Cellular Mechanisms for Amyloid beta -Protein Activation of Rat Cholinergic Basal Forebrain Neurons

Jack H. Jhamandas, Caroline Cho, Balvinder Jassar, Kim Harris, David MacTavish, and Jacob Easaw

Division of Neurology, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Jhamandas, Jack H., Caroline Cho, Balvinder Jassar, Kim Harris, David MacTavish, and Jacob Easaw. Cellular Mechanisms for Amyloid beta -Protein Activation of Rat Cholinergic Basal Forebrain Neurons. J. Neurophysiol. 86: 1312-1320, 2001. The deposition of amyloid beta -protein (Abeta ) in the brain and the loss of cholinergic neurons in the basal forebrain are two pathological hallmarks of Alzheimer's disease (AD). Although the mechanism of Abeta neurotoxicity is unknown, these cholinergic neurons display a selective vulnerability when exposed to this peptide. In this study, application of Abeta 25-35 or Abeta 1-40 to acutely dissociated rat neurons from the basal forebrain nucleus diagonal band of Broca (DBB), caused a decrease in whole cell voltage-activated currents in a majority of cells. This reduction in whole cell currents occurs through a modulation of a suite of potassium conductances including calcium-activated potassium (IC), the delayed rectifier (IK), and transient outward potassium (IA) conductances, but not calcium or sodium currents. Under current-clamp conditions, Abeta evoked an increase in excitability and a loss of accommodation in cholinergic DBB neurons. Using single-cell RT-PCR technique, we determined that Abeta actions were specific to cholinergic, but not GABAergic DBB neurons. Abeta effects on whole cell currents were occluded in the presence of membrane-permeable protein tyrosine kinase inhibitors, genistein and tyrphostin B-44. Our data indicate that the Abeta actions on specific potassium conductances are modulated through a protein tyrosine kinase pathway and that these effects are selective to cholinergic but not GABAergic cells. These observations provide a cellular basis for the selectivity of Abeta neurotoxicity toward cholinergic basal forebrain neurons that are at the epicenter of AD pathology.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Deposition of amyloid protein in the form of diffuse and neuritic plaques is an important pathological hallmark of Alzheimer's disease (AD) (Hardy 1997; Selkoe 1999). The major component of the neuritic amyloid plaques is amyloid beta -protein (Abeta ), a 39-43 amino acid peptide that is generated from a larger protein, the amyloid precursor protein (APP). There is considerable evidence to suggest that Abeta and other peptide fragments derived from APP influence cellular homeostasis and neuronal signaling through modulation of ion channel function (for review see Fraser et al. 1997).

The interactions of Abeta within the membrane occur either with preexisting ionophores or through the formation of de novo ion channels (Arispe et al. 1996; Fraser et al. 1997). Abeta 25-35, an 11 amino acid fragment considered to represent the neurotoxic domain of the parent Abeta peptide, activates large, nonselective cation currents in bullfrog sympathetic and rat hippocampal neurons (Furukawa et al. 1994; Simmons and Schneider 1993). Abeta 25-35 and Abeta 1-40 induce Ca2+ influx through voltage-gated channels in cortical and NIE-115 neuroblastoma cells, respectively (Davidson et al. 1994; Weiss et al. 1994). As yet no receptor for Abeta has been identified, but its neurotoxic effects have been postulated to be mediated via plasma membrane receptors for advanced glycation end products (RAGE), class A scavenger receptor (SR)-related proteins, and/or the 75 kDa-neurotrophin receptor (El Khoury et al. 1996; Kuner et al. 1998; Yan et al. 1996). Although Abeta modulation of ionic conductances has been studied in many neuronal and nonneuronal systems, the linkage of these observations to changes in neuronal excitability is less well understood.

It is now well accepted, that apart from Abeta deposition, certain chemically defined neurotransmitter systems, particularly the cholinergic basal forebrain neurons, display a selective vulnerability and degeneration in AD (Price 1986). Emerging data support a potential link between Abeta peptides and the basal forebrain cholinergic system. Abeta peptides can inhibit the release of endogenous acetylcholine and high-affinity choline uptake from the hippocampus and cortex (Kar et al. 1996, 1998). There is Abeta -mediated inhibition of acetylcholine synthesis in cultured cholinergic neurons (Pedersen et al. 1996). Single injection of Abeta into the basal forebrain, but not the striatum, induces damage to cholinergic neurons (Butcher et al. 1997). These observations indicate that the chemical phenotype of an individual cell is an important feature of Abeta toxicity, a notion that is strengthened by the finding that GABA-containing neurons of the hippocampus exhibit a relative resistance to Abeta in sharp contrast to the vulnerability of cholinergic neurons exposed to this peptide (Pike and Cotman 1993). However, whether the effects of Abeta are selective to cholinergic basal forebrain neurons, which are at the epicenter of AD pathology, is an important but unresolved question.

In this study, we investigated the actions of Abeta on acutely dissociated rat cholinergic basal forebrain neurons from the nucleus of the diagonal band of Broca (DBB) using a combination of whole cell patch-clamp and single-cell reverse transcription polymerase chain reaction (RT-PCR) analysis. Our data show that the blockade of specific potassium conductances is a potential underlying mechanism for the action of Abeta on DBB neurons and may explain its effects in regulating their excitability. Finally, we identify tyrosine phosphorylation as an intracellular signal transduction pathway for these actions.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dissociation procedures

Details of the procedure for acute dissociation of neurons from the DBB are described in Jassar et al. (1999). Briefly, brains were quickly removed from decapitated male Sprague-Dawley rats (15-25 day postnatal) and placed in cold artificial cerebrospinal fluid (ACSF) that contained (in mM) 140 NaCl, 2.5 KCl, 1.5 CaCl2, 5 MgCl2, 10 HEPES, and 33 D-glucose 33 (pH 7.4). Brain slices (350 µm thick) were cut on a vibratome, and the area containing the DBB was dissected out. Although most of the tissue contained the horizontal limb of the DBB, some slices may have included a component of the vertical limb of the DBB. Acutely dissociated neurons were prepared by enzymatic treatment of slice with trypsin (0.65 mg/ml) at 30°C, followed by mechanical trituration for dispersion of individual cells. Cells were then plated on poly-L-lysine (0.005% wt/vol)-coated cover slips and viewed under an inverted microscope (Zeiss Axiovert 35). All solutions were kept oxygenated by continuous bubbling with pure oxygen.

Electrophysiological recordings

Whole cell patch-clamp recordings were performed at room temperature (20-22°C) using an Axopatch-1D amplifier. Series resistance compensation was continuously adjusted to >80% and monitored and readjusted as necessary during the course of each experiment. Junction potential was nulled with the pipette tip immersed in the bath. Internal patch pipette solution contained (in mM) 140 K-methylsulfate, 10 EGTA, 5 MgCl2, 1 CaCl2, 10 HEPES, 2.2 Na2-ATP, and 0.3 Na-GTP (pH 7.2). Putative acutely dissociated DBB neurons were initially identified for recording by visual inspection. Current-voltage relationships and excitability characteristics were used to distinguish neurons from glial or other cell types. Whole cell recordings were also done in the bridge current-clamp mode using an Axoclamp-2B amplifier to examine the effects of Abeta on current-evoked changes in excitability of the acutely dissociated DBB neurons. Action potentials were evoked by brief current injection (0.6-1.5nA, 600 ms duration) through the patch pipette. The resting membrane potential (RMP), number of spikes elicited, and interspike intervals were recorded for comparison under different experimental conditions. The membrane currents (voltage-clamp experiments) or the membrane voltages (current-clamp experiments) were recorded and analyzed on computer using pCLAMP software (version 6.0.3).

After whole cell configuration was established, we waited at least 5 min for steady-state currents to stabilize. The filter was set at 20 kHz during data acquisition. Cells were held in voltage clamp at -80 mV, which was close to the RMP observed in earlier studies on neurons from basal forebrain slices (Alonso et al. 1994; Easaw et al. 1997). A 1-s long hyperpolarizing command to -110 mV was applied to remove inactivation of K+ channels so that the maximum current could be activated during the subsequent slow voltage ramp to +30 mV (20 mV/s) that followed it. No obvious tail currents were observed at the end of the ramp when the command potential was returned to -80 mV, suggesting that the ramp elicited mainly steady-state currents.

Cell size was estimated electronically using the whole cell capacitance compensation circuit on the Axopatch-1D amplifier. Maximum voltage-clamp error in recording a current of 10 nA using a patch electrode with an electrode resistance of 5 MOmega was 10 mV. This reflects the average maximum error since the currents recorded were usually smaller than 10 nA. In figures displaying difference currents, capacitance transients have been truncated.

To examine the effects of Abeta on the contribution of Ca2+ to voltage-dependent ionic currents, we utilized an external solution that was nominally Ca2+-free and contained 50 µM Cd2+. In this solution CaCl2 was replaced with an equimolar concentration of MgCl2. To record currents through calcium channels, we used Ba2+ as a charge carrier as previously described (Easaw et al. 1999). The external solution contained (in mM) 150 tetraethylammonium chloride, 2 BaCl2, 10 HEPES, and 30 glucose (pH to 7.4 with TEA-OH). The internal patch pipette solution consisted of (in mM) 130 Cs-methanesulfonate, 2 MgCl2, 10 HEPES, 10 BAPTA, 4 Mg-ATP, 0.3 Na-GTP, and 0.1 Leupeptin (pH to 7.2 with CsOH). Depolarizing voltage steps from -80 to +70 mV (increment 10 mV/step; 20 ms duration) were applied to voltage-clamped DBB neurons under control conditions and in the presence of Abeta . Leak currents were minimal under our recording conditions. They did not change during the recordings and were not affected by application of Abeta . Therefore we did not subtract these in subsequent measurements of steady-state barium currents.

We also studied the effects of Abeta on INa. To isolate INa, the external solution contained (in mM) 125 NaCl, 20 TEA-Br (to block K+ currents), 2 MgCl2, 5 MnCl2 (to block Ca2+ currents), 10 HEPES, and 20 glucose (pH 7.4 with Tris-OH), and the internal solution consisted of (in mM) 130 cesium-methanesulfonate, 10 HEPES, 10 BAPTA, 5 Mg-ATP, and 0.3 Na2-GTP (pH 7.2 with Cs-OH). The currents were evoked by 10-ms voltage steps from -80 mV to a maximum of +60 mV (increment 10 mV/step; 10 ms duration) in the presence and absence of Abeta .

Drugs and solutions

All chemicals were purchased from Sigma (St. Louis, MO) except the following: Abeta 1-40, Abeta 25-35, Abeta 35-25 (QCB Biosource International, Camarillo, CA); genistein and daidzein (ICN, Costa Mesa, CA); and tyrphostin B-44 (Calbiochem, San Diego, CA). Stock solutions of Abeta peptides were prepared by dissolving the peptides at 1 mM in deionized water and stored in aliquots at -70°C (Ueda et al. 1997). On the day of the experiment, Abeta peptides were diluted in external perfusing medium just before the time of application. All drugs and chemicals were applied via bath perfusion at the rate of 3-5 ml per minute, which allowed complete exchange in less then half a minute. Data are presented as means ± SE. Student's two-tailed t-test (paired when appropriate) was utilized for determining significance of effect.

Single-cell RT-PCR for chemical phenotyping

Where possible, neurons were harvested after electrophysiological recordings were completed and readied for RT-PCR according to a previously described protocol (Surmeier et al. 1996). In brief, contents of the electrode containing the cell and 5 µl of internal solution were expelled into a 0.2-ml PCR tube containing 5 µl sterile water (Sigma water W-4502), 0.5 µl dithiothreitol 0.1 M (DTT), 0.5 µl RNasin (10 U/µl), and 1 µl oligo-dT (0.5 µg/µl). The tube was then placed on ice. Single-stranded cDNA was then synthesized from mRNA by adding a solution containing 1 µl SuperScript II RT (200 U/µl), 2 µl 10 × PCR buffer, 2 µl 25 mM MgCl2, 1.5 µl 0.1 M DTT, 1 µl 10 mM dNTPs, and 0.5 µl RNasin (10 U/µl). The PCR tube was gently mixed and incubated in a Techne Progene thermal cycler at 42°C for 50 min. The process was then terminated by heating to 72°C for 15 min, and the tube cooled to 4°C. Subsequently 2 µl of the RT product was taken and combined with 5 µl 10 × PCR buffer, 5 µl 25 mM MgCl2, 0.5 µl Taq polymerase (5 U/µl), 31.5 µl sterile water (sigma water W-4502), 1 µl 25 mM dNTP mixture, and 1.5 µl of a specific set of primers (15 µM). All reagents were purchased from GIBCO BRL. Primer sequences for choline acetyltransferase (ChAT) and for glutamate decarboxylase (GAD) have been previously described (Surmeier et al. 1996; Tkatch et al. 1998), and that for beta -actin was obtained from GenBank (the lower primer 5'-GAT AGA GCC ACC AAT CCA C, the upper primer 5'-CCA TGT ACG TAG CCA TCC A). All primers were synthesized at the University of Alberta Department of Biochemistry. The contents were mixed together and placed in the thermal cycler. The PCR amplification protocol was as follows: step 1: 94°C 4 min; step 2: 94°C 1 min, 53°C 1 min, 72°C 45 s (step 2 was repeated 35 times); step 3: 72°C 15 min; step 4: held at 4°C. A portion of the product was then run on a 2% TEA agarose gel, and the gel was then placed in a bath containing 2 µg/ml of ethidium bromide, after 10-min DNA bands were visualized with ultraviolet light box and photographed with a Polaroid camera.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Most of the acutely dissociated neurons from the DBB had neuronlike morphology (i.e., large cells with a conspicuous nucleus, nucleolus, and a few blunt processes that were truncated axon/dendrites). Under our recording conditions, the average input conductance measured from the slope of the current-voltage (I-V) relationships between -60 and -110 mV was 1.20 ± 0.16 nS (mean ± SE, n = 51).

Based on the previous observations (Jassar et al. 1999), we utilized a voltage-ramp protocol where the cells were held at -80 mV and subjected to voltage ramps from -110 to +30 mV at the rate of 20 mV/s after conditioning at -110 mV for 1 s.

Effects of Abeta on potassium currents

Whole cell potassium currents were recorded under control conditions and in the presence of Abeta (range of concentration 0.1-2.0 µM). The concentration of Abeta (1 µM) that we used in the present experiments is among the lowest used in previous electrophysiological studies of this peptide in acutely dissociated neurons (range 2-100 µM) (reviewed in Fraser et al. 1997) or in brain slices (range 200 nM to 2 µM) (Pettit et al. 2001; Wu et al. 1995). In 51 DBB neurons, the outward currents in the voltage range from -30 to +30 mV were decreased by both the longer isoform of the peptide (Abeta 1-40) and the truncated active peptide fragment (Abeta 25-35). Maximal effects of Abeta on whole cell currents were observed within 90 s application, and the response did not desensitize with repeated applications of the peptide. Figure 1, A and B, shows the reversible decrease in outward currents caused by application of Abeta 25-35 (1 µM) and Abeta 1-40 (1 µM), respectively. The amplitude of the currents at +30 mV was decreased significantly in the presence of Abeta 25-35 (control = 6.67 ± 0.24 nA, Abeta 25-35 = 5.72 ± 0.23 nA, n = 51, P < 0.001) or Abeta 1-40 (control = 7.52 ± 0.72 nA, Abeta 1-40 = 6.43 ± 0.70 nA, n = 12, P < 0.001). The percent reduction in amplitude of currents at +30 mV was also similar with both peptides (Abeta 25-35 = 14.21 ± 1.34%, Abeta 1-40 = 14.88 ± 4.16%, P = 0.8). The scrambled peptide fragment (reverse fragment, Abeta 35-25) had no effect on the whole cell current-voltage relationships (not illustrated). Since the effects of Abeta 1-40 and Abeta 25-35 on whole cell currents were essentially identical, we therefore utilized active peptide fragment Abeta 25-35 (1 µM) in all subsequent experiments. In 11 DBB neurons, Abeta 25-35 did not evoke any change in whole cell current or caused an increase or decrease in whole cell current that was <5% from control values at +30 mV. An example of a cell that was nonresponsive to Abeta is shown in Fig. 1C.



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Fig. 1. Abeta effects on whole cell currents. A: current-voltage (I-V) plots of whole cell currents from a diagonal band of Broca (DBB) neuron evoked under control conditions, in the presence of 1 µM Abeta 25-35 and 10 min wash out after Abeta application. The voltage protocol used for evoking whole cell currents is shown in the inset. B: I-V plot of whole cell currents from a DBB neuron evoked under control conditions, in the presence of 1 µM Abeta 1-40 and 10 min wash out after Abeta . Both fragments of Abeta (Abeta 25-35 and Abeta 1-40) decreased outward currents in the voltage range from -30 to +30 mV. C: I-V plot from a DBB neuron that did not respond to Abeta 25-35.

The outward potassium currents are a mixture of calcium-activated and noncalcium-activated components. Calcium-activated currents include the voltage-sensitive conductances called maxi gK(Ca) (IC) and the voltage-insensitive ones that underlie action potential afterhyperpolarization (IAHP). Noncalcium-activated conductances consist of the delayed rectifier, M- and A-conductances (IK, IM, and IA, respectively) and sodium currents. In DBB neurons, there was little evidence for IM relaxation in the currents evoked by hyperpolarizing commands (10 mV/step) to -110 mV from a holding potential of -30 mV. Therefore IM was not investigated in detail in this study. We studied the effects of Abeta on IC, IK, and IA as well as on calcium (ICa) and sodium (INa) currents.

EFFECTS OF Abeta ON CALCIUM-ACTIVATED POTASSIUM CURRENTS. Of the two main Ca2+-activated potassium currents, under whole cell recording conditions, IAHP makes little contribution and majority of the currents flow through the voltage-sensitive Ca2+-activated potassium channels, IC (Jassar et al. 1999). To elucidate the contributions of these conductances to Abeta effects, we examined Abeta actions under conditions where the external perfusion solution was replaced with the one that was Ca2+-free (0 mM Ca2+) and contained 50 µM Cd2+. Figure 2A shows the average of current-voltage relationships obtained from 17 neurons under control conditions, with 0 mM Ca2+ external medium, and with Abeta in the presence of 0 mM Ca2+ external. Replacing the external solution with 0 mM Ca2+ decreased the currents by 18.58 ± 2.46% (control = 6.94 ± 0.47 nA, 0 mM Ca2+ = 5.62 ± 0.40 nA, n = 17). Application of Abeta under these conditions further reduced the currents by 8.77 ± 1.72% (0 mM Ca2+ Abeta  = 5.16 ± 0.41 nA, n = 17, P < 0.001). We also studied Abeta effects in the presence of charybdotoxin (CTX) and iberiotoxin, specific blockers of IC channels. Figure 2B shows the average of I-V relationships obtained from 19 neurons under control conditions, in the presence of CTX (25 nM) and Abeta application in the presence of CTX. CTX reduced the outward currents at +30 mV by 12.89 ± 2.56% (control = 6.62 ± 0.37 nA, CTX = 5.76 ± 0.37 nA, n = 19). Application of Abeta in the presence of CTX further reduced the currents by 7.81 ± 1.83% (CTX + Abeta  = 5.31 ± 0.33 nA, n = 19, P < 0.001). The percent reduction of currents by Abeta in the presence of CTX was not significantly different from that obtained by omitting Ca2+ from the external perfusate (P = 0.09). In seven cells, we also examined the effects of Abeta in the presence of iberiotoxin (50 nM). Abeta reduction of whole cell currents with iberiotoxin was similar (10.2 ± 2.5%, not illustrated) to that observed with CTX.



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Fig. 2. Effects of Abeta on whole cell potassium currents. A: I-V plots of mean whole cell currents from DBB neurons (n = 17) evoked under control conditions, in 0 mM Ca2+, and in 1 µM Abeta 25-35 in 0 mM Ca2+. B: I-V plots of mean whole cell currents from DBB neurons (n = 19) evoked under control conditions, in 25 nM charybdotoxin, and in 1 µM Abeta 25-35 in charybdotoxin. C: I-V relationships of mean barium currents (IBa) in DBB neurons (n = 9) under control conditions, in the presence of 1 µM Abeta 25-35, and on wash out of Abeta . D and E: the effects of Abeta 25-35 (1 µM) on IK and IA in a DBB neuron, respectively. D: voltage protocol for recording IK is depicted on the left with holding potential of -80 mV and a 150-ms conditioning pulse to -40 mV. In this protocol, outward currents are mediated through IK (delayed rectifier) and IC (calcium-activated potassium conductance). E: in the same neuron, Abeta causes a decrease in transient outward K+ currents (IA). IA was obtained as difference currents by subtracting the currents obtained by the voltage protocol shown in D from that obtained by applying the voltage protocol shown in E where cells were held at -80 mV and a 150-ms conditioning pulse to -120 mV was applied.

EFFECTS OF Abeta ON CALCIUM CURRENTS. Since Abeta reduction of whole cell currents is attenuated by approximately 50% in the presence of CTX or by the removal of external Ca2+, Ca2+-activated currents appear to play an important role in the response of DBB neurons to Abeta application. This can result from either an effect on Ca2+-dependent conductances, i.e., IC or, a more upstream effect on Ca2+ channels that in turn may activate potassium conductances. To examine this issue, we recorded barium currents (IBa) flowing through Ca2+ channels. Figure 2C shows the average I-V relationships of IBa recorded from nine neurons. Abeta did not significantly affect the IBa (control = 3.32 ± 0.23 nA, Abeta  = 3.19 ± 0.25 nA, n = 9, P = 0.12, at -10 mV).

EFFECTS OF Abeta ON TRANSIENT OUTWARD (IA) AND THE DELAYED RECTIFIER (IK) POTASSIUM CURRENTS. IA and IK are voltage-sensitive currents, and their activation and inactivation are strongly voltage dependent. IA requires the holding potential to be relatively hyperpolarized (approximately -110 mV) for removal of its inactivation, whereas it is inactivated at -40 mV. On the other hand, IK is not inactivated at -40 mV. These biophysical properties of IA and IK can thus be utilized to isolate these currents. Therefore a conditioning pulse to -40 mV will activate IK without any significant contamination by IA (Connor and Stevens 1971; Easaw et al. 1999). A conditioning pulse to -120 mV will activate both IA and IK. The difference currents obtained by subtracting the currents evoked by depolarizing pulses following a conditioning pulse to -40 mV from those evoked following a conditioning pulse to -120 mV provide an accurate estimate of IA (Connor and Stevens 1971; Easaw et al. 1999). Figure 2D shows the currents recorded from a neuron with a conditioning pulse to -40 mV for 150 ms, representing mainly IK, under control conditions, in the presence of Abeta and recovery on wash out of Abeta . Abeta reduced IK by 10.93 ± 2.4% (control = 7.14 ± 0.42 nA, Abeta  = 6.41 ± 0.45 nA, n = 34, P < 0.001 at +30 mV). Figure 2E shows the difference currents recorded from the same neuron representing mainly IA, under control conditions, in the presence of Abeta , and on wash out. IA was reduced significantly by Abeta by 16.96 ± 3.89% (control = 5.42 ± 0.58 nA, Abeta  = 4.58 ± 0.55 nA, n = 34, P < 0.001). We have previously shown that the residual sustained current remaining at the end of the 100-ms test pulse (shown in Fig. 3B) consists mainly of IK and IC (Easaw et al. 1999), both of which are also reduced by Abeta .



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Fig. 3. Abeta effects on TEA-sensitive potassium (IK) and sodium currents. A: I-V plots of mean whole cell currents from DBB neurons (n = 7) evoked under control conditions, in 5 mM TEA, 1 µM Abeta 25-35 in TEA, and 10 min wash out after Abeta . B: I-V relationships of mean sodium currents (INa) in DBB neurons (n = 11) under control conditions, in the presence of 1 µM Abeta 25-35, and on wash out of Abeta .

Tetraethylammonium (TEA) ions at a concentration of 5 mM block IK and IC (Jassar et al. 1999). Figure 3A shows the average I-V relationships obtained from seven neurons under control conditions, with 5 mM TEA alone, and Abeta in the presence of TEA. TEA blocked 87.25 ± 2.97% of the outward current at +30 mV (control = 6.30 ± 0.92 nA, TEA = 0.87 ± 0.26 nA, n = 7). Abeta failed to produce any significant effect on the remaining currents in the presence of TEA (TEA + Abeta  = 0.80 ± 0.23 nA, n = 7, P = 0.22).

EFFECTS OF Abeta ON SODIUM CURRENTS. Sodium currents are involved in the fast depolarizing phase of the action potential. Enhancement of these currents can increase the excitability and vice versa. We recorded sodium currents in isolation to assess if Abeta has any effects on these currents. The sodium currents were TTX sensitive. Figure 3B shows the average I-V relationships obtained from 11 cells under control conditions, in the presence of Abeta , and recovery on wash out of Abeta . Abeta did not influence sodium currents in DBB neurons (control = -6.34 ± 0.59 nA, Abeta  = -6.08 ± 0.57 nA, P = 0.12 at +20 mV).

Effects of Abeta on excitability of DBB neurons

Application of Abeta results in depolarization of the RMP, increase in excitability, and loss of accommodation. Figure 4A depicts a DBB neuron showing accommodation under control conditions. On application of Abeta , the number of spikes evoked by injecting the same amount of current as under control conditions in the same neuron was increased, indicating an increase in excitability (Fig. 4B). Under control conditions, the average RMP was -65.5 ± 2.2 mV, which depolarized to -56.4 ± 1.8 mV (n = 21, P < 0.001) on Abeta application and recovered to 72.0 ± 3.3 mV on wash out. The average number of spikes elicited by current injection was 8.9 ± 1.5 under control conditions, 13.9 ± 1.4 in the presence of Abeta , and 6.4 ± 1.8 on wash out (n = 21, P < 0.001). In addition to the increase in excitability, Abeta also caused a loss of accommodation. The interspike interval between the first two and the last two action potentials provides a measure of accommodation. Under control conditions the first interspike interval was 46.5 ± 2.3 ms, and the last interspike interval was 90.1 ± 16.3 ms (n = 6, Fig. 4C). In the presence of Abeta , the first interspike interval was 33.3 ± 1.8 ms, and the last interspike interval was 42.1 ± 2.9 ms. On recovery the first interspike interval was 48.1 ± 1.5 ms, and the last interspike interval was 93.6 ± 18.1 ms.



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Fig. 4. Abeta actions on neuronal excitability of DBB neurons. Action potentials evoked by sustained current injection (600 ms, 1.0 nA) under control conditions (A) and in the presence of 1 µM Abeta 25-35 (B and C). Bar histograms comparing the mean interspike interval between the 1st 2 and the last 2 action potentials (APs) within a train of APs evoked by sustained current injection in DBB neurons (n = 6) under control conditions and in the presence of Abeta (1 µM). Note that in the presence of Abeta , the difference between the 1st and last interspike intervals is diminished consistent with a loss of accommodation. * Significant difference of the last interspike interval from 1st interval (under control conditions) at P < 0.005.

A larger difference between the first and last interspike intervals indicates a greater degree of accommodation. Abeta significantly reduced the difference between the first and the last interspike intervals consistent with a loss of accommodation (P < 0.005).

Chemical phenotype of the Abeta responsive neurons

There are two main chemical neurotransmitter phenotypes represented in the DBB neurons: GABAergic and cholinergic. Whole cell recordings were made from a heterogeneous population of DBB neurons. Definitive determination of the chemical phenotype was done by single-cell RT-PCR analysis. ChAT was used as a specific marker for cholinergic neurons, and GAD was used as a specific marker for GABAergic neurons. Figure 5 shows the photograph of a gel indicating RT-PCR products from an Abeta -responsive cell shown in Fig. 1A and also an Abeta -nonresponsive neuron (Fig. 1C). The Abeta responsive cell on the left reveals a band corresponding to the molecular weight of the ChAT primer, and the Abeta nonresponsive cell in the middle of the gel shows a band corresponding to the molecular weight of GAD primer. Results from 81 DBB neurons that were recorded in either voltage- or current-clamp modes and in which PT-PCR reaction was unequivocal are summarized in Fig. 5, bottom. All cells that responded Abeta with a reduction in whole cell currents or an increase in excitability were ChAT positive (n = 63) and GAD negative. On the other hand, all the Abeta -nonresponsive neurons were GAD positive (n = 18) and ChAT negative.



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Fig. 5. Single-cell RT-PCR analysis of DBB neurons. Photograph of a gel showing 2 cells: one that is Abeta responsive (the same cell as shown in Fig. 1A), which shows a band corresponding to the molecular weight (MW) of the choline acetyltransferase (ChAT) primer, and another that was Abeta nonresponsive (the same cell as in Fig. 1C) and shows a band corresponding to the MW of the GAD primer. The beta -actin lane on the gels serves as a control as does the whole rat brain mRNA. MW for beta -actin is ~625, ChAT ~308, and GAD ~400. Table at bottom shows summary of data on cells characterized for the response to Abeta .

Previous studies have suggested that larger dissociated cells from the basal forebrain are more likely to be cholinergic (Griffith et al. 1994; Jassar et al. 1999). In the present study the average membrane capacitance estimated electronically was 16.7 ± 0.5 pF (n = 171; range 12-29 pF). ChAT-positive cells had an average membrane capacitance of 16.4 ± 0.7 pF (range, 13-21 pF), whereas GAD-positive cells were found to have a capacitance of 18.2 ± 1.4 pF (range, 14-22 pF). Therefore, on the basis of our results, it would seem that cell size correlates poorly with the chemical identity of a particular cell as determined by single cell RT-PCR.

Involvement of protein tyrosine phosphorylation in Abeta response

Protein tyrosine phosphorylation modulates voltage- and ligand-gated channels to influence neuronal function (Raymond et al. 1993; Wang and Salter 1994). In PC 12 cells and olfactory neuroblasts, application of Abeta 1-40 and Abeta 25-35 induces a rapid and dose-dependent tyrosine phosphorylation that is accompanied by a rise in cytosolic Ca2+ (Luo et al. 1995). The DBB cells are enriched with protein tyrosine kinase (PTK) activity, and we have previously shown that GABA responses in these cells are modulated by PTK phosphorylation (Jassar et al. 1997). We investigated whether PTK may play a role in Abeta -evoked responses in DBB neurons. Genistein and tyrphostin B-44 are relatively specific membrane-permeable blockers of PTK (Valenzuela et al. 1995; Wang and Salter 1994). Figure 6A shows the average I-V relationships from 7 neurons under control conditions, in the presence of genistein (100 µM), and Abeta in the presence of genistein. Figure 6B shows the average I-V relationships from six neurons under control conditions, in the presence of tyrphostin B-44 (50 µM), and Abeta in the presence of tyrphostin. These compounds decreased the whole cell outward currents by 60.3 ± 4.97% (control = 6.76 ± 0.55 nA, genistein = 2.66 ± 0.42 nA, n = 7) and 55.01 ± 3.04% (control = 5.87 ± 0.39 nA, tyrphostin = 2.63 ± 0.21 nA, n = 6), respectively. However, Abeta failed to affect these outward potassium currents in the presence of genistein or tyrphostin (genistein + Abeta  = 2.44 ± 0.34 nA, 63.48 ± 4.06%, P > 0.05; tyrphostin + Abeta  = 2.48 ± 0.18 nA, 57.54 ± 2.30%, P > 0.07). The response to Abeta , a reduction of 11.45 ± 0.82% of the outward currents at +30 mV (n = 6), was not significantly affected in the presence of daidzein (100 µM), an inactive analogue of genistein (Fig. 6C, control = 6.85 ± 0.32 nA, daidzein = 6.44 ± 0.36 nA, daidzein + Abeta  = 5.71 ± 0.34 nA, n = 6, P < 0.001).



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Fig. 6. Effects of PTK inhibitors and analogues on Abeta response. A: I-V plot of mean currents evoked by voltage ramps under control conditions, in the presence of 100 µM genistein, and in the presence of 1 µM Abeta with genistein (n = 7). B: I-V relationship of mean currents evoked by voltage ramps under control conditions, with application of 50 µM tyrphostin B-44, and with application of 1 µM Abeta in the presence of tyrphostin (n = 8). C: I-V plot of mean currents obtained from voltage ramps under control conditions, in the presence of 100 µM daidzein, and in the presence of 1 µM Abeta with daidzein (n = 8).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

These experiments demonstrate four major findings. First, Abeta reduces a suite of potassium currents in basal forebrain neurons, including calcium-activated potassium (IC), the delayed rectifier (IK), and transient outward potassium (IA) conductances, but not calcium or sodium currents. Second, under current-clamp conditions, application of Abeta evoked an increase in excitability and a loss of accommodation in cholinergic DBB neurons. Third, using single-cell RT-PCR analysis, we show that Abeta actions are specific to cholinergic, but not GABAergic DBB neurons. Finally, the Abeta effects on cholinergic DBB neurons appear to be mediated via activation of protein tyrosine kinase signaling pathway.

Abeta modulation of ionic conductances and neuronal excitability

Abeta and related amyloidogenic metabolic fragments have been shown to alter cellular ionic conductances with existing channels or by de novo channel formation (Fraser et al. 1997). Such alteration in ionic homeostasis has been linked to the ability of Abeta to induce cell death and may provide a molecular mechanism for neurodegeneration seen in AD (Mattson et al. 1992; Yu et al. 1998). In hippocampal neurons, sAPP, a larger fragment of the parent APP, activates K+ channels and reduces intracellular Ca2+ through cGMP production and protein dephosphorylation (Furukawa et al. 1996). Suppression of excitability and membrane hyperpolarization by such mechanisms have been advanced to support, in part, a "neuroprotective" role for sAPP. However, although Abeta has been shown to activate a wide variety of K+ and Ca2+ conductances, there is limited information on its role in influencing neuronal excitability. Our data show that Abeta decreases IC currents without influencing calcium currents and leads us to conclude that Abeta has a direct effect on IC channels. Since IC is responsible, in part, for the repolarization phase of the action potential and plays an important role in the process of spike frequency adaptation (accommodation) (Vergara et al. 1998), Abeta -induced blockade of IC that we have observed could explain the increase in excitability and loss of accommodation seen with Abeta . We have previously shown that in DBB neurons at RMP, inhibition of IC with either charybdotoxin or iberiotoxin results in an increase in excitability and loss of accommodation similar to that observed for Abeta in the present study (Easaw et al. 1999; Jassar et al. 1999).

Fast-inactivating potassium channels (IA) are also important in modulating neuronal excitability. Abeta -induced reduction of IA observed in our study is consistent with a similar effect observed in rat hippocampal neurons (Good et al. 1996). Blockade of IA by Abeta could lead to increased duration of depolarization during an action potential, which in turn could increase Ca2+ influx. However, unlike several previous reports, we did not observe an effect of Abeta on Ca2+ currents (Brorson et al. 1995; Mattson et al. 1992; Ueda et al. 1997) or Na+ currents. Although Abeta did not influence Ca2+ currents, prolonged depolarization resulting from a blockade of K+ currents (both IA and IK) could still lead to increased total Ca2+ influx. In the present study we noted that Abeta decreases the TEA-sensitive IK conductance, which is different from the observation in septal cell line (SN56) and murine cortical cultures where Abeta exposure resulted in an enhancement of IK (Colom et al. 1998; Yu et al. 1998). However, in the latter studies, no acute effects of Abeta on IK were noted, but 7-11 h after Abeta exposure, the IK was enhanced, and this effect could be blocked by TEA. These delayed effects of Abeta on potentiation of IK have been postulated to explain the ability of Abeta to induce apoptotic cell death in cultures (Yu et al. 1998).

Selective effects of Abeta on cholinergic neurons

Our data indicate that cell size is not a reliable index for distinguishing cholinergic from GABAergic neurons in the DBB. However, single-cell RT PCR offers an effective and reliable means to make a distinction between these two major chemical phenotype of cells in the basal forebrain. At present, the cause of preferential degeneration of forebrain cholinergic neurons remains unclear. The neurotoxic potential of Abeta peptides could possibly mediate the degeneration of cholinergic neurons in AD brains. This notion is supported by data that a single injection of Abeta peptide into the septal nucleus induces damage to cholinergic but not parvalbumin-containing (presumably GABAergic) neurons (Harkany et al. 1995). Cholinergic neurons in hippocampal cultures seem particularly susceptible to injury following exposure to Abeta , whereas GABA-containing neurons are relatively resistant to Abeta -induced neurotoxicity (Pike and Cotman 1993). Our experiments show that the effects of Abeta in modulating K+ channel conductances and neuronal excitability are specific to cholinergic and not GABAergic neurons. This finding apart from demonstrating, at a cellular level, a link between Abeta peptides and cholinergic function, may help explain the selective vulnerability of cholinergic neurons to Abeta that is observed in vivo and in cell cultures. Abeta 's ability to render cholinergic neurons hyperexcitable through loss of accommodation could result in prolonged depolarization and eventual cell death resulting from excessive Ca2+ influx. Indeed Abeta has previously been shown to markedly potentiate glutamate-induced cell death in human cortical neurons through an increase in Ca2+ influx (Mattson et al. 1992).

Abeta and protein tyrosine phosphorylation

In the present study we have observed that Abeta -mediated reduction in whole cell K+-currents in DBB neurons is occluded by bath application of PTK inhibitors genistein and tryphostin B-44(-), but unaffected by daidzein, which is structurally similar to genistein but has no effect on PTK activity. It is possible that the effects of genistein, apart from its properties as a PTK inhibitor, could be also be attributed to its ability to directly block voltage-gated K+ conductances (Ogata et al. 1997; Smirnov and Aaronson 1995). However, Abeta effects were also blocked by tyrphostin B-44(-), another PTK inhibitor, which has not been shown to block K+ conductances. These results suggest that endogenous PTKs may play an important role in coupling the Abeta effects on K+ conductances in cholinergic forebrain neurons. There is considerable evidence that protein tyrosine kinases and phosphatases, acting on potassium channels, can regulate neuronal excitability (Jonas and Kaczmarek 1996).

Conclusions

The fact that Abeta is constitutively produced in the brain (Shoji et al. 1992) and is capable of influencing ion channel function of cholinergic neurons as demonstrated in the present study suggests that this peptide may have a neuromodulatory role within the DBB in vivo apart from its neurotoxic effects in the context of AD. The underlying mechanism for the specificity of Abeta response toward cholinergic, and not GABAergic, basal forebrain cells is an important question that remains unresolved. It is possible that the difference in responsiveness of cholinergic versus GABAergic cells to Abeta that we have observed in the present study may be due to the presence of a receptor for Abeta , which has yet to be identified, on cholinergic but not GABAergic neurons. Further studies of the receptor and molecular mechanisms underlying the coupling of Abeta effects to potassium channels through PTK signaling pathways in cholinergic basal forebrain neurons may shed additional important insights into cholinergic hypofunction seen in AD.


    ACKNOWLEDGMENTS

We thank Dr. C. W. Bourque for helpful comments and suggestions and C. Krys for assistance with preparation of the manuscript.

This research was supported by grants from the Canadian Institutes of Health Research (MT-10473) and the University Hospital Foundation. C. Cho, J. Easaw, and B. Jassar were supported by training awards from the Alberta Heritage Foundation for Medical Research.


    FOOTNOTES

Address for reprint requests: J. H. Jhamandas, Div. of Neurology, 530 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada (E-mail: jack.jhamandas{at}ualberta.ca).

Received 7 February 2001; accepted in final form 24 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society