacology and †Cellular Biology and Pathology, School of Medicine, University of Salamanca, 37007 Salamanca, Spain Abstract––In the present study, a cationic membrane conductance activated by the acetylcholine agonist carbachol was characterized in vitro in neurons of the basolateral amygdala. Extracellular perfusion of the K+ channel blockers Ba2+ and Cs+ or loading of cells with cesium acetate did not affect the carbachol-induced depolarization. Similarly, superfusion with low-Ca2+ solution plus Ba2+ and intracellular EGTA did not affect the carbachol-induced depolarization, suggesting a Ca2+-independent mechanism. On the other hand, the carbachol-induced depolarization was highly sensitive to changes in extracellular K+ or Na+. When the K+ concentration in the perfusion medium was increased from 4.7 to 10 mM, the response to carbachol increased in amplitude. In contrast, lowering the extracellular Na+ concentration from 143.2 to 29 mM abolished the response in a reversible manner. Results of coapplication of carbachol and atropine, pirenzepine or gallamine indicate that the carbachol-induced depolarization was mediated by muscarinic cholinergic receptors, but not the muscarinic receptor subtypes M1, M2 or M4, specifically. These data indicate that, in addition to the previously described reduction of a time- and voltageindependent K+ current (IKleak), a voltage- and time-dependent K+ current (IM), a slow Ca2+-activated K+ current (sIahp) and the activation of a hyperpolarization-activated inward rectifier K+ current (IQ), carbachol activated a Ca2+-independent non-selective cationic conductance that was highly sensitive to extracellular K+ and Na+ concentrations.  1998 IBRO. Published by Elsevier Science Ltd. Key words: slices, intracellular, receptors, Ca2+ independent.

It has been suggested that the amygdala plays a critical role in the processes that encode emotion, motivation, learning and memory.1 In rats, Whitelaw et al.27 demonstrated the importance of the basolateral amygdaloid nucleus in the processes by which previously neutral stimuli gain motivational relevance. Studies carried out in monkeys23 suggested that the neuronal activity in amygdaloid neurons partially depends on the emotional meaning of the stimulus for the animal. In humans, amygdaloid lesions are associated with deficits in the interpretation of emotional components of language, a reduction in the frequency and intensity of facial expressions, and impairment of the judgement of mood, facial expressions and emotional situations.3 The unquestioned behavioral importance of the amygdala is mediated by its relationship with other structures of the nervous system, some of which are included within the cholinergic system. Acetylcholine is a putative neurotransmiter that is found throughout the mammalian CNS. Quantitative ‡To whom correspondence should be addressed. Abbreviations: ACSF, artificial cerebrospinal fluid; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; RMP, resting membrane potential; TEA, tetraethylammonium chloride.

microassay for choline acetyltranferase14 revealed that the nuclei contained within the basolateral amygdaloid complex serve as the major targets of this cholinergic input. Immunohistochemical analyses for catechol acetyltransferase indicate that cholinergic input to cells in the basolateral complex is derived from extrinsic afferents that originate from the substantia innominata,6,29 but the function of these projections is not known at present. Characterization of the effects of acetylcholine in the basolateral complex may provide insight into the function of this nucleus. The cholinergic agonist carbachol has been shown to evoke muscarinic receptor-mediated depolarizing responses in many structures of the nervous system.8,13,16,17,19,21,24,28 Carbachol-induced depolarization is due to (i) suppression of the voltage- and time-dependent K+ current (IM), (ii) suppression of the slow Ca2+-activated K+ current (sIahp), (iii) suppression of the voltage-independent K+ leak current (IKleak), or (iv) activation of the hyperpolarizationactivated inward rectifier current (IQ). In addition, previous work has shown that muscarinic receptors mediate the activation of non-selective cationic conductances in hippocampal neurons,4,8,24 locus coeruleus neurons,10,25 neocortical neurons12,16 and

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sympathetic ganglion cells.15 Previous results obtained in our laboratory demonstrate that carbachol causes a slow depolarization, sometimes with decreased or unchanged membrane resistance, that persists even with perfusion with 1–5 mM Ba2+, a specific inhibitor of IM and IKleak,5,30 and at resting membrane potentials (RMPs;
636 mV) at which IM and IQ are inactivated. These results give rise to the possibility that the carbachol-induced depolarization observed in amygdala pyramidal neurons is mediated by another mechanism that involves an increase in Na+ and/or Ca2+ conductances. To examine this possibility and further characterize the carbachol-induced depolarization, the effect of carbachol on rat basolateral amygdala neurons was measured using intracellular recording. EXPERIMENTAL PROCEDURES

Amygdala slices were prepared for intracellular recording as described previously.30 Briefly, male or female Wistar rats raised in the animal colony of the University of Salamanca (120–150 g) were anesthetized with ether. After anesthesia, the animals were decapitated and a block of tissue containing amygdaloid regions was immersed in oxygenated ice-cold (6–10C) artificial cerebrospinal fluid (ACSF) in which the NaCl (117 mM) had been omitted and replaced with sucrose (234 mM) to maintain the osmolarity. Coronal slices (400 mm thick) were cut on cold (4–6C) oxygenated Ringer using a Vibratome, transferred to an incubation chamber and incubated for approximately 1–2 h at room temperature. For recording, a single slice containing the amygdaloid complex was transferred to an interface recording chamber (Medical Systems Corp.) and perfused continuously with ACSF composed of (mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4 and 11 glucose. The ACSF was bubbled with 95% O2/5% CO2 and maintained at 322C (meanS.E.M.). Where indicated, in the ACSF containing Ba2+ or Cs+, Ca2+ was replaced with Mg2+, or NaCl was replaced with N-methyl--glucamine. All drugs were applied to the preparation dissolved in the perfusion medium. Intracellular recordings were obtained from the basolateral amygdaloid complex using borosilicate glass (W.P.I.) microelectrodes (tip resistance 140–180 MÙ) filled with a 3 M potassium acetate solution and connected to the headstage of an intracellular recording amplifier (Bio-Logic VF 180). Data were acquired and stored as analog signals on video cassettes using a modified video recorder (Cibertec Physiorec-3). Data were transferred to a microcomputer using an analog-to-digital converter interface (CED 1401) for off-line analysis using a custom software application. Only data from those neurons that exhibited stable RMPs more negative than
55 mV in the absence of direct holding current and that generated overshooting action potentials were included for analysis. Where indicated, 2% biocytin in 2 M potassium acetate was included in the recording solution and injected into the cell to allow for cellular identification. At the end of the experiment, slices were fixed by immersion in 0.1 M phosphate buffer with 1.25% glutaraldehyde. The fixed slices were embedded in a 2% solution of agar, cryoprotected with 30% sucrose in phosphate buffer, and 35- to 40-µm sections were cut using a freezing microtome. Cells injected with biocytin were visualized with a standard avidin–biotin incubation and diaminobenzidine reaction with nickel intensification, according to previously described methods.18,30 Representative neurons were reconstructed from serial sections to determine the cell type.

All the electrophysiological data are expressed as means (S.E.M.); in all cases n is the number of neurons. Data were analysed statistically using a paired Student’s t-test and, where indicated, one-way ANOVA. Statistical significance was determined at a level of P<0.05. Atropine, barium chloride, tetraethylammonium chloride (TEA), pirenzepine, flunarizine, gallamine and biocytin were purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.), and carbachol was purchased from Research Biochemicals Inc. (Natick, MA, U.S.A.).

RESULTS

Intracellular recordings were obtained from a total of 132 neurons located in the anterior basolateral nucleus (n=92), posterior basolateral nucleus (n=26), the ventrolateral part of the lateral nucleus (n=9) and the dorsolateral part of the lateral nucleus (n=5) of the amygdala.2 Consistent with previous studies in the amygdala,7,22,26 neurons in this region could be subdivided into broad classes according to their electrophysiological properties and firing pattern. The vast majority of neurons activated by injection of depolarizing current below 0.2 nA generated a short series (two to three) of action potentials (bursts), with the second and third impulses exhibiting decreased amplitude and increased duration. Cells exhibiting this type of response were classified as type I (burst) cells.30 Occasionally, recordings were obtained from multiple spike neurons, which were not studied further. Previous studies have established that neurons identified as burst cells correspond morphologically to pyramidal cells.30 To examine whether this correspondence also held for the neurons analysed in the present study, some cells were filled with biocytin following the recording session and the tissue was then processed for histochemistry. All of the cells classified electrophysiologically as burst cells were also morphologically identified as pyramidal neurons; therefore, in the remaining sections of this report, burst cells are referred to as pyramidal cells. Because we did not find morphological or electrophysiological differences in relation to the different location of recorded cells, all recordings have been grouped. Application of 10–20 µM carbachol resulted primarily in membrane depolarization (6.81.14 mV; n=56; Fig. 1). The carbachol-induced depolarization was slow in onset and was sometimes accompanied by the induction of action potential firing. This response was similar to that described by Dutar and Nicoll9 in hippocampal pyramidal cells. This effect, which did not diminish during sustained superfusion with carbachol, was accompanied by a small decrease or no change in membrane input resistance, consistent with our previous observations30 and previous observations in rat hippocampal cells.24 Atropine (1.5 µM) abolished the carbachol-induced depolarization in all of eight experiments, indicating that the response was mediated by muscarinic receptors.

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Fig. 1. Effect of carbachol on membrane potential and input resistance of rat amygdala pyramidal neurons. In this cell, superfusion with carbachol (Cch) produced a membrane depolarization not accompanied by action potential firing, but with a small but significant decrease in membrane input resistance. In this and the subsequent figures, drug application is represented by the traces at the top of the figure. The middle traces represent voltage and the lower trace represents current. The downward deflections in the voltage trace represent the electrotonic response of the cell to injection of square-wave current pulses of 200 ms. The arrows indicate the points at which these traces were expanded in time.

Atropine inhibition was irreversible after extensive washing (10–15 min; data not shown). The carbachol-induced depolarization was also blocked by 5 mM TEA, suggesting the involvement of selective K+ channels. The mechanism of carbachol-induced depolarization Extracellular perfusion with Ba2+ (1–5 mM; n=9), an IM and IKleak blocker, evoked a slow depolarization of a few millivolts (51.3 mV), accompanied by a small increase in membrane input resistance (control, 473 MÙ; under Ba2+ superfusion, 564 MÙ; n=8). In these conditions, superfusion with carbachol still induced a slow depolarization.30 This response was similar to that described previously by Colino and Halliwell8 in the hippocampus. To prevent activation of IQ, which is also activated by carbachol,8 recordings were made with extracellular Ba2+ (1–5 mM) and Cs+ (2 mM) (n=11) present. Superfusion with this solution evoked a depolarization of 41.8 mV, accompanied by an increase of input membrane resistance (73 MÙ) compared with the control situation. Even in these conditions, carbachol-induced depolarization persisted (Fig. 2). Thus, the carbachol-induced depolarization appeared to be mediated by a K+ conductance other than IM, IKleak or IQ.

The carbachol-induced depolarization and calcium To test the possible role of Ca2+ conductances and the slow Ca2+-activated K+ current sIahp in the carbachol-induced depolarization, Ca2+ currents were blocked using the Ca2+ antagonist flunarizine (10 mM; n=12). After 10-min superfusion with flunarizine, the carbachol-induced depolarization remained unaffected. This depolarizing effect was generated without appreciable changes in the membrane input resistance (Fig. 3). To determine whether carbachol acted by releasing other transmitters, some experiments were performed in low-Ca2+ solution. After 6 min superfusion with low-Ca2+ solution, the carbachol-induced depolarization did not show significant changes. The carbachol-induced depolarization was also unaffected by the addition of 1 mM Ba2+ to the lowCa2+ solution, to prevent the possible effects of carbachol-induced IM inhibition (n=6). The records in Fig. 4 illustrate such an effect; in this case, the changes in RMP mediated by the addition of Ba2+ were previously compensated with direct current. We examined whether intracellular Ca2+ was required for the carbachol-induced depolarization. Recordings were obtained using low-Ca2+ solutions and microelectrodes filled with 25 mM EGTA. In these conditions, the carbachol-induced

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Fig. 2. Records from a cell bathed in ACSF containing 1 mM Ba2+ and 2 mM Cs+ to block potassium channels. Carbachol application is illustrated by the trace at the top of the figure. In order to assess changes in the input membrane resistance, the depolarization generated by cations was compensated by direct current.

Fig. 3. The carbachol-induced depolarization was not prevented by pretreatment with flunarizine. With co-application of carbachol and flunarizine, the carbachol-induced depolarization was still evident. To determine changes in input resistance, the depolarization was compensated with direct current until the membrane potential was equal to the control resting potential.

depolarization was unchanged (n=5). These results indicate that neither a rise in intracellular Ca2+, influx of Ca2+ nor transmitter release were required to elicit the carbachol-induced depolarization. The carbachol-induced depolarization and sodium and potassium We examined the effect of changing the external K+ concentration on the carbachol-induced

depolarization to determine whether it was mediated by a non-selective cationic conductance. Increase of K+ concentration evoked a depolarizing effect (73 mV), concomitant with a decrease in the input membrane resistance (41.7 MÙ), compared with the control situation. Injection of direct current was used to compensate for the change in resting potential. The amplitude of the carbachol-induced depolarization was significantly (P<0.05) augmented

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Fig. 4. Superfusion with low-Ca2+ solution containing Mg2+ and Ba2+, to prevent trans-synaptic effects and changes in IM and IKleak, respectively, did not prevent the carbachol-induced depolarization.

Fig. 5. The carbachol-induced depolarization (10 µM) was dependent on extracellular K+. Superfusion with a high concentration of K+ (10 mM) evoked a slow depolarization that was compensated with direct current. The carbachol-induced depolarization was accompanied by action potentials and was more pronounced than with perfusion of control solution containing 4.7 mM K+.

(17.22.46 mV; n=18) versus control (6.8 1.14 mV; n=56) when the K+ concentration in the bathing medium was increased from 4.7 to 10 mM (Fig. 5). These results are consistent with the hypothesis that the effect is mediated by an increase in a non-selective cationic conductance. If the carbachol-induced depolarization is mediated by an increase in a non-selective cation conductance, then it should also be modified by changes in extracellular Na+ concentration. Replacement of 80% of the extracellular Na+ with N-methyl--

glucamine, which reduced the extracellular concentration of Na+ from 143.2 to 29 mM, elicited a hyperpolarization (72 mV), concomitant with an increase in the input membrane resistance (31.2 MÙ). Both parameters slowly returned to the control values (Fig. 6). In these conditions, the carbachol-induced depolarization was blocked (n=12). This series of experiments was performed at normal RMPs; thus, the reduction in the carbacholinduced depolarization was not associated with changes in voltage-dependent conductances.

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Fig. 6. The carbachol-induced depolarization was dependent on extracellular Na+. Lowering the Na+ concentration from 143.2 to 29 mM produced a hyperpolarization that recovered slowly. Note that depolarizing pulses applied in the middle of the experiment evoked action potentials with lower amplitudes. Addition of carbachol did not evoke a depolarization. The resting potential recovered to control values after washing with control solution.

Fig. 7. The carbachol-induced depolarization under continuous superfusion with pirenzepine. In this example, carbachol evoked a depolarizing response similar to that observed in control conditions.

Muscarinic receptor subtype mediating the carbachol response To characterize the subtype of muscarinic receptor involved in the carbachol-induced depolarization, the effects of pirenzepine, an M1 receptor antagonist,20 and gallamine, an M2 and M4 antagonist, were examined. After 15-min superfusion with pirenzepine (500 nM, a concentration at which pirenzepine selectively blocks M1 receptors), we did not observe changes in the membrane input resistance. In these condictions, carbachol-induced depolarization was only partially reduced (depolarization in control conditions, 6.81.14 mV; under pirenzepine, 4.3 2.2 mV; n=6; Fig. 7), suggesting that the majority of

the effect of carbachol was not due to activation of M1 receptors. Superfusion with gallamine (10–20 µM) did not change the cells’ biophysical parameters, such as RMP or input membrane resistance, but still induced depolarization (52.3 mV; n=10; Fig. 8). In these experiments, presynaptic effects were prevented by using low-Ca2+ solution. Thus, it appears that a muscarinic receptor subtype other than M1, M2 or M4 mediates the carbachol-induced depolarization. DISCUSSION

The present experiments characterize a cationic conductance activated by muscarinic cholinergic

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Fig. 8. The carbachol-induced depolarization with perfusion with gallamine in low-Ca2+ solution. Carbachol evoked a similar depolarizing response as in control conditions.

agonists in the basolateral amygdaloid complex. This conductance is voltage independent, presents a high sensitivity to changes in extracellular Na+ and K+ concentration, and is Ca2+ independent. The depolarization elicited by carbachol in the pyramidal neurons of the amygdala26,28 and in other areas of the nervous system19,21,24 has been suggested to be mediated by the blockage of some potassium current. However, our previous data30 show that the depolarization was independent of the RMP value, occurred with a small decrease or no change in the input membrane resistance and was blocked by TEA. In addition, the depolarization could be evoked even after superfusion with Ba2+, a blocking agent of the M-current,5 and Cs+, to block IQ mediated by potassium. In view of these findings, we suggest that in amygdaloid neurons the depolarization evoked by carbachol could be the result of changes in other conductances. Firstly, we studied the dependence of the depolarizing effects on Ca2+. The present results show that carbachol’s effects were observed even in the presence of flunarizine, a calcium channel blocking agent, and were independent of external Ca2+ concentration, because the same effect was observed with low Ca2+ and EGTA. These facts clearly differentiate carbachol’s effect in the amygdala from that observed in the hippocampus by Colino and Halliwell,8 which was Ca2+ dependent. Calciumindependent effects generated by carbachol have also been described by Shen and North25 in the locus ceruleus and by Haj-Dahmane and Andrade12 in the cortex.

Secondly, we have studied the possible involvement of an independent non-selective cation conductance, as occurred in some other structures of the nervous system.8,11,12,25 The present experiments demonstrate the dependence of the carbachol effect on the external sodium and potassium concentration, suggesting the involvement of these ions in the response mediated by the muscarinic agonist. The responses of amygdaloid neurons to carbachol are voltage independent at RMP values of
63 6 mV, are not blocked by barium or cesium, are calcium independent, and their amplitude depends on extracellular potassium and sodium concentrations. For all these reasons, we suggest that the changes in RMP observed because of the effects of carbachol may be related to changes in cationic conductances. The non-selective cationic conductance observed in amygdaloid neurons under carbachol superfusion was blocked by TEA. Similar effects of TEA on nonselective cationic conductance have recently been described in cortical neurons12 and the hippocampus.11 Although the compounds currently available are insufficiently selective to be able to classify the subtype of muscarinic receptor responsible for the effect of cholinomimetics in the excitable tissues, some data support the hypothesis that M1 and M3 receptors couple to inhibit Im and activate sIahp, and that M2 and M4 receptors couple to inhibit ICa. The fact that carbachol in the amygdala evokes a response in the presence of pirenzepine and gallamine, M1, M2 and M4 muscarinic channel blocking agents, suggests that the depolarizing effect of carbachol should be independent of changes in the

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conductance coupling to these receptors, suggesting that carbachol’s effects could be mediated by the activation of a different receptor subtype (M3, M5), associated in this case with the control of the non-specific cationic conductance. There is evidence, obtained in the hippocampus, that the response to carbachol is mediated through non-selective cationic channels that are not coupled to G-proteins.11 Although the intracellular transduction mechanism that links muscarinic receptors to cationic channels in the amygdala requires additional study, the present study provides evidence that the carbachol-induced depolarization is Ca2+ independent, suggesting that the carbachol effect is not mediated through Ca2+ coupling to a second messenger pathway.

CONCLUSION

The responses of amygdaloid neurons to carbachol are voltage independent at RMP values of
63 6 mV, are not blocked by barium or cesium, are calcium independent and their amplitude depends on extracellular potassium and sodium concentrations. For all these reasons, we suggest that the depolarizing effect of carbachol may be related to changes in cationic conductances mediated by activation of M3/M5 muscarinic receptors. Acknowledgements—We are grateful to A. Colino for perceptive comments, to I. Plaza for technical assistance and to G. H. Jenkins for revising the English version of the manuscript. This research is sponsored by grant D.G.I.C.Y.T. PB 96-1285.

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