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

ORIGINAL RESEARCH ARTICLE Excessive cerebrocortical release of induced by NMDA antagonists is reduced by GABAergic ␣ and 2- SH Kim1, MT Price2, JW Olney2 and NB Farber2

1Department 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) antagonists (eg MK-801, , [PCP]) injure cerebrocortical in the posterior cingulate and retrosplen- ial 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 inhi- bition resulting in excessive release of acetylcholine (ACh). Previously we have found that ␣ the systemic of GABAergic agents and 2-adrenergic agonists can block this neuro- toxicity. 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 , and to modify MK-801-induced ACh release) was studied in adult female rats using in vivo . Both MK-801 and keta- mine 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 indi- cated 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; ; Alzheimer’s disease; posterior cingulate; retrosplenial cortex; MK-801; ketamine; microdialysis;

Introduction degeneration that occurs in Alzheimer’s disease.4,8,11 Based on these findings and on the observation that the Cerebrocortical neurons in the adult rat are NMDA receptor system becomes markedly hypofunc- injured by systemic administration of , such as tional in old age12–15 and is even more hypofunctional MK-801, ketamine, and phencyclidine (PCP), that in patients with Alzheimer’s disease,16 NRHypo has block N-methyl-d-aspartate (NMDA) glutamate (Glu) been proposed as a candidate mechanism to help receptors.1 Although the neurotoxic action was explain in Alzheimer’s dis- initially described as reversible and limited to specific ease.11,17,18 In addition, because NMDA antagonists, neurons in the posterior cingulate/retrosplenial cortex such as PCP and ketamine, cause a schizophrenia-like (PC/RSC), it was soon learned that it can be irreversible psychotic reaction in adult humans, NRHypo is and can affect many neuronal populations, depending increasingly being recognized as a mechanism that may on the length of time NMDA receptors are maintained play a role in either the psychotic manifestations or in a profoundly hypofunctional state.2–4 Administering structural brain changes (or both) that occur in schizo- PCP in high dosage or by continuous infusion for sev- phrenia.18–26 Thus, research aimed at further clarifying eral days induces a prolonged NMDA receptor hypo- the receptor mechanisms and circuitry underlying function (NRHypo) state resulting in a widespread pat- NRHypo-induced neurotoxic effects in the brain may tern of neuronal degeneration.4–8 Low or intermediate have both theoretical and practical importance. doses of PCP trigger an abnormal heat shock protein In a series of studies it has been shown that the (Kd 72) reaction affecting the same widely distributed PC/RSC neurotoxic effects of NMDA antagonists are populations of neurons that are killed by higher blocked by systemic administration of several classes doses.4,9,10 This disseminated pattern of neurodegener- of transmitter receptor-specific agents, including agon- ation closely resembles the pattern of neurofibrillary ␣ ists of GABAA and 2-adrenergic receptors and antag- onists of muscarinic and non-NMDA receptors.10,27,28 Based on these and related Correspondence: NB Farber, MD, Washington University, Depart- 10,11,18,21 ment of Psychiatry, 4940 Children’s Place, St Louis, MO 63110– findings, we have postulated that the neuro- 1093, USA. E-mail: farbernȰpsychiatry.wustl.edu toxic action of NRHypo is mediated by a complex poly- Received 29 October 1998; revised and accepted 6 January 1999 synaptic mechanism involving an interference in Role of ACh, GABA and NE in NRHypo neurotoxicity SH Kim et al 345 GABAergic inhibition resulting in excessive release of clonidine, or the GABAA agonists, pentobarbital and acetylcholine (ACh) and Glu, respectively, at muscar- diazepam, to modify MK-801 effects on PC/RSC ACh inic and non-NMDA Glu receptors on PC/RSC neurons. levels. ␣ To explain the finding that 2-adrenergic agonists block NRHypo-induced neurotoxicity, we have proposed that Materials and methods they do so by counteracting the postulated ACh-releas- ing action of NRHypo.27 Additional findings corrobor- Animal preparation ating the proposed involvement of muscarinic recep- Adult female Sprague–Dawley rats, weighing between tors on PC/RSC neurons include the demonstration 250–320 g, were used. Seventy-two hours prior to the that microinjection of , a muscarinic microdialysis experiment, the rats were anesthetized antagonist, into the PC/RSC of rats treated systemically with isoflurane, placed in a Kopf stereotaxic apparatus with MK-801, prevents the neurotoxic reaction in the and a guide cannula (20-gauge stainless steel; Small PC/RSC ipsilateral to the injection.29 Parts, Miami, FL, USA) introduced into the brain The circuitry and receptor mechanisms that we pos- through a burr hole that was made in the occiput. The tulate may mediate NMDA antagonist neurotoxicity are guide cannula was inserted so that it coursed horizon- depicted in Figure 1. Although consistent with a large tally from posterior to anterior in a plane parallel with body of pharmacological evidence, this circuit diagram the dorsum of the skull with the tip of the cannula has not been corroborated by direct measurement of being placed at AP −5.5; ML −0.6; DV −1.5 relative to transmitter release at the points in the circuitry where Bregma30 so that the dialysis membrane would be the diagram would predict such release would occur. maximally exposed to the PC/RSC (Figure 2). The can- For example, the interpretation that NRHypo triggers nula was cemented in place with dental acrylic excessive release of ACh in PC/RSC and that GABAA attached to two stainless steel screws anchored in the ␣ agonists or 2-adrenergic agonists are neuroprotective skull. A stainless steel obturator (25 gauge; Small Parts) because they prevent such release, has not been con- was inserted into the cannula to maintain patency until firmed by direct measurement of ACh levels in the microdialysis probe was inserted. PC/RSC. To provide such data, we undertook the present experiments in which in vivo microdialysis Microdialysis procedure methods were used to study the effect of MK-801 and Concentric microdialysis probes (5 mm tip length) ketamine on ACh levels in PC/RSC of freely-moving were constructed according to methods described by ␣ 31 rats, and to test the ability of the 2-adrenergic , Robinson and Whishaw. On the eve of the dialysis

Figure 1 Circuitry proposed to mediate NRHypo-induced neurotoxicity and excessive ACh release. To explain NRHypo- induced excessive ACh release, we propose that Glu, acting through NMDA receptors on GABAergic and noradrenergic neurons, maintains tonic inhibitory control over a cholinergic that projects to and innervates certain PC/RSC neurons. Systemic administration of an NMDA (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 non- NMDA 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 = acetyl- = ␣ = ␣ = = ; NE ; 2 2 subtype of ; 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. Role of ACh, GABA and NE in NRHypo neurotoxicity SH Kim et al 346

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 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 CMA/110 liquid switch (CMA, Stockholm, Sweden) through the guide cannula, and the animal placed in was used to allow the instantaneous change form the a round plastic bowl (BAS Bee Keeper System, West artificial CSF to the -containing artificial CSF sol- Lafayette, IN, USA). The probe extended 5 mm beyond ution without risking the introduction of air into the the guide cannula thus allowing an extensive amount microdialysis probe. Samples were stored at −80°C of PC/RSC to be sampled. After insertion, the probe prior to analysis by high pressure liquid chromato- was perfused continually at a flow rate of 0.5 ␮l min−1 graphy (HPLC). Animals were killed immediately fol- with artificial cerebral spinal fluid (CSF, 145 mM NaCl, lowing the microdialysis experiments, perfused with

2.7 mM KCl, 1.2 mM CaCl2, 1.0 mM MgCl2 and 2.0 mM 4% paraformaldehyde, and their sectioned and Na2HPO4) containing 100 nM (an acetyl- examined histologically to verify microdialysis probe inhibitor) at pH 7.4. On the day of the placement. Only animals with correctly implanted experiment, the probe was perfused at a rate of 2.0 ␮l probes were included in the data analysis. min−1 for 2 h prior to sample collection. Three samples (40 ␮l) were collected every 20 min to assess basal con- ACh analysis centrations of ACh. Then samples were collected at 20- Extracellular concentrations of ACh were quantified in min intervals following drug administration. For those dialysates using HPLC coupled with electrochemical studies in which the drug (MK-801 or clonidine) was detection. A 10-␮l sample of the perfusate was injected perfused into the PC/RSC over a period of time, a directly into an ESA integrated HPLC system, equipped Role of ACh, GABA and NE in NRHypo neurotoxicity SH Kim et al 347 with a CMA/200 refrigerated microsampler (CMA, Stockholm, Sweden), an ESA 5200A Coulochem elec- trochemical 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 −1 min 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 peroxide by a post-col- umn solid phase reactor (ACH-SPR 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 out- put. 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 absol- ute level by the calculated basal output and multiply- ing the result by 100 to obtain a percentage. Percent- ages for each time point were averaged over all the animals in the given experimental condition. Differ- ences between each experimental condition and con- trol were evaluated by two way ANOVAs seeking a sig- nificant interaction between treatment condition and Figure 3 Effect of NMDA antagonists on ACh output in time. Significant ANOVAs were subsequently evalu- PC/RSC. Dialysate samples were obtained over 20-min per- ated by further F-tests to determine the specific time iods. Mean ACh output was calculated as described in the points that accounted for the significant interaction. Methods section and expressed as a percentage of basal out- put (mean ± SEM). Error bars are not visible at some time points due to their small magnitude. Systemic injection of − − Results 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 - To test the hypothesis that NRHypo induces release of line (MK-801: F[11,153] = 3.58, P Ͻ 0.0005; ketamine: ACh in the PC/RSC, MK-801 was administered subcut- F[11,93] = 7.40, P Ͻ 0.0001). The significant treatment con- aneously (sc) at a dose (0.5 mg kg−1) that reliably causes dition by time interaction for both MK-801 and ketamine was neurotoxic changes in PC/RSC neurons in 100% of the explained by significant differences in ACh output at all time Ͻ animals,1 and ACh was measured in the PC/RSC points after the injection (P 0.05 for all points). Application ␮ dialysate at 20-min intervals for 1 h prior to and 3 h of MK-801 (20 M) into the PC/RSC by perfusion in the dialy- after MK-801 treatment. The ACh concentration rose sis probe (MK-801 perfusion) did not result in any change in ACh output (treatment × time interaction: F[11,132] = 1.36; P abruptly (Figure 3) to a level approximately three times Ͼ 0.1), indicating that the site of action of NMDA antagonists higher than baseline by 20 min after MK-801 adminis- with respect to controlling ACh release is outside of the tration, then continued to rise gradually to a peak value PC/RSC. Arrow indicates the time of sc injection of test agent. at 3 h (the last measurement) that was 400% of base- In the experiment where MK-801 was perfused via the dialy- line. Similar effects on ACh release (Figure 3) were sis probe into the PC/RSC, the solid bar indicates the period seen with ketamine (50 mg kg−1 sc) confirming that during which MK-801 was present in the perfusate. antagonism of NMDA receptors (ie NRHypo) is the likely mechanism leading to the increase in ACh out- put. Saline injections alone did not lead to an increase is not likely due to a local action of MK-801 in the in ACh output above basal levels (Figure 3). PC/RSC. ␣ To determine whether the ACh releasing action of To test the hypothesis that 2-adrenergic agonists MK-801 can be attributed to an action of MK-801 in block the PC/RSC neurotoxic activity of NRHypo by the PC/RSC, MK-801 (20 ␮M) was locally infused into interfering with NRHypo-induced ACh release, we ␣ the PC/RSC via the dialysis probe. Direct introduction administered the 2-, clonidine of MK-801 into PC/RSC resulted in no significant (0.05 mg kg−1 sc), either by itself or in combination change in ACh levels in PC/RSC at any time over the with MK-801 and found that by itself clonidine does 3-h period of infusion (Figure 3), which signifies that not influence PC/RSC ACh levels, but in combination the increased release stimulated by systemic MK-801 with MK-801 it substantially suppresses the MK-801 Role of ACh, GABA and NE in NRHypo neurotoxicity SH Kim et al 348

Figure 4 Effect of clonidine on NRHypo-induced ACh release in PC/RSC. Dialysate samples were obtained over 20- Figure 5 Effect of GABAergic agonists on NRHypo-induced min periods. Mean ACh output was calculated as described ACh release in PC/RSC. Dialysate samples were obtained over in the Methods section and expressed as a percentage of basal 20-min periods. Mean ACh output was calculated as ± output (mean SEM). Error bars are not visible at some time described in the Methods section and expressed as a percent- points due to their small magnitude. The systemic injection age of basal output (mean ± SEM). Error bars are not visible −1 of clonidine (0.05 mg kg sc) when given with systemic MK- at some time points due to their small magnitude. The injec- −1 801 (0.5 mg kg sc) resulted in significant decrease in ACh tion of pentobarbital (25 mg kg−1 sc) when given with MK-801 output in the PC/RSC as compared to that seen with MK-801 (0.5 mg kg−1 sc) resulted in a significant decrease in ACh out- × = Ͻ alone (treatment time interaction: F[14,234] 2.51, P put in the PC/RSC (treatment × time interaction: F[14,180] = 0.005). The significant treatment condition by time interac- 7.31, P Ͻ 0.0001). The significant treatment condition by time tion was explained by significant differences in ACh output interaction was explained by significant differences (P Ͻ Ͻ at all time points after the injection of MK-801 (P 0.05). 0.005) in ACh output at all time points after 140 min (ie in ␮ The application of clonidine (100 M) into the PC/RSC by samples collected between 141 [21 min post pentobarbital perfusion in the dialysis probe without exposing the animals injection] and 240 min). Diazepam (6 mg kg−1 sc) had a similar to MK-801, resulted in no significant change in ACh output effect on MK-801-induced ACh release (treatment × time × as compared to that seen with saline injections (treatment interaction: F[14,255] = 4.82, P Ͻ 0.0001). The significant = Ͼ time interaction: F[11,87] 0.56, P 0.1; saline data not treatment condition by time interaction was explained by sig- graphed for presentation clarity). Application of clonidine nificant differences (P Ͻ 0.05) in ACh output at all time points ␮ (100 M) into the PC/RSC by perfusion in the dialysis probe beginning 21 min after the injection of diazepam (ie time −1 in animals that received MK-801 systemically (0.5 mg kg sc) points 160–240). Filled arrow indicates the time of sc injec- did not result in any significant change in ACh output tion of MK-801. Open arrow indicates when pentobarbital or × = Ͼ (treatment time interaction: F[12,196] 0.98, P 0.1) as diazepam was injected. compared to MK-801 alone. Filled arrow indicates the time of sc injection of MK-801. Open arrow indicates when clonid- ine was injected in those animals that received MK-801. In the PC/RSC, MK-801 was administered systemically the experiment where clonidine was perfused via the dialysis and clonidine (100 ␮M) was administered directly into probe into the PC/RSC the solid bar indicates the period dur- the PC/RSC via the dialysis probe. Infusion of cloni- ing which clonidine was present in the perfusate. dine 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 effect, limiting it to 200% of basal output instead of not in the PC/RSC. 400% (Figure 4). To determine whether clonidine- To test the hypothesis that GABAergic inhibition induced suppression of the ACh releasing action of plays a major role in the mechanism by which NRHypo MK-801 can be attributed to an action of clonidine in induces excessive release of ACh in the PC/RSC, pento- Role of ACh, GABA and NE in NRHypo neurotoxicity SH Kim et al 349 barbital (25 mg kg−1 sc) was administered to rats that which contributes to injury of the PC/RSC neuron. −1 had received MK-801 (0.5 mg kg sc) 2 h previously. Pentobarbital acts directly at the GABAA receptor to Pentobarbital caused a precipitous fall in the PC/RSC restore inhibitory control over the concentration of ACh from 350% of basal output to a and returns the ACh releasing activity to normal. This level 50% of basal output (Figure 5). To further evalu- provides a logical explanation for the finding that ate the role of GABAA receptors we administered pentobarbital, if applied in a timely manner, prevents diazepam systemically (6 mg kg−1 sc) to rats treated 2 h PC/RSC neuronal injury.10,32 Further study will be previously with MK-801 and found it reduced the elev- needed to determine if this simple mechanistic expla- ation to 150% of basal output (Figure 5). These results nation is correct or whether another, more compli- are consistent with our hypothesis that loss of GABA- cated, mechanism is involved. ergic inhibition plays a major role in NRHypo-induced Diazepam at a dose of 6 mg kg−1 sc decreased the

ACh release, and that restoring GABAA receptor magnitude of the MK-801-induced ACh release but, activity by administration of pentobarbital or diazepam unlike pentobarbital, diazepam did not totally abolish reinstated inhibitory control over ACh release, thereby the ACh releasing action or decrease ACh levels to returning ACh to normal or near normal levels. below baseline. This observation is consistent with the earlier finding10 that diazepam in the same dose range provided substantial but not complete protection Discussion against the PC/RSC neurotoxic action of MK-801. We 10 Based on the initial finding that GABAA agonists and have proposed that the greater efficacy of pentobarbital muscarinic cholinergic antagonists protect against compared to diazepam may be due to a difference in NRHypo-induced neurodegeneration, it was proposed the mechanism by which these agents interact at 10 that NMDA receptor blockade abolishes GABAergic GABAA receptors. ␣ inhibitory control over ACh release, thereby causing To explain the finding that 2-adrenergic agonists excessive release of ACh at muscarinic receptors on block NRHypo-induced neurotoxicity we have pro- PC/RSC neurons, as a proximal mechanism contribu- posed that they do so by counteracting the ACh-releas- ␣ ting to the injury of these neurons. The more recent ing action of this state, and that the 2-adrenergic observation29 that direct injection of the muscarinic receptor through which norepinephrine (NE) normally antagonist, scopolamine, into PC/RSC protects PC/RSC regulates ACh release in this circuit may either be on neurons against the neurotoxic action of systemically the dendrosomal surface of the cholinergic neuron or administered MK-801, further corroborates that hyper- on its terminal in the PC/RSC region. The present ␣ activation of muscarinic receptors in the PC/RSC finding that the 2-adrenergic agonist, clonidine, when region is a critical component of the neurotoxic mech- administered systemically, strongly suppresses the anism. ACh releasing action of MK-801, confirms that NE does Our present finding that systemic application of normally regulate ACh release in this circuit, that NMDA antagonists induces a sustained increased NMDA receptor blockade abolishes this regulatory release of ACh in the PC/RSC region confirms that the mechanism and that clonidine restores it. This finding basis for the hyperactivation of PC/RSC muscarinic is consistent with the proposed circuit diagram (Figure receptors is that these receptors are being persistently 1) depicting the NE neuron functioning as an inhibitory flooded with excessive ACh. The fact that the excessive neuron that is driven by Glu to inhibit the release of release was sustained and showed no signs of abating ACh from a basal forebrain cholinergic neuron. Failure during the 3-h sampling period is consistent with the of clonidine to completely suppress the ACh releasing assumptions of our circuit diagram (Figure 1) that ACh action of MK-801 is consistent with the fact that the release in this network is normally held under constant dose used is the ED50 dose for protecting against MK- (tonic) restraint and that blockade of NMDA receptors 801 neurotoxicity, ie, it is a dose that suppresses the removes this tonic restraining mechanism, thereby neurotoxic reaction by 50%.27 allowing a pattern of sustained ACh release that would The present experiments were not designed to deter- never be encountered under normal circumstances. mine definitively where the NMDA receptor-bearing The observation that pentobarbital effectively arrests GABAergic neuron or the cholinergic neuron that it the excessive ACh release and returns ACh concen- inhibits are located. However, the finding that systemic trations in PC/RSC to normal or subnormal levels, is administration of MK-801 triggered ACh release consistent with the interpretation10,18,21 that GABA- whereas local PC/RSC application of MK-801 via the ergic inhibitory neurons and GABAA receptors are dialysis probe did not, signifies that the NMDA recep- critically involved, and that Glu, acting at a NMDA tor-bearing GABAergic neuron with which MK-801 receptor on the GABAergic neuron drives the tonic interacts to trigger ACh release, is not located in the inhibitory mechanism by which release of ACh in the PC/RSC region. In addition, we found that systemic PC/RSC is controlled. According to this interpretation administration of clonidine suppressed the ACh releas- MK-801 blockade of the NMDA receptor prevents Glu ing action of MK-801 but local application of clonidine ␣ from driving the GABAergic neurons and this inacti- into PC/RSC did not. This signifies that the 2-adre- vates the GABAA receptor on the cholinergic neuron nergic receptor with which clonidine interacts is not and releases the cholinergic neuron from inhibition, in the PC/RSC region, ie it is not on a cholinergic axon thereby causing excessive release of ACh in PC/RSC terminal in the PC/RSC region nor on the dendrosomal Role of ACh, GABA and NE in NRHypo neurotoxicity SH Kim et al 350 surface of an intrinsic cholinergic neuron in the able data suggest an interesting and readily testable PC/RSC region. Cell bodies in the diagonal band region hypothesis—that it may be a general principle that of the basal forebrain provide the main extrinsic source release of ACh from axon terminals of basal forebrain of cholinergic fibers to the PC/RSC.33–35 Thus the diag- cholinergic neurons which project to various portions onal band region is the most likely location of the chol- of the , and other limbic inergic neurons that contribute to the PC/RSC neuro- brain regions, may be regulated by Glu acting via ␣ toxic action triggered by systemic MK-801, and the 2- NMDA receptor-bearing GABAergic neurons which are adrenergic receptor probably is located on the dendro- co-localized with the cholinergic neurons in the basal somal surface of this neuron. Confirming this con- forebrain. clusion we have found that the direct injection of clon- While the present findings pertain specifically to the idine into the diagonal band attenuates MK-801- regulation of ACh release in a single brain region, the induced neurotoxicity in the PC/RSC while direct PC/RSC, the NRHypo mechanism has the potential to injections into the PC/RSC itself or the produce widespread corticolimbic neurodegeneration are ineffective.36 Since it is known that there is a rela- which follows a distribution pattern resembling the tively dense population of GABAergic pattern of cholinergic innervation of cerebrocortical that are colocalized with and have the same irregular and related limbic brain regions. Since excessive distribution as the magnocellular basal forebrain chol- release of ACh could be an important factor in inergic neurons,37,38 it is likely that the NMDA recep- determining whether a specific region undergoes tor-bearing GABAergic neuron, that regulates the NRHypo-induced neurodegeneration, it will be release of ACh in the PC/RSC, is an coloca- important in the future to study whether the effects of lized with the cholinergic neuron that it regulates in NMDA antagonists on ACh release in a given brain the diagonal band region of the basal forebrain. region are correlated with the region’s suceptibility to Although other more complicated interpretations NRHypo-induced neurodegeneration. could be conceived, the interpretation we have In addition to excessive release of ACh we have pro- adopted (depicted schematically in Figure 1) is the posed that the NRHypo state also causes the excessive most parsimonious one that fits all available data. release of Glu.21 On-going work indicates that it is the Previously we have proposed, and here we present excessive stimulation of both muscarinic cholinergic microdialysis data consistent with a mechanism by and non-NMDA glutamatergic receptors on the vulner- which Glu, acting at NMDA receptors on GABAergic able pyramidal neuron which leads to the observed neurons in the basal forebrain, regulates release of ACh through a more complex circuitry than in the posterior cingulate and retrosplenial regions of we have depicted in Figure 1.29,36 Preliminary results the cerebral cortex. Several years ago, Giovaninni et with microdialysis indicate that in addition to the al39 described a similar mechanism by which the abnormal release of ACh, NMDA antagonists also release of ACh in the hippocampus is regulated. These induce abnormal release of excitatory amino acids in authors demonstrated that when an NMDA antagonist PC/RSC46 as well as prefrontal cortex.26,47 In view of is injected directly into the septal region where the the important roles that both the cholinergic and Glu cholinergic neurons that project to the hippocampus transmitter systems have in , cognition and are located, it triggers a striking release of ACh in the related mental functions, it is important from both a hippocampus. If they coinjected into the septal region health and disease perspective to develop a better

both an NMDA antagonist and the GABAA agonist, understanding of normal mechanisms by which the , the ACh releasing action of the NMDA Glu system regulates activity in the ACh system, and antagonist was prevented. Their interpretation was as abnormal mechanisms by which the two systems can follows: Glu, acting at NMDA receptors on GABAergic act in concert to produce deranged thinking (if mildly neurons that upon cholinergic neurons in the abnormal) or widespread excitotoxic neurodegener- medial septum, regulates the release of ACh in a dis- ation (if severely abnormal). tant brain region, the hippocampus. Our findings and To fully appreciate how blockade of NMDA recep- interpretation are identical except that in the system tors can trigger a neurodegenerative syndrome, it is we are studying the cholinergic neurons, which are necessary to begin thinking of Glu in a new light, as regulated by NMDA receptor-bearing GABAergic neu- an agent that performs major inhibitory functions. By rons, are located in the diagonal band region (slightly tonically activating NMDA receptors on GABAergic subjacent and caudal to the medial septum) and the neurons (Figure 1), Glu regulates inhibitory tone and distant brain region where release of ACh is being ordinarily protects neurons against the brain’s own monitored is the PC/RSC region of the cerebral cortex. self-destructive potential; removing this inhibitory There are no prior studies directly focusing on mech- mechanism from certain networks unleashes excito- anisms regulating release of ACh in the PC/RSC, but toxic forces that wreak self destruction within the net- there is evidence that intraperitoneal administration of work. The excitotoxic forces are complex and include MK-801 (non-competitive NMDA antagonist), intracer- glutamatergic, cholinergic and other less well defined ebroventricular administration of CPP (competitive components, but the key to understanding this neuro- NMDA antagonist) or inhalation of toxic process is to recognize that Glu is not only an (laughing , another NMDA antagonist40,41) evokes excitotoxic contributor to the pathological outcome, it release of ACh in other cortical areas.42–45 Thus, avail- is the driver of the inhibitory mechanism that normally Role of ACh, GABA and NE in NRHypo neurotoxicity SH Kim et al 351 holds the excitotoxic forces in check. Inhibition failure Alzheimer’s disease. In: Giacobini E, Becker R (eds). Alzheimer Dis- (failure of Glu to maintain inhibition) is the basic prin- ease: Therapeutic Strategies. Birkhauser: Boston, 1994, pp 293– 298. ciple underlying NRHypo neurodegeneration. We have 18 Farber NB, Newcomer JW, Olney JW. The glutamate synapse in postulated that this inhibition failure mechanism may neuropsychiatric disorders: focus on schizophrenia and Alzhei- play a central role in two major neuropsychiatric dis- mer’s disease. In: Ottersen OP, Langmoen I, Gjerstad L (eds). The orders, namely schizophrenia and Alzheimer’s disease. Glutamate Synapse as a Therapeutical Target: Molecular Organiza- While it is beyond the scope of the present writing to tion and Pathology of the Glutamate Synapse. Elsevier: Amster- dam, 1988, pp 421–437. discuss in detail the putative role of NRHypo in either 19 Javitt DC, Zukin SR. Recent advances in the phencyclidine model of these diseases, each of these topics has been of schizophrenia. Am J Psychiatry 1991; 148: 1301–1308. reviewed elsewhere.11,17,18,21,25,48,49 20 Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD et al. 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