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NEUROPHARMACOLOGY
Departments of Psychiatry (M.W., S.S.N., J.B.A., R.S.D., M.A.) and Neurobiology (M.A.), Yale University School of Medicine and the Ribicoff Research Facilities, Connecticut Mental Health Center (C.X.), New Haven, Connecticut
Received April 17, 2003; accepted July 28, 2003.
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Abstract |
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). Both the cortex and the hippocampus progressively lose their supply of ACh in AD as well as in Parkinson's disease, Lewy body dementia, and Down's Syndrome due to a continuing atrophy of their ACh source, the cholinergic cell bodies of the nucleus basalis and the medial septum/diagonal band of Broca (MSDB), respectively (Whitehouse et al., 1982; Mufson et al., 1989; Arendt et al., 1995).
Acetylcholinesterase (AChE) inhibitors, which increase synaptic levels of available ACh by preventing its degradation, are currently the most prescribed drugs for the treatment of AD, albeit of limited effectiveness. The AChE inhibitors physostigmine and tacrine (tetrahydroaminoacridine; THA) slow the decline of cognitive function and memory in patients with mild or moderate AD (Weinstock, 1995). Beneficial effects of tacrine on the slowing of the electroencephalogram have also been reported in mild cases of AD patients both after a single dose and up to 3 months of treatment, following which they may become ineffective (Alhainen et al., 1991; Perryman and Fitten, 1991; Jelic et al., 1998). The limited effectiveness of AChE inhibitors has been attributed to several factors, which includes reduced availability of ACh as the neurodegeneration progresses, as well as to molecular changes that may occur following long-term treatment with AChE inhibitors, such as an up-regulation of the AChE enzyme and a down-regulation of the ACh-synthesizing enzyme (Kaufer et al., 1998).
In animals, systemically administered AChE inhibitors produce hippocampal theta waves (Teitelbaum et al., 1975; Olpe et al., 1987; Valjakka et al., 1991; Yamamoto, 1998; Ikonen and Tanila, 2001) and improve learning and memory. These effects are blocked by the muscarinic receptor antagonist, scopolamine, as well as by lesions of the MSDB (Teitelbaum et al., 1975; Olpe et al., 1987; Ennaceur and Meliani, 1992; Ennaceur, 1998; Ikonen and Tanila, 2001), thereby indicating a critical role for muscarinic receptors and the MSDB in mediating the effects of AChE inhibitors.
The MSDB, which via the septohippocampal pathway maintains hippocampal learning and memory functions and the associated theta rhythm, is also considered a critical locus for the well described memory-impairing effects of scopolamine (Stewart and Fox, 1990; Givens and Olton, 1994; Givens and Olton, 1995). Scopolamine-induced amnesia has been likened to early symptoms of Alzheimer's disease and is reversed by AChE inhibitors in rats as well as in subhuman and human primates (Ye et al., 1999). At the cellular level, scopolamine has been shown to act within the MSDB by disrupting impulse flow in the septohippocampal GABAergic pathway. Moreover, the effects of scopolamine can be observed in vitro in a brain slice preparation of the MSDB (Alreja et al., 2000), suggesting that the muscarinic tone, which is critical for learning and memory, originates locally within the MSDB, presumably via axon collaterals of septohippocampal cholinergic neurons (Bialowas and Frotscher, 1987; Leranth and Frotscher, 1989).
Based on the above, we hypothesize that AChE inhibitors, by preventing destruction of available ACh within the MSDB, would lead to an enhancement of the muscarinic tone within the MSDB, a mechanism that may contribute or even be critical to the mnemonic effects of AChE inhibitors. In addition, in a recent study, we found that exogenous ACh also excites septohippocampal GABAergic neurons via nicotinic cholinergic receptors (Wu et al., 2003). A second goal of this study was to test for the presence of a nicotinic tone in septohippocampal neurons. Knowledge of the cellular mechanisms by which AChE inhibitors work may help design new treatments that could restore cognitive functions when AChE inhibitors cease to be effective.
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Materials and Methods |
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). Briefly, rats were anesthetized with chloral hydrate (400 mg/kg i.p.) and killed by decapitation. The ACSF (pH 7.35-7.38), equilibrated with 95% O2-5% CO2, contained 126 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 10 mM D-glucose, 25 mM NaHCO3, 2 mM CaCl2, and 2 mM MgSO4. Following decapitation, the brain was removed and placed in a Petri dish containing ACSF and trimmed to yield a small block containing the MSDB. Two coronal slices of 
300-µm thickness containing the MSDB were cut with a vibrating-knife microtome (FHC Inc., Bowdoinham, ME) and transferred to a Plexiglas recording chamber (1.5 ml volume) on the fixed stage of an Olympus BX50WI microscope (Olympus Optical Co., Ltd., Tokyo, Japan). The slice was kept in place with a grid and maintained at 33 ± 0.5°C. One to two hours later, the slice was used for recording. The chamber was continuously perfused with normal ACSF at a rate of 1 to 2 ml/min.
Labeling of Septohippocampal Cholinergic Neurons Using Cy3-192IgG
Rats were anesthetized using the following cocktail: 75 mg/kg ketamine, 4 mg/kg Xylazine, and 0.075 mg/kg acepromazine. Cy3-192IgG (3-5 µl; 0.4 mg/ml) was stereotaxically injected unilaterally or bilaterally into the lateral ventricle of each rat with a Hamilton syringe (22-gauge needle) at a rate of 0.5 µl/min. The coordinates used were 0.8 mm posterior from bregma, 1.2 mm lateral from midline, and 3 to 4 mm below the dura. Two to five days later, slices were prepared from Cy3-192IgG-injected rats and used for electrophysiological recordings. Recordings from unlabeled neurons were restricted to slices taken from rats injected biventricularly with the marker.
Retrograde Labeling of Septohippocampal Neurons
Retrograde labeling of septohippocampal neurons was performed by pressure injecting 50 to 100 nl of rhodamine-labeled fluorescent latex microspheres (Lumafluor Inc., Naples, FL) at several sites within the hippocampus using a glass micropipette (40-50-µm tip diameter). Rhodamine microspheres (0.02-0.2-µm diameter) show little diffusion and consequently produce small, sharply defined injection sites. Once transported back to neuronal somata, the label persists for at least 10 weeks in vivo and 1 year after fixation. Microspheres have been reported to possess no obvious cytotoxicity or phototoxicity as assessed by intracellular recording and staining of retrogradely labeled cells in brain slice preparation (Katz et al., 1984). The stereotaxic coordinates were (-2.8, -1.4, -2.8), (-4, -1.4, and -2.8), and (-5.8, -4.5, and -3.5 to -6-mm track) for anteroposterior, lateral, and ventral, respectively. Two or more days later, the injected rats were used to prepare brain slices. Injection sites were confirmed for each experiment.
Fluorescence and Infrared Imaging
Infrared, differential interference contrast imaging was performed to visualize neurons for extracellular or patch-clamp recording using an Olympus BX-50 microscope (Olympus) equipped with a 60x water immersion objective (numerical aperture, 0.9; Olympus). Images were detected with a CCD-300-RC camera (Dage-MTI, Michigan City, IN) and displayed on a standard black and white video monitor (Dage-MTI; HR 120). The images were transferred to the hard disk of a personal computer using an LG-3 scientific frame grabber (Scion Corp., Frederick, MD) and processed further with Adobe Photoshop. Cy3-192IgG-labeled and rhodamine-labeled neurons were visualized using the appropriate fluorescence filters. A neuron viewed with infrared optics was considered to be the same as that viewed with fluorescence optics when the infrared image and the fluorescent image of the neuron had the same position and orientation with the two imaging systems.
Electrophysiology Recordings
The image of the cells in the slice was displayed on a video monitor, and glass pipettes used for electrophysiological recordings were visually advanced through the slice to the surface of the cell from which recordings were made. Extracellular recordings were performed with glass micropipettes filled with 2 M NaCl (5-10 M
). Whole-cell patch-clamp recordings were performed using previously described methods (Alreja and Liu, 1996). In brief, low-resistance (2.5-3.5 M) patch pipettes were filled with a solution containing 120 mM K methylsulfonate/KCl, 10 mM HEPES, 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid K4, 20 mM sucrose, 2.38 mM CaCl2, 1 mM MgCl2, 1 mM K2ATP, and 0.1 mM GTP (pH 7.32-7.35). All recordings were made using an Axoclamp-2B amplifier (Axon Instruments Inc., Union City, CA). Current-clamp recordings were made in the bridge mode, and the output signal was filtered at 10 KHz. The cells selected for study had spike amplitudes of 70 to 100 mV. Spike durations were measured at half-spike amplitude. In spontaneously firing cells, these measurements were done at the resting potential, and in quiescent cells, firing was induced by injecting a small amount of depolarizing current. Voltage-clamp recordings were performed using the continuous single electrode voltage-clamp mode. The firing rate, current, and voltage signals were amplified and continuously recorded on a chart recorder (Gould 2200).
Combined Immunocytochemistry and In Situ Hybridization
Immunocytochemistry. Rat brain cryocut sections (16-µm thick) were fixed in 4% paraformaldehyde for 10 min followed by a 5-min 1% hydrogen peroxide treatment and two rinses of 5 min each in PBS. Sections were blocked with 2.5% bovine serum albumin and 3000 U/ml heparin in PBS for 30 min to prevent nonspecific binding of antibody. Slides were then rinsed three times in PBS. Sections were then incubated for 48 h in an antibody solution (0.25% Triton X, 1% bovine serum albumin, 3000 U/ml heparin, and 10 U/ml anti-RNase in PBS) containing a 1:1000 dilution of monoclonal anti-parvalbumin antibody. Unbound antibody was removed by three PBS rinses and one rinse in PB. Subsequently, sections were incubated in biotinylated anti-mouse IgG at a 1:200 dilution for 1 h at room temperature. Unbound secondary antibody was removed by three 5-min rinses in PBS. Sections were incubated in preformed avidin and biotinylated horseradish peroxidase macromolecular complex (ABC reagent; Vector Laboratories, Burlingame, CA). Following this incubation, sections were rinsed three times in PB and then stained with the DAB staining kit (Vector Laboratories) according to the directions of the manufacturer. Slides were air dried and processed as follows for in situ hybridization.
In Situ Hybridization (ISH). ISH was performed as per standard protocols for cryocut sections. Briefly, sections were fixed for 10 min in cold 4% paraformaldehyde, followed by a 1-min rinse in PBS and another 1-min rinse in 100 mM triethanolamine buffer and acetylated in 100 mM triethanolamine buffer containing 0.25% acetic anhydride. After rinsing twice in 2x standard saline citrate buffer, sections were then dehydrated in an alcohol series. Antisense cDNA template for muscarinic receptor 3 (Bonner et al., 1987) with an upstream Sp6 polymerase site was used to generate 35 S radio-labeled riboprobes. Hybridization was overnight at 55°C. Following posthybridization washes and Rnase A treatment, slides were air dried and exposed to film to determine success of ISH and duration of exposure to emulsion. Slides were then dipped in nuclear track emulsion (NTB2; Eastman Kodak, Rochester, NY), dried for 4 h, and then stored in a dark, dry box. After 4 weeks, sections were developed using Kodak D-19 developer (4 min at 16°C) and fixed in Kodak Fixer. Slides were counterstained in cresyl violet, dehydrated in an alcohol series, cleared in Histochoice (15 s), and coverslipped with DPX mountant. Sections were observed using a Carl Zeiss Axioplan brightfield microscope (Jena, Germany) and photographed with a digital videocassette camera.
Specificity of signal was ascertained by first exposing sections to autoradiographic film and only then dipped in emulsion. A cell was considered colocalizing paravalbumin protein and M3 mRNA when emulsion grains overlaying a parvalbumin-positive cell had a higher density (50%) than background (determined by counting emulsion grains in a brain region not showing specific signal on film). Four fields were examined from each section, and four sections were used from three animals.
Materials
The various AChE inhibitors, acetylcholine chloride, muscarine chloride, atropine sulfate, (-)-scopolamine hydrobromide, and methoctramine tetrahydrochloride, as well as nicotinic receptor agonists and antagonists were obtained from Sigma-Aldrich (St. Louis, MO). Muscarinic toxin 7 (MT-7) was obtained from Peptide Institute, Inc. (Osaka, Japan). All drugs were diluted in ACSF from previously prepared stock solutions that were prepared in water and stored at -20°C. Rhodamine microspheres were obtained from Lumafluor Inc., and Cy3-192IgG was custom synthesized by Advanced Targeting Systems (San Diego, CA).
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Results |
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; Shaw et al., 1985), a wide array of AChE inhibitors with different primary mechanisms of action were chosen. The AChE inhibitors tested included the long-acting, clinically used physostigmine, tetrahydroaminoacridine (THA/tacrine), and galanthamine, as well as the irreversible cholinesterase inhibitor, diisopropylfluorophosphate. The effects of short-acting inhibitors such as neostigmine, ambenonium, and edrophonium were also tested. Physostigmine and neostigmine are carbamates and act as metabolic inhibitors of acetylcholinesterase. Edrophonium is a quaternary compound that blocks the AChE enzyme competitively by binding to its active site, whereas ambenonium is a rapidly reversible non-covalent inhibitor of acetylcholinesterase. Tacrine causes a long-lasting but reversible allosteric inhibition of AChE by binding to a hydrophobic region near the anionic 
or 
sites on its surface, and diisopropylfluorophosphate is an irreversible phosphorylating inhibitor. In all, we tested the effect of 7 AChE inhibitors on 80 unidentified MSDB neurons. The effects of multiple AChE inhibitors were tested on the same cell. As shown in Fig. 1, a qualitatively similar response was obtained in all cases, albeit, as expected, the magnitude and duration of the effect varied among different AChE inhibitors. In extracellular recordings, 77.3% of the neurons tested were excited by exogenous ACh/muscarine (58/75), 17.3% (13/75) were inhibited, and 5.4% (4/75) of neurons were not affected by ACh/muscarine. Of the 58 neurons excited by ACh/musc, 49 (84.5%) responded to AChE inhibitors with a strong excitation, suggesting the presence of a cholinergic tone. A similar effect was obtained with all of the AChE inhibitors (Fig. 1, a-e). Of the 13 neurons inhibited by ACh/muscarine, 2 responded to AChE inhibitors with an inhibition and the remaining neurons showed no response (data not shown). The four neurons not affected by ACh/muscarine showed no response to AChE inhibitors. Cumulative concentration-response studies performed in five cells with the clinically most prescribed AChE inhibitor, tacrine, revealed an EC50 of 3.2 µM (Fig. 1d).
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The effect of the short-acting AChE inhibitors on GABA-type MSDB neurons was also tested using intracellular voltage-clamp recordings. GABA-type neurons were identified by their electrophysiological characteristics and their excitatory response to muscarine (Wu et al., 2000). Of neurons tested, 85.7% (12/14) responded to AChE inhibitors with an excitation, and both neostigmine and ambenonium mimicked the effect of muscarine and produced inward currents (95.4 ± 22.7 pA; n = 12). The magnitude of the AChE inhibitor response at the concentration tested was significantly lower than the effect of a near-maximal concentration of muscarine (254.2 ± 47.9 pA; Fig. 1, f and g).
AChE Inhibitor-Induced Excitation of MSDB Neurons Involves Muscarinic but Not Nicotinic Receptors. Since ACh excites septohippocampal GABA-type neurons via both nicotinic and muscarinic receptors (Wu et al., 2000), we determined the contribution of these two receptor subtypes to the AChE-induced excitation. As shown in Fig. 1, the broad-spectrum muscarinic receptor antagonists, atropine or scopolamine, significantly reduced basal firing (Alreja et al., 2000) and also blocked both the excitatory (n = 19) and the inhibitory (n = 3) effects of AChE inhibitors (see below).
The contribution of nicotinic receptors to the AChE inhibitor response was determined using several nicotinic receptor antagonists. Within the MSDB, nicotinic responses are blocked by the broad-spectrum nicotinic receptor antagonist, d-tubocurarine, and by the 42-subtype-selective antagonist, dihydro--erythroidine, but not by the 7-selective antagonists, methyllycaconitine and -bungarotoxin (Fig. 2, a and b). Note that, in contrast to the muscarinic receptor antagonists, which reduce basal firing, nicotinic receptor antagonists did not produce any change in basal firing rates in the population of cells tested, suggesting that MSDB neurons are under a muscarinic but not a nicotinic excitatory tone under resting conditions. Similarly, nicotinic receptor antagonists also had no effect on the AChE inhibitor-induced excitation, suggesting that AChE inhibitor-induced excitation of MSDB neurons involves activation of muscarinic but not nicotinic receptors (Fig. 2, c and d). These findings are consistent with the behavioral effects of systemic AChE inhibitors on septohippocampal functions that are blocked by muscarinic receptors (see Introduction).
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Effect of AChE Inhibitors on Identified Septohippocampal Cholinergic and Noncholinergic Neurons. Having ascertained the presence of AChE inhibitor effects on MSDB neurons, we next conducted a systematic study to determine the effect of AChE inhibitors on MSDB neurons that project to the hippocampus, as it is these septohippocampal neurons that control the activity of the hippocampus and facilitate performance in hippocampus-associated learning and memory tasks. Based on our earlier studies, in which we have shown that neurons that are excited by ACh/muscarine belong to the noncholinergic subpopulations (presumably, GABAergic) and project to the hippocampus (Wu et al., 2000), we hypothesized that AChE inhibitor-excited neurons would belong to the septohippocampal GABAergic neuronal population. Septohippocampal cholinergic neurons, on the other hand, are either inhibited or not affected by muscarine and were found to be under a minimal muscarinic tone as tested using muscarinic receptor antagonists (Alreja et al., 2000).
Septohippocampal cholinergic neurons were identified in the living state using the fluorescent marker Cy3-192IgG as previously described (Alreja et al., 2000; Wu et al., 2000). This technique exploits the fact that septohippocampal cholinergic neurons but not the GABAergic neurons of the MSDB exclusively express the low-affinity nerve growth factor receptor, p75. Thus, this marker, which is composed of an antibody against the p75 receptor (192IgG) conjugated to the Cy3 fluorochrome, is taken up only by cholinergic terminals and therefore exclusively labels only the cholinergic subpopulation. The specificity of this marker and its inert nature has been thoroughly confirmed by us (Alreja et al., 2000; Wu et al., 2000) and others (Hartig et al., 1998) in earlier studies.
Consistent with our earlier findings (Wu et al., 2000), septohippocampal cholinergic neurons, identified using Cy3-192IgG, were either inhibited or not affected by exogenous ACh/muscarine and lacked a depolarizing sag in response to hyperpolarizing pulses (Fig. 3, a1-a3). We tested the effect of two short-acting AChE inhibitors, neostigmine and ambenonium, and the clinically used AChE inhibitor, tacrine, on 29 septohippocampal cholinergic neurons that were inhibited by ACh/muscarine. AChE inhibitors mimicked the effect of ACh/muscarine in 24% of cholinergic neurons tested (7/29) (Fig. 3, a, c, d, and e) and had no effect on the remaining 76% of neurons. The mean magnitude of the response to AChE inhibitors was 8.6 ± 2.4 mV, whereas the response to muscarine was 11.6 ± 2.4 mV in magnitude. The inhibitory effects of AChE inhibitors were blocked by atropine (n = 3), suggesting involvement of muscarinic receptors (Fig. 3, a3). Thus, AChE inhibitors can modulate the activity of a small subpopulation of septohippocampal cholinergic neurons.
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In contrast to the Cy3-192IgG-labeled neurons mentioned above, a vast majority of the unlabeled neurons (94%; 15/16) recorded from brain slices taken from rats injected biventricularly with Cy3-192IgG responded to ACh/musc with an excitation and exhibited a depolarizing sag in response to hyperpolarizing pulses that was absent in the Cy3-192IgG-labeled neurons (Fig. 3, b1-b3). It should be mentioned that biventricular injections of Cy3-192IgG label at least 90% of choline acetyltransferase-positive neurons and the unlabeled population is composed primarily of the noncholinergic neurons that colocalize the calcium-binding protein, parvalbumin. Parvalbumin is uniquely expressed by the septohippocampal GABAergic neurons in this nucleus that exhibit a depolarizing sag in response to hyperpolarizing current steps (Morris et al., 1999; Wu et al., 2000). All 15 unlabeled neurons responded to AChE inhibitors with a profound excitation, and atropine blocked the excitatory effects in the three neurons tested (Fig. 3, b-e).
AChE Inhibitors Excite Septohippocampal GABAergic Neurons via M3 Receptors. Septohippocampal neurons were retrogradely labeled following injection of rhodamine beads into the hippocampus. Rhodamine beads label both the septohippocampal cholinergic and GABAergic neurons. However, if a rhodamine-labeled septohippocampal neuron responded to ACh/musc with an excitation and displayed the appropriate electrophysiological signature, which included the presence of a depolarizing sag (Morris et al., 1999), then it was classified as a septohippocampal GABAergic neuron, as septohippocampal cholinergic neurons are not excited by muscarine (Wu et al., 2000). Five of five septohippocampal GABA-type neurons identified using these criteria were excited by AChE inhibitors, with the short-acting inhibitor, neostigmine, producing a 215 ± 15% increase in firing rate (Fig. 4, a-f). Thus, AChE inhibitors enhance the activity of septohippocampal GABAergic neurons in vitro.
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Since the M3 receptor mediates the excitatory effects of muscarine on septohippocampal GABAergic neurons, and M3 receptor antagonists also mimic the effects of scopolamine on MSDB neurons (Liu et al., 1998; Alreja et al., 2000; Wu et al., 2000), we tested the effect of an M3 receptor antagonist on AChE inhibitor-induced activation. Low nanomolar concentrations of 4-DAMP mustard, an irreversible antagonist that selectively inactivates M3 receptors but has no effect on M1, M2, M4, and M5 receptors (see Liu et al., 1998), produced statistically significant reductions in the excitatory response to exogenous ACh/muscarine and neostigmine in seven of eight neurons tested (Fig. 4, c and g). Consistent with our previous findings, 4-DAMP mustard also reduced basal firing rates. In contrast, the M2/M4 receptor antagonist, methoctramine, and the highly selective M1 receptor toxin, MT-7 (Olianas et al., 2000), did not reduce the excitatory response to AChE inhibitors in the three neurons tested (Fig. 4, d, e, and g). Thus, the excitatory effects of AChE inhibitors in the MSDB are mediated primarily via M3 muscarinic receptors (Fig. 4g).
We next used immunolabeling techniques in combination with in situ hybridization to examine whether septohippocampal GABAergic neurons colocalize the message for the M3 muscarinic receptor. It should be mentioned that presence of M3 receptor message has already been reported in about 80% of septohippocampal neurons; however, the phenotype of the M3 mRNA-expressing septohippocampal neurons was not determined (Rouse and Levey, 1996). Septohippocampal GABAergic neurons were identified by an immunopostive reaction to an antibody against the calcium-binding protein, parvalbumin, which is exclusively expressed by the septohippocampal GABAergic neurons in the MSDB (Freund, 1989) and has therefore become an established marker for this neuronal subpopulation. As shown in Fig. 4h, parvalbumin-containing MSDB neurons were found to express M3 receptor mRNA, thus supporting the pharmacological findings.
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AChE Inhibitors Activate MSDB Neurons via Muscarinic but Not Nicotinic Receptors. The profound effects of low micromolar concentrations of AChE inhibitors observed in this study are in support of behavioral studies that suggest that the MSDB may be a key locus in mediating the effects of AChE inhibitors (see Introduction). These effects presumably result from an increase in the availability of locally released ACh as a result of reduced degradation of ACh following inhibition of the AChE enzyme.
A blockade of AChE inhibitor effects by muscarinic but not nicotinic receptor antagonists suggests the presence of a muscarinic but not nicotinic tone within the MSDB slice preparation, which is consistent with published literature showing that AChE inhibitor effects on learning and memory functions and on theta rhythm are blocked by scopolamine (see Introduction). The presence of a muscarine tone supports our recent findings wherein bath application of muscarinic receptor antagonists reduces basal firing rates of GABA-type MSDB neurons in brain slices devoid of extrinsic cholinergic inputs (Alreja et al., 2000). The absence of a nicotinic component in the AChE inhibitor responses may reflect a rapid desensitization of nicotinic receptors due to the augmented concentrations of ACh with AChE inhibitors. A lack of effect of nicotinic receptor antagonists on AChE inhibitor responses also suggests that ACh release within the MSDB is not under the influence of nicotinic receptors.
Interestingly, the number of MSDB neurons found to be under the influence of an excitatory muscarinic tone using AChE inhibitors is much larger (94%) than we previously estimated using muscarinic receptor antagonists (54%). A likely explanation for this difference may be that under basal conditions, the amount of ACh available at some synapses may not be high enough to elicit a physiological response, possibly due to inadequate release and/or high levels of local cholinesterase activity. AChE inhibitors, by preventing degradation, may allow enough ACh to build up so as to have a noticeable effect on basal cell firing of MSDB neurons. Alternately, the increased number of neurons responding to AChE inhibitors may reflect spillover of released ACh from the synaptic clefts, which in the absence of degradation may diffuse long enough distances to activate receptors located outside the synaptic cleft or even in neighboring synapses. Both of these factors may also contribute to the AChE inhibitor effects that were detected in 25% of septohippocampal cholinergic neurons in this study. Previously, using muscarinic receptor antagonists, an inhibitory muscarinic tone was detected only in 7% (1/14) of septohippocampal cholinergic neurons tested.
A third possibility is that some of the actions of AChE inhibitors may be attributable to noncholinesterase activity, albeit muscarinic receptor-dependent, as AChE inhibitor responses were blocked by muscarinic receptor antagonists in 100% of the neurons tested. This seems unlikely for the following reasons: 1) the effects of AChE inhibitors but not of exogenous ACh/muscarine on basal firing rates are significantly reduced following blockade of synaptic transmission, although AChE inhibitors continue to potentiate the effects of exogenous ACh; and 2) similar results were obtained with the seven AChE inhibitors tested even though these AChE inhibitors inhibit AChE via different mechanisms. It is unlikely that all of the AChE inhibitors tested shared a common nonspecific mechanism, that of behaving as an agonist at the muscarinic M3 receptor (see below). Moreover, nonspecific actions of AChE inhibitors are mostly attributed to activation of nicotinic receptors. In the present study, nicotinic receptor antagonists did not reduce the effects of AChE inhibitors.
It should also be mentioned that the effects of one of the AChE inhibitors used in this study, THA, have previously been studied in guinea pig MSDB slices, albeit at concentrations 100- to 200-fold higher than those used in the present study; effects were observed on potassium channels of several populations of electrophysiologically characterized MSDB neurons. However, THA effects on basal properties of neurons, as well as the question of whether they involved activation of muscarinic receptors, were not addressed (Griffith and Sim, 1990).
AChE inhibitor effects, similar to those described in the present study, have been reported only in the rat dorsal cochlear nucleus slices, wherein comparable concentrations of AChE inhibitors induced atropine-sensitive burst firing in a synaptically dependent manner (Chen et al., 1998).
AChE Inhibitors Increase Impulse Flow in the Septohippocampal GABAergic Pathway. The finding that increased impulse flow in the septohippocampal GABAergic pathway but not in the septohippocampal cholinergic pathway may underlie the mnemonic effects of AChE inhibitors further supports the emerging role of the septohippocampal GABAergic pathway in learning and memory. Thus, accumulating data from in vivo microdialysis studies, theta rhythm studies, and behavioral studies as well as in vitro cellular studies indicates that, contrary to current wisdom, manipulations that improve learning and memory and/or facilitate the hippocampal theta rhythm are often associated with a decrease in hippocampal ACh release and an increase in septohippocampal GABA release (see Alreja et al., 2000; Wu et al., 2000). Septohippocampal GABAergic neurons exclusively innervate the GABAergic interneurons of the hippocampus, which, in turn, contact 500 to 1200 pyramidal neurons, connectivity, which results in a powerful disinhibition of hippocampal pyramidal cells following stimulation of septohippocampal GABAergic afferents (see Toth et al., 1997). Thus, although scopolamine disrupts ongoing impulse flow in the septohippocampal GABAergic pathway, AChE inhibitors, like muscarinic agonists, recruit the septohippocampal disinhibitory pathway. This may be a key mechanism by which AChE inhibitors work in the brain, as lesions of the MSDB can prevent both the memory-enhancing effects of AChE inhibitors (Teitelbaum et al., 1975; Olpe et al., 1987; Ennaceur, 1998) and the facilitatory effects of AChE inhibitors on the hippocampal theta rhythm (Ikonen and Tanila, 2001).
AChE Inhibitors Decrease Impulse Flow in the Septohippocampal Cholinergic Pathway. The finding that AChE inhibitors actually decrease the activity of a small subpopulation of septohippocampal cholinergic neurons is also significant and is supported by the finding that in the presence of the AChE inhibitor, physostigmine, uptake of tritiated choline is reduced in the MSDB (Disko et al., 1998). This effect would therefore be counterproductive from the therapeutic angle, as it would further reduce the release of ACh, although within the hippocampus, this reduction could be countered by reduced degradation of available ACh. Nevertheless, a blockade of the inhibitory effects of AChE inhibitors on septohippocampal cholinergic neurons may be desirable, especially as the neurodegenerative process escalates and available ACh continues to dwindle. The inhibitory effects of AChE inhibitors on septohippocampal cholinergic neurons could be due to a direct inhibitory effect of local ACh via the inhibitory muscarinic M2/M4 receptor subtypes localized on a subset of septohippocampal cholinergic neurons (Vilaro et al., 1992; Van der Zee and Luiten, 1994; Levey et al., 1995; Rouse and Levey, 1996) or indirect due to an increase in local GABA release following AChE inhibitor-induced excitation of the septal GABAergic neurons.
The results of this study also show that the slice preparation of the MSDB may be useful in evaluating drugs that may be effective for the treatment of cognitive deficits associated with neurodegenerative disorders and, hopefully, in understanding the cellular mechanisms that may limit the effectiveness of treatments over a prolonged period. For example, the MSDB slice preparation can be used in the future to test whether prolonged treatment with AChE inhibitors or acute stress reduce AChE inhibitor effects in septohippocampal neurons, as has been suggested to occur in the cortex via a down-regulation of the ACh-synthesizing enzyme and an upregulation of AChEs (Soreq and Seidman, 2001). In conclusion, it is hoped that the results of this study and future studies will help design better therapies for treatment of cognitive deficits that occur in various neurodegenerative disorders.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS. AD, Alzheimer's disease; AChE, acetylcholinesterase; AChEI, acetylcholinesterase inhibitor; ISH, in situ hybridization; MSDB, medial septum/diagonal band of Broca; MT-7, muscarinic toxin 7; musc, muscarine; THA, tacrine/tetrahydroaminoacridine acetylcholine; ACh, acetylcholine; ACSF, artificial cerebrospinal fluid; PB, phosphate buffer; PBS, phosphate-buffered saline; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine.
Address correspondence to: Dr. Meenakshi Alreja, Department of Psychiatry, CMHC 335A, Yale University School of Medicine, 34 Park Street, New Haven, CT 06508. E-mail: Meenakshi.Alreja{at}yale.edu
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