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1Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam, Amsterdam; 2The Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands; and 3Institute of Molecular Biology and Biotechnology, Heraklion, Greece
Submitted 16 October 2007; accepted in final form 8 December 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). The hormones pass the blood–brain barrier and bind to intracellular receptors in the brain. Low amounts of corticosterone (such as circulate under rest) are sufficient to activate high-affinity mineralocorticoid receptors (MRs), which are abundantly expressed in limbic areas, i.e., all hippocampal subfields and the lateral septum. Under rest the lower-affinity glucocorticoid receptor (GR) is only about 20% occupied; this receptor becomes substantially activated after stress and at the circadian peak of corticosterone release. Both receptor subtypes act as transcriptional regulators (Pascual-Le Tallec and Lombes 2005; Zhou and Cidlowski 2005) and thus slowly change the function of neurons.
Previous studies have shown that in particular voltage-dependent calcium (Ca) currents are sensitive to the stress hormone corticosterone. In the CA1 area, the amplitude of high-voltage–activated Ca currents is relatively small with predominant MR activation (i.e., in nonstressed rats), but large when GRs are activated, for example as a result of stress some hours before recording (Joëls et al. 2003; Karst et al. 1994; Kerr et al. 1992). It was shown that this effect requires binding of GR homodimers to the DNA (Karst et al. 2000) and most likely is due to an increase in the number of available L-type Ca channels in the plasma membrane (Chameau et al. 2007). In association with an increased Ca-current amplitude, corticosterone induced an increased firing frequency accommodation on depolarization as well as a larger amplitude of the slow afterhyperpolarization (sAHP) that is seen when the depolarization is terminated (Joëls and de Kloet 1989; Kerr et al. 1989). Other basal membrane properties, such as resting membrane potential and input resistance, were not affected by GR activation. The interpretation of these effects is that several hours after stress exposure, information flow through the CA1 hippocampal area is attenuated and thus earlier aroused activity normalized, through a GR-mediated mechanism (Joëls et al. 2007).
Glucocorticoids are also known to alter basolateral amygdala (BLA) function. For instance, consolidation of inhibitory avoidance behavior is promoted by glucocorticoids, acting via GRs in the BLA (Roozendaal and McGaugh 1996, 1997; Roozendaal et al. 2006). However, when we started the present investigation little was known about cellular effects of the hormone. One study described that glucocorticoids increase the amplitude of high-voltage–activated calcium currents in a delayed manner, in a very similar extent to that reported for CA1 pyramidal neurons (Karst et al. 2002); at the single-cell level, relative expression of the Cav1.2 subunit was found to be enhanced. In the present investigation we examined how corticosterone affects active and passive membrane properties of principal neurons in the BLA. We tested the hypothesis that corticosterone increases the sAHP amplitude of BLA neurons, but does not affect other passive and active membrane properties, similar to what was reported for the CA1 hippocampal area.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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For the present study we used male Wistar rats (
150 g; Harlan CPB, Zeist, The Netherlands) or male C57/Bl6 mice (40 g; Harlan CPB). All animals were group-housed for 
7 days before the experiment. Food and water were provided without restriction, lights were on from 8:00 am until 8:00 pm, and the temperature and humidity were kept between 20 and 22°C and 55 ± 15%, respectively. The local committee on animal bioethics of the University of Amsterdam approved all experiments.
Slice preparation and corticosterone treatment
Immediately after decapitation, the brain was removed from the skull and chilled (at 4°C) in artificial cerebrospinal fluid (aCSF) containing (in mmol/L): NaCl 120, KCl 3.5, MgSO4 5.0, NaH2PO4 1.25, CaCl2 0.2, D-glucose 10, and NaHCO3 25.0, gassed with 95% O2-5% CO2. Next, coronal slices (400 µm thick) containing the hippocampus and/or the BLA were prepared with a vibroslicer (Leica VT 1000S; Leica Instruments, Nussloch, Germany). Briefly, frontal lobes and cerebellum were removed and the caudal side of the brain was glued onto the slicing plateau. Slices were stored at room temperature in recording aCSF containing (in mmol/L): NaCl 120, KCl 3.5, MgSO4 1.3, NaH2PO4 1.25, CaCl2 2.5, D-glucose 10, and NaHCO3 25. After 1 h, half of the slices from all experimental groups were subjected to vehicle treatment (0.01% ethanol), whereas the other half received corticosterone (100 nM in 0.01% ethanol), for 20 min at 32°C. Previous studies have shown that this treatment is sufficient to observe changes in cellular properties that require homodimerization of the GR, 1–4 h later (Karst et al. 2000).
Recording
One slice at a time was placed in a recording chamber mounted on an upright microscope (Axioskop 2 FS plus; Carl Zeiss, Oberkochen, Germany) with differential interference contrast, water-immersion objective (x40), and x10 ocular to identify the cells. Under both current- and voltage-clamp conditions the slices were continuously perfused with aCSF (flow rate 2–3 ml/min, temperature 32°C, pH 7.4) consisting of (in mM): 120 NaCl, 3.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 25 NaHCO3, 1.25 KH2PO4, and 10 D-glucose. Patch pipettes for recording were pulled from borosilicate glass (OD 1.5 mm; Science Products, Hofheim, Germany; pipette impedance 3–4 M
) on a Sutter micropipette puller (Sutter Instrument, Novato, CA) and filled with (in mM): 140 K-methane-sulfonate, 10 HEPES, 0.1 EGTA, 4 MgATP, and 0.3 NaGTP; pH 7.3 (solution #I, optimal for recording firing frequency accommodation and the sAHP amplitude; see Faber and Sah 2004). Membrane properties (except sAHP and firing frequency accommodation) were also examined with another solution (#II; in mM): 140 K-methane sulfonate, 10 HEPES, 5 EGTA, 2 MgCl2, 2 Na2ATP, and 0.3 NaGTP; this solution allows easy comparison with earlier studies from our laboratory. In some experiments Alexa hydrozin 568 (0.2 mg/ml; Molecular Probes) was added to this solution to stain cells intracellularly. After diffusion of the dye into the cell, the slices were fixed in 4% phosphate-buffered paraformaldehyde at 4°C for 30 min and rinsed three times with phosphate buffer. Slices were mounted on glass slides under a coverslip, with Vectashield (Vector Laboratories). Immunofluorescent cells in fixed sections were evaluated using a Zeiss LSM 510 (Carl Zeiss, Jena, Germany) confocal laser-scanning device equipped with a dry Plan-Neofluar x20/0.75 lens or a fluorescent microscope (Zeiss Axiophot; Carl Zeiss, Oberkochen, Germany). Cells examined under the confocal microscope were morphologically analyzed with Image J [National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/nih-image/ (De Simoni et al. 2003)] in combination with the Neuron_morpho plug-in (G. D'Alessandro, University of Southampton, Southampton, UK) and LMeasure (R. Scorcioni, Krasnow Institute for Advanced Studies, George Mason University, Fairfax, VA).
Neurons in the BLA were selected for recording if they displayed a pyramidal-shaped cell body, in agreement with the morphology of principal neurons in the BLA of various species (Paré et al. 1995; Rainnie et al. 1993; Washburn and Moises 1992; Yajeya et al. 1997). Interneurons, which are usually smaller and display multipolar, spineless dendrites, were avoided. Only cells with a resting membrane potential more negative than –55 mV were included in this study. Signals were recorded using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Foster City, CA). Responses were filtered at 5 kHz and digitized at 10 kHz (Digidata 1322A; Axon Instruments). All data were acquired, stored, and analyzed on a PC using pClamp 9.0 and Clampfit 9.2 (Axon Instruments). To investigate the firing properties and the properties of the afterhyperpolarization (AHP) after corticosterone or vehicle treatment we used current-clamp conditions. Prolonged current steps (100 or 600 ms) were applied from the resting membrane potential in the range of –200 to 400 pA with 40-pA increments. We included only reliable sAHP recordings, i.e., neurons in which the averaged sAHP for currents steps of 320–400 pA was larger than the averaged sAHP for current steps of 0–120 pA. The currents underlying the sAHP (IsAHP) were further examined under voltage-clamp conditions, by giving a 200-ms voltage step to 0 mV from a holding potential of –50 mV.
In situ hybridization
For in situ hybridization experiments, 16 Wistar rats were used. Animals received a single subcutaneous injection with corticosterone (10 mg/100 g body weight; dissolved in arachide oil) (cort group; n = 8) or vehicle solution (veh group; n = 8) in a total volume of 500 µl. One hour after the injection, at 9:30 am, animals were decapitated. Brains were dissected out of the skull and quickly frozen on dry ice. On a cryostat, 12-µm-thick sections containing the hippocampus and BLA were cut, put on SuperFrost Plus slides (Menzel-Glaser, Brunswick, Germany), and stored at –80°C.
Sections were fixed with 4% paraformaldehyde for 30 min and subsequently washed in phosphate-buffered saline. Then, sections were acetylated for 10 min in 0.1 M triethanolamine (pH = 8.0) with 0.25% acetic anhydride, washed in 2x saline sodium citrate (SSC) for 10 min, and dehydrated in an ethanol series (50, 80, 100, and 100%; 1 min each).
[35S]-dATP end-labeled desoxyoligonucleotide probes were used. Sequences were 5'-gtg ggt ggg gat tct cca tct gct gta atg gac ttc agc tca att (perfect match) or 5'-gtt ggt ggt gat tcg cca tcg gct gtc atg gaa ttc aga tca atg (mismatch control) for Cav1.2, and 5'-tgc taa gaa tga aga atg cgc ttc ctt cgg gga tgg gtg caa ttt (perfect match) or 5'-tga taa gca tga ata atg ctc ttc cgt cgg gta tgg ggg caa tgt (mismatch control) for Cav1.3 mRNA expression. After end-labeling, oligonucleotides were purified with chloroform extraction and ethanol-precipitated. Per slide, 100 µl hybridization mix containing 50% formamide, 10% dextran sulfate, 20 mM DTT, 25 mM NaSO4, 1 mM Na-pyrophosphate, 4x SSC, 5x Denhardt's solution, 100 µg/ml poly(A), 100 µg/ml hsDNA, and 1 x 106 cpm of the oligonucleotide probe was added. Sections were then coverslipped and incubated overnight at 42°C. The next day, coverslips were removed and sections were rinsed in 1x SSC at room temperature and subsequently washed in 1x SSC twice for 30 min at 50°C, and once for 5 min at room temperature. Slides were then dehydrated in an alcohol series, air-dried, and exposed to a Kodak Biomax MR film for 6 wk.
Four sections per probe per animal were scanned and loaded into Image J (Image J 1.37v). Gray values of the BLA as well as the hippocampal CA1 cell layer were measured and corrected for background. Per animal, the gray values for each region were averaged. After this, the values of all animals of the same group were averaged.
Computational model
The compartmental models of a CA1 pyramidal neuron and a BLA pyramidal neuron were implemented in the NEURON simulation environment (Hines and Carnevale 1997). The biophysical model of the CA1 pyramidal neuron was previously described (Sidiropoulou et al. 2007). The model consists of 183 compartments and includes a variety of passive and active membrane mechanisms known to be present in CA1 pyramidal cells. We assumed a uniform membrane resistance of Rm = 40 k·cm2, a uniform intracellular resistivity Ra = 70 ·cm, and a specific membrane capacitance of 1.0 µF·cm–2. The resting membrane potential of the model neuron was set at –66 mV. Active mechanisms included two types of Hodgkin–Huxley-type Na+ currents (axonal: INaa; dendritic: INad); three voltage-dependent K+ currents (IKdr, IA, IM); a fast Ca2+- and voltage-dependent K+ current (IfAHP); a slow Ca2+-dependent K+ current (IsAHP); a hyperpolarization-activated nonspecific cation current (Ih); a low-voltage–activated calcium current (ICaT); a persistent sodium current (INap); and four types of Ca2+- and voltage-dependent calcium currents (ICaN, ICaR, ICaL-1.3, ICaL-1.2). The ICaL-1.2 models of the L-type Ca2+ current carried by channels are composed of the Cav1.2 subunits, whereas the ICaL-1.3 models of the L-type Ca2+ current carried by channels are composed of the Cav1.3 subunits. Based on the available data (Marrion and Tavalin 1998), the ICaL-1.3 was colocalized with the IsAHP as previously described (Markaki et al. 2005). Channel equations, distributions, and densities of INa, IKdr, and IA are described in more detail elsewhere (Poirazi et al. 2003).
The reconstructed BLA pyramidal neuron was based on a three-dimensional confocal representation of an intracellularly stained neuron and constituted 48 compartments (one somatic, 47 dendritic, and one axonic compartment). We assumed a uniform membrane resistance Rm = 15 k·cm2 and a uniform intracellular resistivity Ra = 70 ·cm. The resting membrane potential was set at –66 mV and the input resistance (measured at –100 pA) at 150 M. Active mechanisms included one type of Hodgkin–Huxley-type fast Na+ current; a persistent sodium current (INap); three voltage-dependent K+ currents (IKdr, IA, IM); a fast Ca2+- and voltage-dependent K+ current (IfAHP); a slow Ca2+-dependent K+ current (IsAHP); a hyperpolarization-activated nonspecific cation current (Ih); a low-voltage–activated calcium current (ICaT); and three types of Ca2+- and voltage-dependent calcium currents (ICaN, ICaR, ICaL-1.3, ICaL-1.2). Based on the in situ hybridization data, the ICaL-1.3 was included in negligible amounts, and thus not colocalized with IsAHP. Both CA1 and BLA models were heavily validated with respect to passive and active properties of the neurons (for CA1: Markaki et al. 2005; Poirazi et al. 2003; for BLA: this study) as well as stress-induced changes in excitability (for CA1: Sidiropoulou et al. 2007; for BLA: this study).
Statistical analysis
Results are expressed as means ± SE and analyzed for statistical significance (P < 0.05) using a Student's t-test. In the case of the sAHP amplitude and the number of spikes evoked by a depolarizing pulse, data were analyzed with a generalized model for repeated measures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Recordings were made from 68 principal neurons in mouse and rat BLA. Visual inspection under the microscope revealed that these neurons had a pyramidal-shaped cell body, from which several (two to five) spiny dendrites branched in various directions. This was confirmed in part of the neurons (n = 22), which were filled with the intracellular dye Alexa hydrozin 568 (0.2 mg/ml; see typical example in Fig. 1A). Occasionally (n = 3) and on purpose, we filled neurons without an apparent pyramidal-shaped cell body. These neurons typically showed a very high firing frequency on depolarization (means ± SE: 51 ± 4 spikes for a 0.2-nA pulse of 600-ms duration; cf. in pyramidal-shaped BLA cells: 6 ± 1 spikes, n = 11) and no spines on their dendrites.
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(see DISCUSSION). As is evident from Table 1, treatment with 100 nM corticosterone for 20 min, 1–4 h before recording, did not change either the resting membrane potential or the input resistance of mouse principal BLA neurons. Similarly, properties of the action potential were unaffected by hormone treatment: Values for the action potential half-width, threshold, rise time, and amplitude (from threshold) were very comparable for the vehicle- and corticosterone-treated cells (Table 1). This was confirmed in slices from rats, in two sets of experiments using different solutions in the recording pipette (Table 1). Contrary to our expectation, corticosterone also did not significantly affect the amplitude of the sAHP in BLA neurons (Fig. 1). In accordance with the current-clamp data, voltage-clamp recording of the IsAHP revealed no difference between the corticosterone- and vehicle- treated BLA cells (Fig. 1). Also, the number of spikes elicited by a depolarizing pulse (0–400 pA, 600-ms duration) was comparable for the vehicle-treated group and corticosterone-treated cells.
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Expression of calcium channel subunits in the CA1 hippocampal area and BLA
The sAHP is known to be very sensitive to Ca-influx through L-type channels (Bowden et al. 2001; Lima and Marrion 2007; Marrion and Tavalin 1998; but see Pineda et al. 1998). Because corticosterone increases high-voltage–activated sustained (and presumably L-type) calcium currents in both BLA and CA1 principal neurons by about 100%, the lack of any effect on the sAHP in the BLA was unexpected. L-type calcium channels can be composed of Cav1.2 or Cav1.3 subunits, in combination with auxiliary subunits (Arikkath and Campbell 2003; Catterall 2000). Interestingly, there is evidence that channels responsible for the sAHP are colocalized with Cav1.3 rather than Cav1.2 subunits (Bowden et al. 2001). If so, only corticosteroid effects on Cav1.3-containing channels are expected to have functional consequences for the sAHP amplitude. We wondered whether corticosterone targets different calcium channel subunits in the CA1 and BLA. Therefore we investigated the distribution of Cav1.2 and Cav1.3 subunits and their transcriptional regulation by corticosterone with in situ hybridization.
As shown in Fig. 3, Cav1.2 mRNA expression was low to moderate in the CA1 area and the BLA. High expression levels were observed in the CA3 area and the dentate gyrus. Compared with vehicle-treated animals, rats that received corticosterone 1 h before tissue collection displayed similar Cav1.2 expression in the CA1 hippocampal area. In the BLA, a 20% nonsignificant (P > 0.1) up-regulation of Cav1.2 mRNA expression was observed.
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Influence of Cav1.2 and Cav1.3 on sAHP amplitude: a modeling approach
Electrophysiologically, it is not possible to distinguish currents generated by channels containing Cav1.2 versus Cav1.3 subunits. Thus we could not verify by electrophysiological means the subunit composition of L-type calcium channels in the BLA and CA1 hippocampal area or their corticosteroid modulation. Therefore we used a modeling approach to test whether the differential distribution of Cav1.2 and Cav1.3 subunits in the two brain regions and their corticosteroid dependence could explain the observed discrepancy in modulation of the sAHP amplitude.
Compartmental models of a CA1 pyramidal neuron and a BLA pyramidal neuron were implemented in the NEURON simulation environment (Hines and Carnavale 1997; for details see METHODS). The CA1 neuron model included both ICaL-1.2 and ICaL-1.3, whereas the BLA neuron model included ICaL-1.2 and extremely low amounts of ICaL-1.3. We questioned whether transcriptional regulation of the Cav1.2 or Cav1.3—which in the model was simulated by increasing the ICaL-1.2 and the ICaL-1.3, respectively—can explain the discrepancy in sAHP amplitude. To this end, ICaL-1.2 and ICaL-1.3 were increased by 20, 50, or 100%. These numbers were chosen based on the range of corticosteroid-induced changes described in the present and earlier studies, using either in situ hybridization, single-cell RNA amplification, or electrophysiological methods (Chameau et al. 2007; Joëls et al. 2003; Karst et al. 2002). Increasing the ICaL-1.2 by 20, 50, or 100% did not change the sAHP amplitude, either in the CA1 or in the BLA pyramidal model neuron (Fig. 4, A1 and A2). A similar increase in the ICaL-1.3 did enhance the sAHP amplitude in the CA1 model neuron, but only 
20%, whereas the electrophysiologically measured sAHP amplitude was increased by 30–60% (Fig. 2).
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; Joëls et al. 1997). We therefore next examined whether a decrease in the extrusion rate of the Ca pump in the CA1 area could be responsible for the discrepancy between the simulation and electrophysiological data. Figure 4B1 shows the effect of a 20, 50, or 100% increase of the ICaL-1.3 in combination with a decrease in the extrusion rate of the Ca pump in a CA1 neuron. The experimentally observed 30–60% increase in sAHP amplitude could be accurately simulated by, e.g., a 50% increase in ICaL-1.3 in combination with a 20 to 40% decrease in the Ca extrusion as well as by a 20% increase in ICaL-1.3 in combination with 40 to 60% decrease in the extrusion rate of the Ca pump (Fig. 4B1). Applying the same changes in the extrusion rate of intracellular calcium in the BLA neurons in combination with a 20, 50, or 100% increase in ICaL-1.2 leads to much smaller changes in the sAHP peak (Fig. 4B2). Traces comparing the sAHP in the CA1 and BLA model neurons under control (black line) and "stressed" (gray line) conditions are shown in Fig. 4, C1a and C2, a and b, respectively. To determine whether the differential changes in the sAHP amplitude between CA1 and BLA model neurons critically depended on the presence/absence of ICaL-1.3, the amount of ICaL-1.3 in the CA1 model neuron was largely reduced and its colocalization with the IsAHP was removed. Following that change, the sAHP in the CA1 model neuron only marginally increased under the "stressed" condition (Fig. 4C1b). The BLA neuron was also altered, in such a way as to resemble the biophysical mechanisms of the CA1 neuron (i.e., we increased ICaL-1.3 and colocalized it with the IsAHP). This was sufficient to evoke a much larger increase in the sAHP amplitude, under similar stressed conditions (Fig. 4C2c).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Karst et al. 1994, 2000, 2002; Kerr et al. 1992). We here tested the hypothesis that corticosterone—similar to what was reported earlier for the CA1 hippocampal area (Joëls and de Kloet 1989; Kerr et al. 1989)—increases the sAHP amplitude of BLA neurons, but does not affect other passive and active membrane properties. Using whole cell recording in mice, we here confirmed earlier findings (with sharp electrodes) from rat CA1 neurons that corticosterone indeed increases the amplitude of the sAHP. Contrary to our expectations, we found that principal neurons in the BLA do not exhibit a change in the amplitude of the sAHP (and the IsAHP). We argue that this can be (at least partly) explained by a different subunit composition of the calcium channels contributing to the L-type calcium current. Effects of corticosterone on membrane properties of CA1 and BLA neurons
Earlier studies with microelectrodes in rats have shown that administration of corticosteroid hormones does not consistently change the resting membrane potential, input resistance, or action potential properties of rat CA1 pyramidal neurons (Joëls et al. 1989; Kerr et al. 1989). This was confirmed in the present study using whole cell recording in mouse hippocampal slices. Similar to the CA1 hippocampal area, we also did not observe any change in these passive and active membrane properties in principal neurons of the BLA. This was found in mice as well as rats (using two different recording solutions). Contrary to our expectations, we also did not observe any change in the sAHP, IsAHP, or the spike frequency accommodation in the BLA. The sAHP is very sensitive to the recording conditions, more specifically to the composition of the pipette solution. The lack of any corticosteroid effect on the sAHP in BLA neurons, however, was not due to suboptimal recording conditions because we readily confirmed earlier findings in CA1 pyramidal neurons. We not only observed a delayed increase in the sAHP amplitude, but also extended these observations by demonstrating an increased IsAHP amplitude, 1–4 h after corticosterone administration.
We have several reasons to believe that our observations in the BLA were based on a homogeneous population of pyramidal(-like) neurons. First, recordings were confined to cells that under the recording microscope exhibited a pyramidal-shaped cell body, with a restricted number of dendrites branching from the soma. The shape seen under the recording microscope was confirmed in a group of cells that was intracellularly filled after recording and analyzed with a fluorescent or confocal microscope; all of these neurons had appreciable amounts of spines on their dendrites. Second, and importantly, all cells incorporated in the present analysis showed an input resistance well below 400 M and spike frequency accommodation, similar to properties reported earlier for pyramidal neurons and incompatible with interneurons. These morphological and electrophysiological characteristics are largely in line with earlier studies (Paré et al. 1995; Rainnie et al. 1993; Washburn and Moises 1992; Yajeya et al. 1997).
Very recently, a study appeared that reported that corticosterone depolarizes BLA neurons, increases their input resistance, and reduces the spike-frequency accommodation (Duvarci and Paré 2007). Small differences in recording conditions between this and our study, such as the intracellular chloride concentration, may have contributed to the discrepancies, particularly since glucocorticoids were found to shift the reversal potential of chloride-dependent conductances (Duvarci and Paré 2007). Also, it is noteworthy that the intercell variation in this recent study appeared to be larger, which may signify that perhaps various subpopulations of pyramidal neurons were included. Because it cannot be excluded that corticosterone has different effects on various subtypes of BLA neurons, less strict inclusion criteria may have led to a different outcome. The importance of strict inclusion criteria was indeed evident from our own study. Thus three pyramidal cells in the vehicle-treated control group were excluded from the overall analysis since they had a very high input resistance (means ± SE: 507 ± 27 M; each cell was >3SDs removed from the mean of the remaining cells). Their overall morphological features under the recording microscope (more specifically the pyramidal-shaped cell body and number of primary branches) could not be distinguished from those of the other cells. This is reminiscent of projection neurons in the lateral amygdala, which also display two subclasses of pyramidal-shaped neurons, i.e., a prevalent subclass of cells with an average membrane resistance of 270 M and an infrequently encountered subclass of cells with an average membrane resistance of 645 M (Sosulina et al. 2006). If the high-input-resistance cells were included in our analysis, the control group had on average a larger sAHP amplitude and spike-frequency accommodation than those of the corticosterone-treated cells, similar to what was reported in the afore-mentioned recent paper (Duvarci and Paré 2007). This "difference" disappeared completely when more strict inclusion criteria were applied.
Relevance of calcium channel subunit distribution
The sAHP is known to be sensitive to intracellular calcium levels. For instance, the increased sAHP amplitude seen with aging is thought to develop secondary to enhanced calcium influx through L-type channels (Markaki et al. 2005; Power et al. 2002; Thibault et al. 2007). This involves enhanced levels of the Cav1.3 subunit (Herman et al. 1998; Veng et al. 2003), much more so than of the Cav1.2 subunit (Davare and Hell 2003; Herman et al. 1998), although posttranslational modification of Cav1.2 may also play a role (Davare and Hell 2003). Corticosterone has been reported to strongly increase high-voltage–activated sustained calcium currents in CA1 neurons (Karst 1994, 2000; Kerr 1992), by doubling the number of functional L-type channels in the plasma membrane (Chameau et al. 2007). Interestingly, high-voltage–activated calcium currents were increased to a similar extent by GR activation in the BLA (Karst et al. 2002). Although no pharmacological distinction was made in that study between various types of currents, there is suggestive evidence that the increase pertained to the L-type current: First, GR activation increased the sustained component, whereas the low-voltage–activated transient component was in fact decreased in amplitude; second, single-cell Cav1.2 mRNA expression was increased after GR activation and correlated well with the current amplitude. Despite the comparable enhancement in L-type calcium currents induced in CA1 and BLA pyramidal neurons by corticosterone, the sAHP was enhanced in the former but not in the latter cells. In view of the role of the two L-type pore-forming subunits regarding the effect of aging on the sAHP amplitude, we wondered whether corticosterone may target different L-type calcium channel subunits in the CA1 area versus the BLA.
The general distributional pattern of Cav1.2 and Cav1.3 expression in the hippocampus that we observed with in situ hybridization was very comparable to earlier reports (Ludwig et al. 1997; Tanaka et al. 1995), with high expression of Cav1.2 in the CA3 and dentate gyrus and high levels of Cav1.3 primarily in the dentate gyrus. Expression in the CA1 region was comparatively low. The available literature on expression levels of L-type channel subunits is less informative about the BLA, since a subdivision of the amygdala nuclei was not given (Ludwig et al. 1997; Tanaka et al. 1995). A recent immunocytochemical report showed that Cav1.2 is present in BLA principal cells and has a subcellular distribution similar to that in CA1 pyramidal neurons (Pinard et al. 2005). Accordingly, we observed moderately high expression of Cav1.2 in the BLA. However, in this rat strain and the present conditions (e.g., age) Cav1.3 expression in the BLA was low, i.e., below the detection limit with in situ hybridization.
In the CA1 area, Cav1.2 expression was not affected by corticosterone. The Cav1.3 expression was increased by 50% compared with vehicle-injected controls, although the effect was far less prominent when compared with naïve, noninjected controls (<20%; Van Gemert, personal communication). The present observations on Cav1.2 and Cav1.3 expression largely agree with an earlier study where Cav1.2 and 1.3 expression was analyzed with qPCR after treating hippocampal slices (from naïve mice) with 100 nM corticosterone; in that study no significant effects of corticosterone were seen for either subunit (Chameau et al. 2007). We tentatively conclude that in the CA1 area the 100% increase in L-type calcium current is certainly not due to transcriptional regulation of Cav1.2 subunits; possibly, it can be explained by regulation of the Cav1.3 gene, at least under specific circumstances. In the BLA, it was earlier found with single-cell RNA analysis that 1–4 h after application of a selective GR agonist the relative Cav1.2 mRNA expression in single cells is significantly increased (Karst et al. 2002). Here we found—using the mixed agonist corticosterone, and examining the expression level in the overall population of cells—a 20% (although nonsignificant) up-regulation of Cav1.2 expression. The fact that we 1) now looked 1 h after corticosterone application as opposed to 1–4 h after the agonist in the earlier study and 2) did not restrict investigation of transcript level to the principals cells from which electrophysiological recordings were obtained may explain why, presently, the difference did not reach significance. After fear conditioning (a highly stressful situation), Cav1.2 protein level was also reported to be elevated (Shinnick-Gallagher et al. 2003). Thus contrary to the CA1 area, stress levels of corticosterone may affect Cav1.2 expression in the BLA. Since Cav1.3 expression was presently below the detection limit in the BLA, statements about GR regulation of this subunit are premature, although it is clear that corticosterone treatment did not raise Cav1.3 mRNA expression above the threshold for detection.
It is technically not possible to test functionally whether corticosterone targets different calcium channel subunits in the BLA versus CA1 region because these subunits cannot be distinguished with pharmacological tools. We therefore used a modeling approach to examine to what extent differential regulation of the subunits in the two brain areas could explain the diverging hormonal effects on the sAHP amplitude. We introduced three putative levels of transcriptional regulation (i.e., 20, 50, and 100%; see earlier text), based on earlier and current expression and electrophysiological data. For incorporation of ICaL-1.2 and ICaL-1.3 we relied on the present in situ hybridization observations. At the single-cell level, mRNA expression levels for these subunits were indeed found to correlate quite well with the current amplitude (Chen et al. 2000), although we certainly cannot exclude that posttranslational modification of channel subunits also plays a role in their functional relevance (see, e.g., Davare and Hell 2003). In the CA1 model, ICaL-1.2 was fivefold more abundant than ICaL-1.3, supported also by data showing that the L-type calcium current of CA1 neurons is reduced to <20% in Cav1.2 knockout mice (Moosmang et al. 2005). Levels of ICaL-1.3 in our BLA model were extremely low. The ICaL-1.3 in the CA1 model cell was incorporated in the soma and at the base of dendrites, whereas ICaL-1.2 (in both CA1 and BLA) was placed in the soma as well as in clusters along the dendritic tree, as seen in immunohistochemical studies (Hell et al. 1993; Pinard et al. 2005).
As expected, increasing the abundance of Cav1.2 does not change the sAHP amplitude, either in CA1 cells or in the BLA. Doubling the Cav1.3 levels did increase the sAHP amplitude in the CA1 model cell, but only by 20%. Interestingly, glucocorticoids repress the expression of the plasma membrane calcium pump isoform 1 in hippocampal cells (Bhargava et al. 2002). In accordance, corticosterone application to acutely dissociated CA1 neurons delays the extrusion of Ca (Joëls et al. 1997). We therefore considered a combined effect of corticosterone on Cav1.3 expression and Ca-extrusion rate. The simulation data indicate that such combined effects can indeed fully explain the observed changes in the sAHP (see Fig. 5). Similar changes in the Ca extrusion rate in the BLA model neuron resulted in quite smaller increases in the sAHP. Since no change in the sAHP was observed experimentally after glucocorticoid modulation the function of the Ca-extrusion pump may not be altered by glucocorticoids in the BLA. However, this remains to be confirmed experimentally. Alternative explanations, of course, cannot be ruled out. It is possible that corticosterone differentially regulates channels underlying the sAHP in the CA1 region versus the BLA, although qPCR investigation of the SK1 channel—which is colocalized with Cav1.3 subunits (Bowden et al. 2001)—did not reveal any regulation by corticosteroids in the CA1 region (Y Qin, S. Spijker, P. Chameau, A. B. Smit, and M. Joels, unpublished observation). Moreover, it has been disputed whether SK channels indeed underlie the sAHP in CA1 cells (Bond et al. 2004).
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The large sAHP amplitude and increased firing frequency accommodation seen 1–4 h after corticosterone levels peak have been interpreted as a means for CA1 cells to attenuate information flow through the CA1 area and thus to normalize local activity that is enhanced shortly after stress, due to rapid effects of catecholamines, neuropeptides, and steroids acting through nongenomic pathways (Joëls et al. 2007). If corticosterone does not change the sAHP and firing frequency accommodation in BLA principal cells this could signify that enhanced activity in these cells is less efficiently constrained several hours after stress exposure. Information with a strong emotional load, which will heavily activate BLA neurons, may therefore have longer-lasting consequences for limbic activity than neutral information.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Address for reprint requests and other correspondence: M. Joëls, SILS-CNS, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands (E-mail: joels{at}science.uva.nl)
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