Excessive cerebrocortical release of acetylcholine induced by ... - Nature

Molecular Psychiatry (1999) 4, 344–352  1999 Stockton Press All rights reserved 1359–4184/99 $15.00

ORIGINAL RESEARCH ARTICLE

Excessive cerebrocortical release of acetylcholine induced by NMDA antagonists is reduced by GABAergic and ␣2-adrenergic agonists SH Kim1, MT Price2, JW Olney2 and NB Farber2 1

Department of Neuropsychiatry, Korea University College of Medicine, Seoul, Korea; 2Department of Psychiatry, Washington University, St Louis, MO, USA N-methyl-D-aspartate (NMDA) glutamate (Glu) receptor antagonists (eg MK-801, ketamine, phencyclidine [PCP]) injure cerebrocortical neurons in the posterior cingulate and retrosplenial cortex (PC/RSC). We have proposed that the neurotoxic action of these agents is mediated in part by a complex polysynaptic mechanism involving an interference in GABAergic inhibition resulting in excessive release of acetylcholine (ACh). Previously we have found that the systemic injection of GABAergic agents and ␣2-adrenergic agonists can block this neurotoxicity. In the present study we tested the hypothesis that NMDA antagonists trigger release of ACh in PC/RSC and that this action of NMDA antagonists is suppressed by GABAergic agents or ␣2-adrenergic agonists. The effect of MK-801 and ketamine on PC/RSC ACh output (and the ability of pentobarbital, diazepam and clonidine to modify MK-801-induced ACh release) was studied in adult female rats using in vivo microdialysis. Both MK-801 and ketamine caused a significant rise in PC/RSC ACh output compared to basal levels. Pentobarbital, diazepam and clonidine suppressed MK-801’s effect on ACh release. Exploratory studies indicated that the site of action of these agents was outside of the PC/RSC. The microdialysis results are consistent with several aspects of the circuitry proposed to mediate the neurotoxic action of NMDA antagonists. Keywords: NMDA antagonists; schizophrenia; Alzheimer’s disease; posterior cingulate; retrosplenial cortex; MK-801; ketamine; microdialysis; neurotoxicity

Introduction Cerebrocortical neurons in the adult rat brain are injured by systemic administration of drugs, such as MK-801, ketamine, and phencyclidine (PCP), that block N-methyl-d-aspartate (NMDA) glutamate (Glu) receptors.1 Although the neurotoxic action was initially described as reversible and limited to specific neurons in the posterior cingulate/retrosplenial cortex (PC/RSC), it was soon learned that it can be irreversible and can affect many neuronal populations, depending on the length of time NMDA receptors are maintained in a profoundly hypofunctional state.2–4 Administering PCP in high dosage or by continuous infusion for several days induces a prolonged NMDA receptor hypofunction (NRHypo) state resulting in a widespread pattern of neuronal degeneration.4–8 Low or intermediate doses of PCP trigger an abnormal heat shock protein (Kd 72) reaction affecting the same widely distributed populations of neurons that are killed by higher doses.4,9,10 This disseminated pattern of neurodegeneration closely resembles the pattern of neurofibrillary

Correspondence: NB Farber, MD, Washington University, Department of Psychiatry, 4940 Children’s Place, St Louis, MO 63110– 1093, USA. E-mail: farbern얀psychiatry.wustl.edu Received 29 October 1998; revised and accepted 6 January 1999

degeneration that occurs in Alzheimer’s disease.4,8,11 Based on these findings and on the observation that the NMDA receptor system becomes markedly hypofunctional in old age12–15 and is even more hypofunctional in patients with Alzheimer’s disease,16 NRHypo has been proposed as a candidate mechanism to help explain neurodegeneration in Alzheimer’s disease.11,17,18 In addition, because NMDA antagonists, such as PCP and ketamine, cause a schizophrenia-like psychotic reaction in adult humans, NRHypo is increasingly being recognized as a mechanism that may play a role in either the psychotic manifestations or structural brain changes (or both) that occur in schizophrenia.18–26 Thus, research aimed at further clarifying the receptor mechanisms and circuitry underlying NRHypo-induced neurotoxic effects in the brain may have both theoretical and practical importance. In a series of studies it has been shown that the PC/RSC neurotoxic effects of NMDA antagonists are blocked by systemic administration of several classes of transmitter receptor-specific agents, including agonists of GABAA and ␣2-adrenergic receptors and antagonists of cholinergic muscarinic and glutamatergic non-NMDA receptors.10,27,28 Based on these and related findings, we have postulated10,11,18,21 that the neurotoxic action of NRHypo is mediated by a complex polysynaptic mechanism involving an interference in

Role of ACh, GABA and NE in NRHypo neurotoxicity SH Kim et al

GABAergic inhibition resulting in excessive release of acetylcholine (ACh) and Glu, respectively, at muscarinic and non-NMDA Glu receptors on PC/RSC neurons. To explain the finding that ␣2-adrenergic agonists block NRHypo-induced neurotoxicity, we have proposed that they do so by counteracting the postulated ACh-releasing action of NRHypo.27 Additional findings corroborating the proposed involvement of muscarinic receptors on PC/RSC neurons include the demonstration that microinjection of scopolamine, a muscarinic antagonist, into the PC/RSC of rats treated systemically with MK-801, prevents the neurotoxic reaction in the PC/RSC ipsilateral to the injection.29 The circuitry and receptor mechanisms that we postulate may mediate NMDA antagonist neurotoxicity are depicted in Figure 1. Although consistent with a large body of pharmacological evidence, this circuit diagram has not been corroborated by direct measurement of transmitter release at the points in the circuitry where the diagram would predict such release would occur. For example, the interpretation that NRHypo triggers excessive release of ACh in PC/RSC and that GABAA agonists or ␣2-adrenergic agonists are neuroprotective because they prevent such release, has not been confirmed by direct measurement of ACh levels in PC/RSC. To provide such data, we undertook the present experiments in which in vivo microdialysis methods were used to study the effect of MK-801 and ketamine on ACh levels in PC/RSC of freely-moving rats, and to test the ability of the ␣2-adrenergic agonist,

clonidine, or the GABAA agonists, pentobarbital and diazepam, to modify MK-801 effects on PC/RSC ACh levels.

Materials and methods Animal preparation Adult female Sprague–Dawley rats, weighing between 250–320 g, were used. Seventy-two hours prior to the microdialysis experiment, the rats were anesthetized with isoflurane, placed in a Kopf stereotaxic apparatus and a guide cannula (20-gauge stainless steel; Small Parts, Miami, FL, USA) introduced into the brain through a burr hole that was made in the occiput. The guide cannula was inserted so that it coursed horizontally from posterior to anterior in a plane parallel with the dorsum of the skull with the tip of the cannula being placed at AP −5.5; ML −0.6; DV −1.5 relative to Bregma30 so that the dialysis membrane would be maximally exposed to the PC/RSC (Figure 2). The cannula was cemented in place with dental acrylic attached to two stainless steel screws anchored in the skull. A stainless steel obturator (25 gauge; Small Parts) was inserted into the cannula to maintain patency until the microdialysis probe was inserted. Microdialysis procedure Concentric microdialysis probes (5 mm tip length) were constructed according to methods described by Robinson and Whishaw.31 On the eve of the dialysis

Figure 1 Circuitry proposed to mediate NRHypo-induced neurotoxicity and excessive ACh release. To explain NRHypoinduced excessive ACh release, we propose that Glu, acting through NMDA receptors on GABAergic and noradrenergic neurons, maintains tonic inhibitory control over a basal forebrain cholinergic neuron that projects to and innervates certain PC/RSC neurons. Systemic administration of an NMDA receptor antagonist (or NRHypo produced by any mechanism) would abolish inhibitory control over this excitatory input to PC/RSC neurons, resulting in excessive release of ACh. We have proposed 21 that the excessive release of ACh produced by this projection combines with excessive release of Glu that is produced through different neuronal projections to excessively stimulate two distinct receptor populations (muscarinic cholinergic and nonNMDA glutamatergic) on the vulnerable PC/RSC pyramidal neuron, resulting in neurotoxicity. This circuit diagram focuses exclusively on PC/RSC neurons. We hypothesize that a similar but not necessarily identical mechanism mediates damage induced in other corticolimbic brain regions by sustained NRHypo. (+) = excitatory input; (−) = inhibitory input; ACh = acetylcholine; NE = norepinephrine; ␣2 = ␣2 subtype of adrenergic receptor; GA = GABAA subtype of GABA receptor; m3 = m3 subtype of muscarinic cholinergic receptor; non-NMDA = non-NMDA subtype of Glu ionotropic receptor; NMDA = NMDA subtype of Glu receptor.

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Figure 2 Insertion of guide cannula and dialysis probe into the PC/RSC. (a) Drawing of a sagittal section of a rat brain just off of midline (ML +0.4) illustrating the placement of the guide cannula and dialysis probe into the PC/RSC. The vertically oriented box indicates the rostral-caudal level from which the tissue section in (b) is taken. Numbers at the top of the figure indicate distance in mm from Bregma. (b) Coronal tissue section taken from an adult rat demonstrating the location of the dialysis probe. Arrows indicate hole made by dialysis probe in the PC/RSC.

experiment, a microdialysis probe was inserted through the guide cannula, and the animal placed in a round plastic bowl (BAS Bee Keeper System, West Lafayette, IN, USA). The probe extended 5 mm beyond the guide cannula thus allowing an extensive amount of PC/RSC to be sampled. After insertion, the probe was perfused continually at a flow rate of 0.5 ␮l min−1 with artificial cerebral spinal fluid (CSF, 145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, 1.0 mM MgCl2 and 2.0 mM Na2HPO4) containing 100 nM neostigmine (an acetylcholinesterase inhibitor) at pH 7.4. On the day of the experiment, the probe was perfused at a rate of 2.0 ␮l min−1 for 2 h prior to sample collection. Three samples (40 ␮l) were collected every 20 min to assess basal concentrations of ACh. Then samples were collected at 20min intervals following drug administration. For those studies in which the drug (MK-801 or clonidine) was perfused into the PC/RSC over a period of time, a

CMA/110 liquid switch (CMA, Stockholm, Sweden) was used to allow the instantaneous change form the artificial CSF to the drug-containing artificial CSF solution without risking the introduction of air into the microdialysis probe. Samples were stored at −80°C prior to analysis by high pressure liquid chromatography (HPLC). Animals were killed immediately following the microdialysis experiments, perfused with 4% paraformaldehyde, and their brains sectioned and examined histologically to verify microdialysis probe placement. Only animals with correctly implanted probes were included in the data analysis. ACh analysis Extracellular concentrations of ACh were quantified in dialysates using HPLC coupled with electrochemical detection. A 10-␮l sample of the perfusate was injected directly into an ESA integrated HPLC system, equipped


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with a CMA/200 refrigerated microsampler (CMA, Stockholm, Sweden), an ESA 5200A Coulochem electrochemical detector and an ESA ACH-3 polymeric reversed-phase column (particle size 5 ␮m, 15 cm × 3 mm i.d.), which was eluted isocratically at 0.35 ml min−1 with 0.1 M Na2HPO4, 0.5 mM TMACl, 0.005% reagent MB (ESA Inc, Chelmsford, MA, USA), 2 mM OSA (pH 8.0 with HPLC grade 85% phosphoric acid). Following separation by the analytical column, ACh was converted into hydrogen peroxide by a post-column solid phase reactor (ACH-SPR enzyme reactor, ESA Inc). The hydrogen peroxide product was detected at a working electrode set at a potential of +300 mV (model 5040 analytical cell with platinum target, ESA Inc). The limit of detection for ACh was 40 fmol per 10 ␮l injection volume. Data analysis and statistics ACh output was expressed as a percentage of basal output. For each rat a basal output was determined by obtaining the mean for the three samples taken before administration of the test agent. For each rat the ACh in each sample was normalized by dividing the absolute level by the calculated basal output and multiplying the result by 100 to obtain a percentage. Percentages for each time point were averaged over all the animals in the given experimental condition. Differences between each experimental condition and control were evaluated by two way ANOVAs seeking a significant interaction between treatment condition and time. Significant ANOVAs were subsequently evaluated by further F-tests to determine the specific time points that accounted for the significant interaction.

Results To test the hypothesis that NRHypo induces release of ACh in the PC/RSC, MK-801 was administered subcutaneously (sc) at a dose (0.5 mg kg−1) that reliably causes neurotoxic changes in PC/RSC neurons in 100% of the animals,1 and ACh was measured in the PC/RSC dialysate at 20-min intervals for 1 h prior to and 3 h after MK-801 treatment. The ACh concentration rose abruptly (Figure 3) to a level approximately three times higher than baseline by 20 min after MK-801 administration, then continued to rise gradually to a peak value at 3 h (the last measurement) that was 400% of baseline. Similar effects on ACh release (Figure 3) were seen with ketamine (50 mg kg−1 sc) confirming that antagonism of NMDA receptors (ie NRHypo) is the likely mechanism leading to the increase in ACh output. Saline injections alone did not lead to an increase in ACh output above basal levels (Figure 3). To determine whether the ACh releasing action of MK-801 can be attributed to an action of MK-801 in the PC/RSC, MK-801 (20 ␮M) was locally infused into the PC/RSC via the dialysis probe. Direct introduction of MK-801 into PC/RSC resulted in no significant change in ACh levels in PC/RSC at any time over the 3-h period of infusion (Figure 3), which signifies that the increased release stimulated by systemic MK-801

Figure 3 Effect of NMDA antagonists on ACh output in PC/RSC. Dialysate samples were obtained over 20-min periods. Mean ACh output was calculated as described in the Methods section and expressed as a percentage of basal output (mean ± SEM). Error bars are not visible at some time points due to their small magnitude. Systemic injection of MK-801 (0.5 mg kg−1 sc) and ketamine (50 mg kg−1 sc) resulted in a significant elevation in ACh output to 300–400% of baseline (MK-801: F[11,153] = 3.58, P ⬍ 0.0005; ketamine: F[11,93] = 7.40, P ⬍ 0.0001). The significant treatment condition by time interaction for both MK-801 and ketamine was explained by significant differences in ACh output at all time points after the injection (P ⬍ 0.05 for all points). Application of MK-801 (20 ␮M) into the PC/RSC by perfusion in the dialysis probe (MK-801 perfusion) did not result in any change in ACh output (treatment × time interaction: F[11,132] = 1.36; P ⬎ 0.1), indicating that the site of action of NMDA antagonists with respect to controlling ACh release is outside of the PC/RSC. Arrow indicates the time of sc injection of test agent. In the experiment where MK-801 was perfused via the dialysis probe into the PC/RSC, the solid bar indicates the period during which MK-801 was present in the perfusate.

is not likely due to a local action of MK-801 in the PC/RSC. To test the hypothesis that ␣2-adrenergic agonists block the PC/RSC neurotoxic activity of NRHypo by interfering with NRHypo-induced ACh release, we administered the ␣2-adrenergic agonist, clonidine (0.05 mg kg−1 sc), either by itself or in combination with MK-801 and found that by itself clonidine does not influence PC/RSC ACh levels, but in combination with MK-801 it substantially suppresses the MK-801


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Figure 4 Effect of clonidine on NRHypo-induced ACh release in PC/RSC. Dialysate samples were obtained over 20min periods. Mean ACh output was calculated as described in the Methods section and expressed as a percentage of basal output (mean ± SEM). Error bars are not visible at some time points due to their small magnitude. The systemic injection of clonidine (0.05 mg kg−1 sc) when given with systemic MK801 (0.5 mg kg−1 sc) resulted in significant decrease in ACh output in the PC/RSC as compared to that seen with MK-801 alone (treatment × time interaction: F[14,234] = 2.51, P ⬍ 0.005). The significant treatment condition by time interaction was explained by significant differences in ACh output at all time points after the injection of MK-801 (P ⬍ 0.05). The application of clonidine (100 ␮M) into the PC/RSC by perfusion in the dialysis probe without exposing the animals to MK-801, resulted in no significant change in ACh output as compared to that seen with saline injections (treatment × time interaction: F[11,87] = 0.56, P ⬎ 0.1; saline data not graphed for presentation clarity). Application of clonidine (100 ␮M) into the PC/RSC by perfusion in the dialysis probe in animals that received MK-801 systemically (0.5 mg kg−1 sc) did not result in any significant change in ACh output (treatment × time interaction: F[12,196] = 0.98, P ⬎ 0.1) as compared to MK-801 alone. Filled arrow indicates the time of sc injection of MK-801. Open arrow indicates when clonidine was injected in those animals that received MK-801. In the experiment where clonidine was perfused via the dialysis probe into the PC/RSC the solid bar indicates the period during which clonidine was present in the perfusate.

effect, limiting it to 200% of basal output instead of 400% (Figure 4). To determine whether clonidineinduced suppression of the ACh releasing action of MK-801 can be attributed to an action of clonidine in

Figure 5 Effect of GABAergic agonists on NRHypo-induced ACh release in PC/RSC. Dialysate samples were obtained over 20-min periods. Mean ACh output was calculated as described in the Methods section and expressed as a percentage of basal output (mean ± SEM). Error bars are not visible at some time points due to their small magnitude. The injection of pentobarbital (25 mg kg−1 sc) when given with MK-801 (0.5 mg kg−1 sc) resulted in a significant decrease in ACh output in the PC/RSC (treatment × time interaction: F[14,180] = 7.31, P ⬍ 0.0001). The significant treatment condition by time interaction was explained by significant differences (P ⬍ 0.005) in ACh output at all time points after 140 min (ie in samples collected between 141 [21 min post pentobarbital injection] and 240 min). Diazepam (6 mg kg−1 sc) had a similar effect on MK-801-induced ACh release (treatment × time interaction: F[14,255] = 4.82, P ⬍ 0.0001). The significant treatment condition by time interaction was explained by significant differences (P ⬍ 0.05) in ACh output at all time points beginning 21 min after the injection of diazepam (ie time points 160–240). Filled arrow indicates the time of sc injection of MK-801. Open arrow indicates when pentobarbital or diazepam was injected.

the PC/RSC, MK-801 was administered systemically and clonidine (100 ␮M) was administered directly into the PC/RSC via the dialysis probe. Infusion of clonidine locally into the PC/RSC did not significantly influence the MK-801-induced release of ACh (Figure 4), signifying that the locus of action of clonidine is not in the PC/RSC. To test the hypothesis that GABAergic inhibition plays a major role in the mechanism by which NRHypo induces excessive release of ACh in the PC/RSC, pento-


barbital (25 mg kg−1 sc) was administered to rats that had received MK-801 (0.5 mg kg−1 sc) 2 h previously. Pentobarbital caused a precipitous fall in the PC/RSC concentration of ACh from 350% of basal output to a level 50% of basal output (Figure 5). To further evaluate the role of GABAA receptors we administered diazepam systemically (6 mg kg−1 sc) to rats treated 2 h previously with MK-801 and found it reduced the elevation to 150% of basal output (Figure 5). These results are consistent with our hypothesis that loss of GABAergic inhibition plays a major role in NRHypo-induced ACh release, and that restoring GABAA receptor activity by administration of pentobarbital or diazepam reinstated inhibitory control over ACh release, thereby returning ACh to normal or near normal levels.

Discussion Based on the initial finding10 that GABAA agonists and muscarinic cholinergic antagonists protect against NRHypo-induced neurodegeneration, it was proposed that NMDA receptor blockade abolishes GABAergic inhibitory control over ACh release, thereby causing excessive release of ACh at muscarinic receptors on PC/RSC neurons, as a proximal mechanism contributing to the injury of these neurons. The more recent observation29 that direct injection of the muscarinic antagonist, scopolamine, into PC/RSC protects PC/RSC neurons against the neurotoxic action of systemically administered MK-801, further corroborates that hyperactivation of muscarinic receptors in the PC/RSC region is a critical component of the neurotoxic mechanism. Our present finding that systemic application of NMDA antagonists induces a sustained increased release of ACh in the PC/RSC region confirms that the basis for the hyperactivation of PC/RSC muscarinic receptors is that these receptors are being persistently flooded with excessive ACh. The fact that the excessive release was sustained and showed no signs of abating during the 3-h sampling period is consistent with the assumptions of our circuit diagram (Figure 1) that ACh release in this network is normally held under constant (tonic) restraint and that blockade of NMDA receptors removes this tonic restraining mechanism, thereby allowing a pattern of sustained ACh release that would never be encountered under normal circumstances. The observation that pentobarbital effectively arrests the excessive ACh release and returns ACh concentrations in PC/RSC to normal or subnormal levels, is consistent with the interpretation10,18,21 that GABAergic inhibitory neurons and GABAA receptors are critically involved, and that Glu, acting at a NMDA receptor on the GABAergic neuron drives the tonic inhibitory mechanism by which release of ACh in the PC/RSC is controlled. According to this interpretation MK-801 blockade of the NMDA receptor prevents Glu from driving the GABAergic neurons and this inactivates the GABAA receptor on the cholinergic neuron and releases the cholinergic neuron from inhibition, thereby causing excessive release of ACh in PC/RSC

which contributes to injury of the PC/RSC neuron. Pentobarbital acts directly at the GABAA receptor to restore inhibitory control over the cholinergic neuron and returns the ACh releasing activity to normal. This provides a logical explanation for the finding that pentobarbital, if applied in a timely manner, prevents PC/RSC neuronal injury.10,32 Further study will be needed to determine if this simple mechanistic explanation is correct or whether another, more complicated, mechanism is involved. Diazepam at a dose of 6 mg kg−1 sc decreased the magnitude of the MK-801-induced ACh release but, unlike pentobarbital, diazepam did not totally abolish the ACh releasing action or decrease ACh levels to below baseline. This observation is consistent with the earlier finding10 that diazepam in the same dose range provided substantial but not complete protection against the PC/RSC neurotoxic action of MK-801. We have proposed that the greater efficacy of pentobarbital compared to diazepam may be due to a difference in the mechanism by which these agents interact at GABAA receptors.10 To explain the finding that ␣2-adrenergic agonists block NRHypo-induced neurotoxicity we have proposed that they do so by counteracting the ACh-releasing action of this state, and that the ␣2-adrenergic receptor through which norepinephrine (NE) normally regulates ACh release in this circuit may either be on the dendrosomal surface of the cholinergic neuron or on its axon terminal in the PC/RSC region. The present finding that the ␣2-adrenergic agonist, clonidine, when administered systemically, strongly suppresses the ACh releasing action of MK-801, confirms that NE does normally regulate ACh release in this circuit, that NMDA receptor blockade abolishes this regulatory mechanism and that clonidine restores it. This finding is consistent with the proposed circuit diagram (Figure 1) depicting the NE neuron functioning as an inhibitory neuron that is driven by Glu to inhibit the release of ACh from a basal forebrain cholinergic neuron. Failure of clonidine to completely suppress the ACh releasing action of MK-801 is consistent with the fact that the dose used is the ED50 dose for protecting against MK801 neurotoxicity, ie, it is a dose that suppresses the neurotoxic reaction by 50%.27 The present experiments were not designed to determine definitively where the NMDA receptor-bearing GABAergic neuron or the cholinergic neuron that it inhibits are located. However, the finding that systemic administration of MK-801 triggered ACh release whereas local PC/RSC application of MK-801 via the dialysis probe did not, signifies that the NMDA receptor-bearing GABAergic neuron with which MK-801 interacts to trigger ACh release, is not located in the PC/RSC region. In addition, we found that systemic administration of clonidine suppressed the ACh releasing action of MK-801 but local application of clonidine into PC/RSC did not. This signifies that the ␣2-adrenergic receptor with which clonidine interacts is not in the PC/RSC region, ie it is not on a cholinergic axon terminal in the PC/RSC region nor on the dendrosomal

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surface of an intrinsic cholinergic neuron in the PC/RSC region. Cell bodies in the diagonal band region of the basal forebrain provide the main extrinsic source of cholinergic fibers to the PC/RSC.33–35 Thus the diagonal band region is the most likely location of the cholinergic neurons that contribute to the PC/RSC neurotoxic action triggered by systemic MK-801, and the ␣2adrenergic receptor probably is located on the dendrosomal surface of this neuron. Confirming this conclusion we have found that the direct injection of clonidine into the diagonal band attenuates MK-801induced neurotoxicity in the PC/RSC while direct injections into the PC/RSC itself or the nucleus basalis are ineffective.36 Since it is known that there is a relatively dense population of GABAergic interneurons that are colocalized with and have the same irregular distribution as the magnocellular basal forebrain cholinergic neurons,37,38 it is likely that the NMDA receptor-bearing GABAergic neuron, that regulates the release of ACh in the PC/RSC, is an interneuron colocalized with the cholinergic neuron that it regulates in the diagonal band region of the basal forebrain. Although other more complicated interpretations could be conceived, the interpretation we have adopted (depicted schematically in Figure 1) is the most parsimonious one that fits all available data. Previously we have proposed, and here we present microdialysis data consistent with a mechanism by which Glu, acting at NMDA receptors on GABAergic neurons in the basal forebrain, regulates release of ACh in the posterior cingulate and retrosplenial regions of the cerebral cortex. Several years ago, Giovaninni et al39 described a similar mechanism by which the release of ACh in the hippocampus is regulated. These authors demonstrated that when an NMDA antagonist is injected directly into the septal region where the cholinergic neurons that project to the hippocampus are located, it triggers a striking release of ACh in the hippocampus. If they coinjected into the septal region both an NMDA antagonist and the GABAA agonist, muscimol, the ACh releasing action of the NMDA antagonist was prevented. Their interpretation was as follows: Glu, acting at NMDA receptors on GABAergic neurons that synapse upon cholinergic neurons in the medial septum, regulates the release of ACh in a distant brain region, the hippocampus. Our findings and interpretation are identical except that in the system we are studying the cholinergic neurons, which are regulated by NMDA receptor-bearing GABAergic neurons, are located in the diagonal band region (slightly subjacent and caudal to the medial septum) and the distant brain region where release of ACh is being monitored is the PC/RSC region of the cerebral cortex. There are no prior studies directly focusing on mechanisms regulating release of ACh in the PC/RSC, but there is evidence that intraperitoneal administration of MK-801 (non-competitive NMDA antagonist), intracerebroventricular administration of CPP (competitive NMDA antagonist) or inhalation of nitrous oxide (laughing gas, another NMDA antagonist40,41) evokes release of ACh in other cortical areas.42–45 Thus, avail-

able data suggest an interesting and readily testable hypothesis—that it may be a general principle that release of ACh from axon terminals of basal forebrain cholinergic neurons which project to various portions of the cerebral cortex, hippocampus and other limbic brain regions, may be regulated by Glu acting via NMDA receptor-bearing GABAergic neurons which are co-localized with the cholinergic neurons in the basal forebrain. While the present findings pertain specifically to the regulation of ACh release in a single brain region, the PC/RSC, the NRHypo mechanism has the potential to produce widespread corticolimbic neurodegeneration which follows a distribution pattern resembling the pattern of cholinergic innervation of cerebrocortical and related limbic brain regions. Since excessive release of ACh could be an important factor in determining whether a specific region undergoes NRHypo-induced neurodegeneration, it will be important in the future to study whether the effects of NMDA antagonists on ACh release in a given brain region are correlated with the region’s suceptibility to NRHypo-induced neurodegeneration. In addition to excessive release of ACh we have proposed that the NRHypo state also causes the excessive release of Glu.21 On-going work indicates that it is the excessive stimulation of both muscarinic cholinergic and non-NMDA glutamatergic receptors on the vulnerable pyramidal neuron which leads to the observed excitotoxicity through a more complex circuitry than we have depicted in Figure 1.29,36 Preliminary results with microdialysis indicate that in addition to the abnormal release of ACh, NMDA antagonists also induce abnormal release of excitatory amino acids in PC/RSC46 as well as prefrontal cortex.26,47 In view of the important roles that both the cholinergic and Glu transmitter systems have in memory, cognition and related mental functions, it is important from both a health and disease perspective to develop a better understanding of normal mechanisms by which the Glu system regulates activity in the ACh system, and abnormal mechanisms by which the two systems can act in concert to produce deranged thinking (if mildly abnormal) or widespread excitotoxic neurodegeneration (if severely abnormal). To fully appreciate how blockade of NMDA receptors can trigger a neurodegenerative syndrome, it is necessary to begin thinking of Glu in a new light, as an agent that performs major inhibitory functions. By tonically activating NMDA receptors on GABAergic neurons (Figure 1), Glu regulates inhibitory tone and ordinarily protects neurons against the brain’s own self-destructive potential; removing this inhibitory mechanism from certain networks unleashes excitotoxic forces that wreak self destruction within the network. The excitotoxic forces are complex and include glutamatergic, cholinergic and other less well defined components, but the key to understanding this neurotoxic process is to recognize that Glu is not only an excitotoxic contributor to the pathological outcome, it is the driver of the inhibitory mechanism that normally


holds the excitotoxic forces in check. Inhibition failure (failure of Glu to maintain inhibition) is the basic principle underlying NRHypo neurodegeneration. We have postulated that this inhibition failure mechanism may play a central role in two major neuropsychiatric disorders, namely schizophrenia and Alzheimer’s disease. While it is beyond the scope of the present writing to discuss in detail the putative role of NRHypo in either of these diseases, each of these topics has been reviewed elsewhere.11,17,18,21,25,48,49

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Acknowledgements Supported by AG11355, DA05072, SDAC DA00290 (NBF), RSA MH 38894 (JWO), and a Young Investigator Award from NARSAD (NBF).

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