Introduction

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Nociceptin/orphanin FQ (N/OFQ) is an endogenous heptadecapeptide that binds to a receptor (ORL-1), which is homologous to the classical opioid receptors (Henderson and McKnight, 1997). N/OFQ and its receptor play an important role in a variety of physiological functions, including cardiac and renal control (Kapusta, 2000), locomotion (Florin et al., 1996), and nociception (Darland et al., 1998). A role for N/OFQ in memory and learning has also been reported on the basis of in vivo and in vitro observations. Microinjection of N/OFQ into the hippocampus has been shown to result in disruption of spatial learning and memory (Sandin et al., 1997). In addition, knockout mice lacking the ORL-1 receptor perform better at memory acquisition and memory retention tasks, which has been attributed to electrophysiological changes (Manabe et al., 1998).

The nucleus of the diagonal band of Broca (DBB) is one of the basal forebrain nuclei, which play an important role in memory and learning processes. Neurons of the DBB synthesize either acetylcholine or GABA (Paolini and McKenzie, 1993). Degeneration and loss of cholinergic neurons are linked to spatial memory and cognitive deficits seen in Alzheimer's disease (Yankner, 1996). Experimental lesions of DBB in the rat that result in a loss of cholinergic neurons also cause disruption of memory homologous to that seen in human conditions such as Alzheimer's disease (Roman et al., 1993).

Comparative immunohistochemical studies in the rat and human brain have shown that DBB is enriched with N/OFQ and ORL-1-like receptor (Anton et al., 1996; Neal et al., 1999). In other areas of the brain such as the periaqueductal gray, hippocampus, and hypothalamus, N/OFQ plays a predominantly inhibitory role through its actions on activation of an inwardly rectifying K⁺ current (Vaughan et al., 1997; Chiou, 1999; Slugg et al., 1999). N/OFQ also has been shown in the hippocampus and periaqueductal gray to modulate voltage-sensitive Ca²⁺ channels (VSCC; Knoflach et al., 1996; Connor and Christie, 1998). These actions of N/OFQ at a cellular level have been linked to its role in important physiological processes such as pain control and release of neurohormones such as vasopressin (Doi et al., 1998; Pan et al., 2000).

Given that the DBB plays a central role in memory processes and that DBB is enriched with N/OFQ, we examined the ionic mechanisms of N/OFQ actions on acutely dissociated DBB neurons, whose chemical phenotype was determined using single cell RT-PCR technique. We also assessed the effectiveness of recently developed antagonists of N/OFQ in attenuating the N/OFQ-evoked responses in DBB neurons.

	Materials and Methods

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Dissociation Procedures. Details of the procedure for acute dissociation of neurons from the DBB have been described previously (Jassar et al., 1999). Briefly, brains were quickly removed from decapitated male Sprague-Dawley rats (15-25 days postnatal) and placed in cold artificial cerebrospinal fluid that contained 140 mM NaCl, 2.5 mM KCl, 1.4 mM CaCl₂, 5 mM MgCl₂, 10 mM HEPES, and 33 mM D-glucose, pH 7.4. Brain slices (350 µm in thickness) 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 the enzymatic treatment of slices 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 coverslips and viewed under an inverted microscope (Axiovert 35; Carl Zeiss, Thornwood, NY). 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 (Axon Instruments, Union City, CA). Junction potential was nulled with the pipette tip immersed in the bath. Patch electrodes (thin wall with filament, 1.5 mm in diameter; World Precision Instruments, New Haven, CT) were flame polished to yield resistances of 4 to 5 M. Internal patch pipette solution contained 140 mM K-methylsulfate, 10 mM EGTA, 5 mM MgCl₂, 1 mM CaCl₂, 10 mM HEPES, 2.2 mM Na₂-ATP, and 0.3 mM Na-GTP, pH 7.2, while the neurons were perfused with artificial cerebrospinal fluid. Putative acutely dissociated DBB neurons were initially identified for recording by visual inspection. In general, recordings were carried out a minimum of 1 h and up to 10 h post-trypsinization. Current-voltage relationships and excitability characteristics were used to distinguish neurons from glial or other cell types (Jassar et al., 1999). The whole cell patch-clamp recording technique was used to record from these neurons using an Axopatch-1D amplifier and analyzed on computer using pCLAMP software, version 6.0.3 (Axon Instruments).

Single-Cell RT-PCR for Chemical Phenotyping. Neurons were harvested after electrophysiological recordings were completed and readied for RT-PCR according to a protocol described previously (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 of sterile water (W-4502; Sigma-Aldrich, St. Louis, MO), 0.5 µl of 0.1 M dithiothreitol, 0.5 µl of RNasin (10 U/µl), and 1 µl of oligo(dT) (0.5 g/µl). The tube was then placed on ice. Single-stranded cDNA was then synthesized from mRNA by adding a solution containing 1 µl of SuperScript II RT (200 U/µl), 2 µl of 10× PCR buffer, 2 µl of 25 mM MgCl₂, 1.5 µl of 0.1 M dithiothreitol, 1 µl of 10 mM dNTPs, and 0.5 µl of RNasin (10 U/µl). The PCR tube was gently mixed and incubated in a Progene thermal cycler (Techne, Princeton, NJ) 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 of 10× PCR buffer, 5 µl of 25 mM MgCl₂, 0.5 µl of Taq polymerase (5 U/µl), 31.5 µl of sterile water (W-4502; Sigma-Aldrich), 1 µl of 25 mM dNTP mixture, and 1.5 µl of a specific set of primers (15 µM). All reagents were purchased from Invitrogen (Carlsbad, CA). Primer sequences for choline acetyltransferase (ChAT) and for glutamate decarboxylase (GAD) have been described previously (Surmeier et al., 1996; Tkatch et al., 1998) and that for -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 Department of Biochemistry (University of Alberta, Edmonton, AB, Canada). 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; and 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 ethidium bromide. After 10 min, DNA bands were visualized with UV light box and photographed with a Polaroid camera.

Pharmacological Agents Used. The following compounds used in this study were purchased from Sigma-Aldrich: iberiotoxin, TEA-Cl, nimodipine, and -conotoxin GVIA. N/OFQ was purchased from Bachem California (Torrance, CA). CompB (J-113397) and [Phe¹]nociceptin-(1-13)-NH2 (Nphe) were kind gifts from Dr. Satoshi Ozaki (Banyu Pharmaceutical, Okubo, Japan) and Dr. Girolamo Calo (University of Ferrara, Ferrara, Italy), respectively. All the agents were dissolved in distilled water to make 1000× stock solution (stored at 70°C) and 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 to 5 ml/min, which allowed complete exchange in less than half a minute. Data are presented as mean ± standard error of mean. Student's paired two-tailed t test was used for determining significance of effect, with a significance value of p < 0.05.

	Results

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Most of the acutely dissociated neurons from the DBB had neuron-like morphology (i.e., large cells with a conspicuous nucleus, nucleolus, and few blunt processes, which were truncated axon/dendrites). The average membrane capacitance estimated electronically on the Axopatch-1D amplifier was 15.7 ± 0.2 pF (n = 131). 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 1.29 ± 0.12 nanosiemens (n = 98).

N/OFQ Response of Whole Cell Currents in Cholinergic and GABAergic Cells. Based on the previous observations (Jassar et al., 1999), we used 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. N/OFQ inhibited whole cell currents in the 30 to +30 mV range (Fig. 1A). At a voltage of +30 mV, application of 1 M N/OFQ significantly decreased whole cell currents from 7.2 ± 0.8 to 6.0 ± 0.6 nA, a reduction of 15.4 ± 1.7% (n = 14, p < 0.05; Fig. 1B). The average whole cell current at +30 mV after washout of 1 µM N/OFQ was 6.9 ± 0.5 nA, which represented a partial recovery of the responses. We did not observe any differences in N/OFQ responses obtained either 1 or up to 10 h post-trypsinization. The average input conductance before N/OFQ treatment was 1.19 ± 0.23 nanosiemens, which was not significantly different from the input conductance of 1.21 ± 0.24 nS observed during N/OFQ application (n = 14, p < 0.05). N/OFQ inhibited peak whole cell currents of DBB neurons in a dose-dependent manner with an EC₅₀ value of 1.2 nM (Fig. 1C). Thus, to ensure maximal responses, either 100 nM or 1 µM N/OFQ was used in all subsequent experiments.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   N/OFQ effects on whole cell currents in chemically identified DBB neurons. A, I-V plot of whole cell currents for an individual DBB neuron evoked under control conditions (before application of N/OFQ), in the presence of 100 nM N/OFQ, and after washout. Inset shows voltage protocol whereby voltage-activated currents were evoked in DBB neurons by applying a voltage ramp (20 mV/s). Holding potential was 80 mV, and the cells were held at 110 mV for 1 s for conditioning before the depolarizing ramp-clamp protocol. B, I-V plot of average whole cell currents evoked from 14 DBB neurons under control conditions (before application of N/OFQ), in the presence of 1 µM N/OFQ and after washout. C, dose-response curve for N/OFQ inhibition of peak whole cell currents. D, photograph of a gel showing the results of single-cell RT-PCR on three cells. Cells 1 and 2 show bands corresponding to the molecular weight (MW) of the ChAT primer (lanes 3 and 6) and no bands for GAD primer (lanes 4 and 7). Cell 3 shows a band corresponding to the MW of the GAD primer (lane 10) and no band for the ChAT primer (lane 9). The -actin lane on the gels serves as a control (lanes 2, 5, and 8). MW (in kDa) for -actin is ~515, ChAT is ~308, and GAD is ~400. Lanes 1 and 11 show marker bands from the top corresponding to 516, 394, 344, and 298 kDa as identified by the arrows.

Chemical Identity of N/OFQ Responsive DBB Neurons. The neurons of DBB can be divided into two major chemical phenotypic groups based upon whether they synthesize and release the neurotransmitter acetylcholine or GABA (Paolini and McKenzie, 1993). Determination of the chemical phenotype was done by RT-PCR analysis, which provides information on the presence of mRNA in a particular cell that has been recorded from. ChAT was used as a specific marker for cholinergic neurons and GAD was used as a specific marker for GABAergic neurons. Figure 1D shows the photograph of a gel indicating RT-PCR product from three N/OFQ-responsive cells. The two cells on the left are cholinergic (band corresponding to the molecular weight of the ChAT primer) and the neuron on the right is GABAergic (band corresponding to the molecular weight of GAD primer). Forty-four cells that responded to N/OFQ were collected for single cell RT-PCR analysis and of these, 20 cells displayed a positive -actin band. Of the 20 cells where the RT-PCR reaction was successful (as judged by the presence of a -actin band), nine cells were identified as cholinergic and three cells were GABAergic. The remaining eight cells did not show a band for either ChAT or GAD. The lack of detectable RT-PCR product in these cells may be due to prolonged whole cell recordings in many cells, which could result in a dialysis of the intracellular contents of the cell, thereby diminishing or degrading detectable mRNA for ChAT or GAD.

Pharmacological Antagonism of N/OFQ Response in DBB Neurons. The N/OFQ antagonist Nphe attenuated the N/OFQ response to whole cell currents (Fig. 2A). This attenuation was found to be dose-dependent (Fig. 2B). At a concentration of 1 µM Nphe reduced the N/OFQ response to nearly zero (n = 7; Fig. 2B). The IC₅₀ value for Nphe was 16 pM (Fig. 2B). However, Nphe also seemed to act as a partial agonist because it depressed whole cell currents (Fig. 2A). The maximal reduction of whole cell currents evoked by Nphe (11.3 ± 1.8%; n = 5) was not significantly different from that induced by 1 µM N/OFQ (p > 0.05).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Dose-response relationship of N/OFQ antagonists. A, sample I-V plots of whole cell currents from a sample cell evoked under control conditions, in the presence of 1 µM N/OFQ, during the application of 1 µM Nphe, and after washout. B, dose-response curve for Nphe using an application of 100 nM N/OFQ. Each data point represents the average of three to seven cells. C, dose-response curve for CompB in 100 nM N/OFQ. Each data point represents the average of three to seven cells. D, I-V relationship for whole cell currents evoked under control conditions, in the presence of 50 nM IBTX, 1 µM N/OFQ, and both IBTX and N/OFQ. Inset shows graph showing 1 µM IBTX block of N/OFQ.

N/OFQ Effects on Whole Cell Currents Are Attenuated by a Blockade of BK Channels. Under our recording conditions, currents that are activated in the voltage range in which N/OFQ exerts its effects, i.e., 30 to +30 mV, include Ca²⁺-activated K⁺ currents (I_K,Ca). We have previously shown that in DBB neurons the majority of the current flowing through the I_K,Ca channels is attributed to the larger conductance I_K,Ca channels, which are referred to as I_C or BK channels (Jassar et al., 1999; Jhamandas et al., 2001). Iberiotoxin (IBTX) is a selective inhibitor of the family of I_C or BK channels and was used determine whether the effects of the N/OFQ in DBB neurons are in fact mediated through this species of Ca²⁺-activated K⁺ conductances. Figure 2D shows the I-V relationship under control conditions, in the presence of 50 nM IBTX, and N/OFQ (1 µM) in the presence of IBTX. The amplitude of outward currents at +30 mV was significantly reduced from 5.7 ± 0.5 nA under control conditions to 4.8 ± 0.7 nA in the presence of IBTX, which is a 23.1 ± 5.0% reduction (n = 6; p < 0.05). Subsequent application of N/OFQ in the presence of IBTX did not result in a further significant reduction in the mean whole cell current (4.9 ± 0.7 nA; n = 6; p > 0.05). This occlusion of the N/OFQ response in the presence of IBTX results suggests that effects of N/OFQ on whole cell currents are mediated by BK channels.

Effect of N/OFQ on Voltage-Dependent Ca²⁺ Channels. Because N/OFQ blocks BK channels, one possible target of its action can be the VSCC through which the Ca²⁺ influx responsible for the activation of BK channels may occur. To address this possibility, we examined whether N/OFQ affected voltage-dependent Ca²⁺ channels. Currents through Ca²⁺ channels were recorded using barium (I_Ba) as a charge carrier. Previous studies have shown that N-type and L-type Ca²⁺ conductances account for the majority of current flowing through the Ca²⁺ channels in DBB neurons (Easaw et al., 1999). Therefore, we examined the effects of N/OFQ on Ca²⁺ conductances using -conotoxin, a selective blocker of N-type Ca²⁺ channel channels and nimodipine, which blocks the dihydropyridine-sensitive L-type Ca²⁺ channels. Figure 3A shows a sample trace and the voltage protocol used to measure the I_Ba current. Under control conditions, peak I_Ba current of 3.4 ± 0.3 nA was observed at a voltage of 20 mV (Fig. 3B). In the presence of N/OFQ, peak I_Ba current was significantly reduced to 2.2 ± 0.2 mV, which is a decrease of 34.3 ± 2.2% (n = 12; p < 0.05; Fig. 3B). Application of -conotoxin (100 nM), resulted in a 50.6 ± 4.5% decrease in peak I_Ba current (n = 6; Fig. 3C). In the presence of both -conotoxin and N/OFQ, there was a further 6.6 ± 1.4% decrease in peak I_Ba current, which is significantly lower than that observed when N/OFQ was applied without the blocker (n = 6; p < 0.05). In the presence of nimodipine (10 µM), there was a 47.9 ± 2.5% reduction in peak I_Ba current (n = 4; Fig. 3D). When both nimodipine and N/OFQ were applied concurrently, the peak Ca²⁺ current was inhibited by 12.6 ± 2.0% (n = 5; Fig. 3E). Application of -conotoxin and nimodipine simultaneously resulted in a reduction of the peak I_Ba by 63.8 ± 4.9%. When N/OFQ was added in the presence of both blockers, there was only an additional 2.6 ± 3.3% reduction in I_Ba, which was significantly less than that observed when N/OFQ had been applied alone (n = 5; p < 0.05; Fig. 3E).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of N/OFQ on N- and L-type voltage-dependent Ca2+ channels. A, sample trace of Ba2+ currents evoked under control conditions. B, I-V plot for Ba2+ currents evoked under control conditions, in the presence of 1 µM N/OFQ, and upon recovery. C to E, sample traces showing 100 nM -conotoxin, 10 µM nimodipine, and a combination of 100 nM -conotoxin and 10 µM nimodipine, respectively, blocking N/OFQ response in peak Ba2+ currents. On the right, histograms depict percentage of reductions in the presence of the blocker and the blocker plus N/OFQ.

Effects of N/OFQ on Transient Outward (I_A) and Delayed Rectifier (I_K) K⁺ Currents. I_A and I_K are voltage-sensitive currents, and their activation and inactivation are strongly voltage-dependent. I_A 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, I_K is not inactivated at 40 mV. These biophysical properties of I_A and I_K can, thus be used to isolate these currents. Therefore, a conditioning pulse to 40 mV will activate I_K without any significant contamination by I_A (Connor and Stevens, 1971; Jhamandas et al., 2002). A conditioning pulse to 120 mV will activate both I_A and I_K. The difference currents obtained by subtracting the currents evoked by depolarizing pulses after a conditioning pulse to 40 mV from those evoked after a conditioning pulse to 120 mV provide an accurate estimate of I_A. Figure 4A shows the currents recorded from a DBB neuron with a conditioning pulse to 40 mV for 150 ms, representing mainly I_K, under control conditions, in the presence of N/OFQ and recovery on washout of N/OFQ. N/OFQ depressed peak I_K from 9.6 ± 2.0 to 8.4 ± 2.0 nA which is a decrease of 12.8 ± 4.0% (n = 6; p < 0.05). Figure 4B shows the different currents recorded from the same neuron representing mainly I_A, under control conditions, in the presence of N/OFQ, and on washout. In the presence of N/OFQ, peak I_A currents were decreased from 3.7 ± 0.6 to 3.2 ± 0.6 nA, which is a decrease of 16.9 ± 4.0% (n = 6; p < 0.05). We have previously shown that the residual sustained current remaining at the end of the 100-ms test pulse (Fig. 4B) consists mainly of I_K and I_C (Easaw et al., 1999), both of which are also reduced by N/OFQ. Figure 4, C and D, shows the current-voltage relationships of averaged peak I_K (n = 6) and I_A (n = 6), respectively, using the voltage step protocol shown in Fig. 4, A and B. 

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effects of N/OFQ on IK and IA currents. A and B, effects of N/OFQ (100 nM) on IK and IA in a DBB neuron. A, 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 and IC. B, in the same neuron, N/OFQ causes a decrease in transient outward K+ currents obtained by subtracting the voltage protocol shown in A from that obtained by applying the voltage protocol shown in B where cells were held at 80 mV and a 150-ms conditioning pulse to 120 mV was applied. C and D, voltage dependent (40 to +40 mV) changes in mean IK (n = 6) and IA (n = 6) currents, respectively, under control conditions and in the presence of N/OFQ.

	Discussion

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In the present study, we demonstrate that N/OFQ inhibits whole cell currents in both cholinergic and GABAergic acutely dissociated neurons of the DBB, a basal forebrain nucleus that is important in the context of memory and learning mechanisms. The N/OFQ effects on whole cell currents were antagonized by two N/OFQ antagonists used in this study. N/OFQ blocks currents through N- and L-type Ca²⁺ channels and the IBTX-sensitive Ca²⁺-activated K⁺ channels. In addition, N/OFQ also inhibited I_A and I_K.

Pharmacological Antagonism of N/OFQ Effects on Whole Cell Currents. Under voltage-clamp conditions, N/OFQ caused a dose-dependent decrease in outward currents in the voltage range from 30 to +30 mV. Although trypsinization has been reported to inhibit activity of certain receptor types, the time-independent preservation of N/OFQ responses in our study suggests that N/OFQ receptors on DBB cells are not affected by the enzymatic dissociation procedure. The EC₅₀ value for N/OFQ in inhibiting whole cell currents in our study was 1.2 nM and is consistent with previous data from acutely dissociated periaqueductal gray neurons, where an EC₅₀ value of 5 nM was reported for Ca²⁺ conductances (Connor and Christie, 1998). We chose to use higher concentrations of N/OFQ to ensure maximal effects on other ionic conductances (I_K, I_A, and I_C) that may display different sensitivities to the peptide. One of the difficulties in studying the actions of N/OFQ at a cellular level is the lack of an antagonist that does not also exhibit partial agonist activity. Previously, a tridecapeptide analog of N/OFQ, [Phe¹(CH₂-NH)Gly₂]N/OFQ-(1-13)-NH₂, was identified as a potential selective antagonist for N/OFQ on the basis of its actions in guinea pig ileum (Guerrini et al., 1998). However, electrophysiological studies examining the effects of N/OFQ on the periaqueductal gray and locus coeruleus neurons revealed that [Phe¹(CH₂-NH)Gly₂]N/OFQ-(1-13)-NH₂ also displayed partial agonist activity by inhibiting inwardly rectifying K⁺ currents (Chiou, 1999; Connor et al., 1999).

N/OFQ Modulation of Ca²⁺ and K⁺ Conductances. In the locus coeruleus, the supraoptic nucleus, and hippocampal CA1 neurons, N/OFQ has been shown to increase inwardly rectifying K⁺ conductance (Connor et al., 1996; Madamba et al., 1999; Slugg et al., 1999). Acutely dissociated DBB neurons do not display an inwardly rectifying K⁺ conductance (Jassar et al., 1999). However, in this study we report, for the first time, the ability of N/OFQ to block voltage- and Ca²⁺-activated K⁺ conductance (BK channels). BK channels are composed of a pore-forming  subunit and modulatory  subunits (Meera et al., 2000). The 4 subunit is abundantly expressed in the brain and renders the BK channel subunit resistant to IBTX (Meera et al., 2000). The BK channels we observed are sensitive to IBTX, suggesting that 4 subunit was not contributing to channel formation in DBB neurons. Since BK channels are involved in repolarization phase of the action potential and the phenomenon of accommodation (Vergara et al., 1998), N/OFQ may through its effects on this Ca²⁺-dependent K⁺ conductance play an important role in governing the excitability of DBB neurons. Because activation of BK channels requires an influx of Ca²⁺ into the cell, we also examined the actions of N/OFQ on VSCC.

Functional Considerations. N-type Ca²⁺ channels play a significant role in neurotransmitter release (Meir et al., 1999). Single cell RT-PCR analysis of DBB neurons that responded to N/OFQ revealed a majority of these to be cholinergic. Loss of cholinergic tone in basal forebrain neurons is linked to spatial memory and cognitive deficits observed in Alzheimer's disease (Yankner, 1996). An inhibition of N- and L-type Ca²⁺ channels by N/OFQ would result in a diminished release of acetylcholine from terminals of DBB neurons that project densely to the hippocampus.