D3 Antagonism Prevents NMDA Antagonist Neurotoxicity

Acute D2/D3 Dopaminergic Agonism but Chronic D2/D3 Antagonism Prevents NMDA Antagonist Neurotoxicity Nuri B. Farber, Brian Nemmers, and Kevin K. Noguchi Background: Antagonists of the N-methyl-D-aspartate (NMDA) glutamate receptor, most likely by producing disinhibtion in complex circuits, acutely produce psychosis and cognitive disturbances in humans, and neurotoxicity in rodents. Studies examining NMDA Receptor Hypofunction (NRHypo) neurotoxicity in animals, therefore, may provide insights into the pathophysiology of psychotic disorders. Dopaminergic D2 and/or D3 agents can modify psychosis over days to weeks, suggesting involvement of these transmitter system(s). Methods: We studied the ability of D2 /D3 agonists and antagonists to modify NRHypo neurotoxicity both after a one-time acute exposure and after chronic daily exposure. Results: Here we report that D2 /D3 dopamine agonists, probably via D3 receptors, prevent NRHypo neurotoxicity when given acutely. The protective effect with D2 /D3 agonists is not seen after chronic daily dosing. In contrast, the antipsychotic haloperidol does not affect NRHypo neurotoxicity when given acutely at D2 /D3 doses. However, after chronic daily dosing of 1, 3, or 5 weeks, haloperidol does prevent NRHypo neurotoxicity with longer durations producing greater protection. Conclusions: Understanding the changes that occur in the NRHypo circuit after chronic exposure to dopaminergic agents could provide important clues into the pathophysiology of psychotic disorders.

Key Words: Antipsychotics, D2/D3 dopaminergic agents, disinhibition, psychosis, neurotoxicity, NMDA antagonists

A

ntagonists of the N-methyl-D-aspartate (NMDA) glutamate receptor acutely produce a psychotic state that is very similar to that seen in patients with idiopathologic psychotic disorders like schizophrenia (Domino and Luby 1981; Javitt and Zukin 1991), indicating the possibility that NMDA receptor hypofunction (NRHypo) might underlie the cognitive and behavioral symptoms which are seen in schizophrenia and related disorders (Javitt and Zukin 1991; Olney and Farber 1995). Further support that NRHypo is involved in schizophrenia comes from reports tying several NRHypo-producing genetic mutations to the disorder (Cloninger 2002). In rodents, NMDA antagonists produce a hypermetabolic state (Kurumaji and McCulloch 1989; Meibach et al 1979; Nelsom et al 1980) that appears to be the result of inadequate GABAergic, serotonergic, and noradrenergic inhibition of specific excitatory transmitter pathways in certain complex multisynaptic circuits (Farber 2003). The apparent loss of inhibition results in the excessive release of acetylcholine (Giovannini et al 1994; Kim et al 1999) and glutamate (Moghaddam et al 1997) in multiple corticolimbic brain regions. When the simultaneous excessive stimulation of the associated postsynaptic receptors is of a significant enough degree and duration neurotoxicity ensues (Farber et al 2003; Farber, Jiang et al 2002; Farber, Kim et al 2002; Olney et al 1989). If moderate doses of NMDA antagonists are used, the neurotoxicity is reversible and restricted mainly to the restrosplenial cortex (RSC)(Fix et al 1993; Olney et al 1989). Higher doses result in irreversible neurotoxicity in the RSC and other brain regions, suggesting that the disinhibition syndrome is likely active in multiple corticolimbic brain regions (Allen and From the Department of Psychiatry, Washington University, St. Louis, Missouri. Address reprint requests to Nuri B. Farber, M.D., Department of Psychiatry, Washington University, Campus Box 8134, 660 S. Euclid Ave., St. Louis, MO, USA 63110-1093; E-mail: [email protected]. Received August 23, 2005; revised January 3, 2006; accepted February 8, 2006.

0006-3223/06/$32.00 doi:10.1016/j.biopsych.2006.02.019

Iversen 1990; Corso et al 1997; Ellison 1994; Ellison and Switzer 1993; Fix et al 1993; Horvath et al 1997; Wozniak et al 1998). The NRHypo state also produces an activation of several immediate early genes and their products (c-fos, c-Jun, Jun-B, NGFI-A [a.k.a. zif268, krox-24], NGFI-B, NGFI-C and Nurr1) (Dragunow and Faull 1990; Gao et al 1998; Gass et al 1993; Hughes et al 1993; Nakao et al 1996; Nakki et al 1996) and the expression of heat shock protein and BDNF (Castren et al 1993; Hughes et al 1993; Olney et al 1991; Sharp et al 1991). Several of these changes have been proposed to be involved in psychosis. Several agents that reverse or ameliorate the NRHypo disinhibited state and its associated effects in rodents also reverse the drug-induced psychotic state in humans (Farber 2003). These and other findings suggest that the same NRHypo disinhibited state that underlies the observed effects in rodents also produces the cognitive and behavioral symptoms in humans (Olney and Farber 1995). The specific consequences observed depends on the severity of the NRHypo disinhibition state (Farber 2003). Mild disinhibition produces only cognitive and behavioral symptoms. Moderate disinhibition produces reversible neurotoxicity and changes in several immediate early genes along with their associated proteins. Only with severe disinhibition is irreversible degeneration observed. Given the proposal that a similar mechanism underlies these different effects, results from studying the mechanism underlying the reversible neurotoxicity produced by moderate degrees of NRHypo may provide insights into psychosis. In studying the ability of serotonergic agents to prevent NRHypo neurotoxicity (Farber et al 1998) we had noted that S-lisuride tended to be more potent at preventing the neurotoxicity than one would have predicted based on its serotonergic activity alone. Because S-lisuride is also a D2/D3 agonist we were interested in whether agonist activity at D2/D3 receptors would be protective. One goal of this study was to determine whether selective D2/D3 agonists could prevent NRHypo neurotoxicity. The role of dopamine and the D2 family of receptors in the production and treatment of psychosis has been long established. While the D2 receptor is commonly accepted as the likely dopamine receptor involved in treating psychosis, the closely related D3 receptor can still not be conclusively ruled out as being involved (Levant 1997; Richtand et al 2001; Strange 2001). BIOL PSYCHIATRY 2006;60:630 – 638 © 2006 Society of Biological Psychiatry

N.B. Farber et al Antipsychotics probably treat psychosis by blocking D2/D3 receptors (Seeman et al 1976; Strange 2001). This antipsychotic effect is not an immediate one (i.e., within minutes of drug binding to receptors). While some antipsychotic effect can be seen early on (Agid et al 2003; Kapur et al 2005; Leucht et al 2005), slow improvement is usually observed over several weeks of drug administration (Kane et al 1994; Leucht et al 2005; Rabinowitz et al 2001). Similarly, the psychosis seen with D2/D3 agonists is not usually an immediate effect (as is the case with NMDA antagonists) but rather emerges over time and in such a manner that the individual is supersensitive to subsequent agonist exposure (Bell 1973; Brady et al 1991; Ellinwood et al 1973; Richtand et al 2001; Satel et al 1991; Sato et al 1983; Sax and Strakowski 2001). Given the apparent importance of duration of treatment, a second aim of this study was to determine whether chronic administration of dopaminergic agents would alter the ability of NMDA antagonists to produce NRHypo neurotoxicity.

Methods and Materials Animals and Housing Adult female Sprague Dawley rats (Harlan, Indianapolis, Indiana) are more sensitive to the neurotoxic effects of NMDA antagonists (Olney et al 1989) probably due to differences in hepatic metabolism. We, therefore, restricted our study to this gender. All animals were housed in a temperature- and humiditycontrolled animal facility and were maintained on a 12/12-hour light/dark cycle with free access to food and water. Experimental procedures were approved by Washington University’s IACUC. Drug Administration and Experimental Groups Acute Studies. Three separate acute studies were conducted. In the first, adult female Sprague Dawley rats were injected intraperitoneally (ip) with a D2/D3 dopaminergic agonist, followed 15 min later by an injection of the competitive NMDA antagonist, CGS-19755, (20 mg/kg) into the tail vein. Control animals (n ⫽ 80) received an injection (ip) of either dimethyl sulfoxide (DMSO) or water (pH 7.4). For each test agent, at least 47 rats were used and at least 4 doses tested. The animals were sacrificed 4 hours after exposure to the NMDA antagonist drug. Tissue was processed and analyzed for the severity of neurotoxicity in the retrosplenial cortex as described previously (Farber, Jiang et al 2002). Percent reduction in neurotoxicity was calculated by dividing the number of vacuolated neurons in a given experimental animal by the mean number in the CGS-19755-treated controls. The result was subtracted from one and multiplied by 100. Regression analysis was conducted with the four-parameter logistic equation (Hill equation) model of Prism (Graphpad Software Inc., San Diego, CA) in order to mathematically estimate an ED50 (dose of a given compound that reduced the mean number of vacuolated neurons to 50% of the control mean for that agent). To explore whether the D2 or D3 subtype of dopamine receptor was mediating the observed neuroprotective effect, we compared the order of potencies of the test agents for blocking NMDA antagonist neurotoxicity (i.e., ED50 values converted to ␮moles/kg) with their in vitro EC50 functional potencies at these two receptor subtypes (Sautel et al 1995). To judge statistical significance of these comparisons, we used the conservative Spearman correlation coefficients test, which does not make assumptions about the underlying data. In a second set of experiments designed to further clarify the receptor mediating the action of the neuroprotective drugs, adult female rats (n ⫽ 70) received CGS-19755 (20 mg/kg, iv) plus the

BIOL PSYCHIATRY 2006;60:630 – 638 631 D2/D3 agonist quinpirole (ip) at one of several doses (10, 1, 0.1, .01, .003, 0 mg/kg) and a fixed dose of the D2/D3 antagonist U99194A (20 mg/kg ip). While quinpirole has only a 3-fold selectivity for the D3 compared to the D2 receptor (Sautel et al 1995), U99194A has a 10 –20 fold selectivity for the D3 receptor (Audinot et al 1998; Waters et al 1993). In rats, 20 mg/kg of U99194A would be expected to strongly antagonize the D3 receptor but have minimal effects on the D2 receptor (Audinot et al 1998). A minimum of 5 animals was used for each dose condition. Percent protection was determined as described in the first study. Results were analyzed by a 2-way ANOVA with dose of quinpirole and treatment condition (quinpirole vs. quinpirole⫹U99194A) being independent factors and severity of injury being the dependent measure. The third study consisted of two separate experiments in which the ability of the D2/D3 agonist, N,N-propylnorapomorphine (NPA), to prevent CGS-19755 neurotoxicity was compared to NPA’s ability to prevent neurotoxicity produced by the noncompetitive NMDA antagonist, MK-801. In the first experiment, the protective effect of varying doses of NPA (10, 1, 0.3, 0.1, 0.01, 0.001, and 0 mg/kg; n ⱖ 6 for each dose) was studied for their protective ability against fixed doses of CGS-19755 (20 mg/kg, iv) or MK-801 (0.5 mg/kg, sc). In the second experiment, a fixed dose of NPA (0.1 mg/kg, ip) was studied for its protective ability against varying doses of MK-801 (0.5, 0.4, 0.3, 0.25, 0.22, 0.2, and 0.15 mg/kg) or CGS-19755 (100, 50, 25, 20, 18, 15, 12.5, 10, and 3 mg/kg). A minimum of 6 animals was used at each dose. Severity of injury was measured as described elsewhere (Farber, Jiang et al 2002). Chronic Studies. Two separate studies employed chronic dosing of a dopaminergic agonist or antagonist. The first utilized bromocriptine (10 mg/kg each day) for 0 (i.e. one acute injection) or 1 week and the second utilized, haloperidol (1 mg/kg each day) for 0 (i.e. one acute injection), 1, 3, or 5 weeks. For each study, adult female Sprague Dawley rats (n ⱖ 12 for each group) were injected ip once daily with a test agent or vehicle control. On the last day, 15 min after the injection of the test agent, MK-801 was injected and the animal sacrificed 4 hours later. Severity of injury was measured as before. ANOVA models were used to determine whether chronic exposure to a specific agent could alter sensitivity to NRHypo neurotoxicity. For the bromocriptine study, a 1-way ANOVA model with treatment condition (i.e., control, acute bromocriptine, chronic bromocriptine) as the independent factor and number of injured neurons as the dependent factor was used. For the chronic haloperidol study a 2-way ANOVA with duration of treatment (e.g., 4 hrs, 1, 3, and 5 weeks) and agent (DMSO or haloperidol) as the betweensubjects independent factors was employed. Number of injured neurons was the dependent measure. Significant effects were further analyzed with planned post-hoc comparisons (Fisher’s PLSD). An alpha level of 0.05 was used to judge significance. Pharmacological Agents NPA (R(⫺)-10, 11-dihydroxy-n, n-propylnorapomorphine hydrochloride), quinelorane (quinelorane dihydrochloride), talipexole (B-HT 920 dihydrochloride), U99194A (U99194A maleate), and MK-801 ((⫹)-MK-801 hydrogen maleate) were purchased from Sigma-Aldrich (St Louis, Missouri) and dissolved in distilled water and the pH adjusted to 7.4. Apomorphine (R-(⫺)-apomorphine hydrochloride hemihydrate), bromocriptine (bromocriptine methanesulfonate salt) and haloperidol were also purchased from Sigma-Aldrich but were dissolved in DMSO. Quinpirole (quinpirole hydrochloride) was purchased from Tocwww.sobp.org/journal

632 BIOL PSYCHIATRY 2006;60:630 – 638 ris-Cookson (Ellisville, MO) and dissolved in pH-adjusted water. 7-OH-DPAT (7-hydroxy-2-(di-n-propylamino)tetralin hydrobromide) was purchased from Research Biochemicals International (Natick, Masachusetts) and dissolved in DMSO. CGS-19755 was a kind gift from Novartis and was dissolved in pH-adjusted water.

Results D2/D3 Agonists Prevent NRHypo Neurotoxicity when Given Acutely To initially probe the D2/D3 receptor, bromocriptine, a partial D2/D3 agonist, was studied. It dose dependently prevented the neurotoxic action of CGS-19755 (20 mg/kg, iv) with an ED50 of 0.52 mg/kg (Figs. 1A–E; Table 1). Consistent with bromocriptine

N.B. Farber et al being a partial agonist, the amount of protection offered reached a plateau of ⬇60%. NPA, a highly potent and selective full D2/D3 agonist, could also prevent NRHypo neurotoxicity in a dose dependent fashion. NPA was substantially more potent than bromocriptine, having an ED50 of 0.020 mg/kg and full protection was obtained at higher doses (Fig. 1F; Table 1). Similar dose-dependent protection was provided by several other D2/D3 agonists (Table 1). In order to explore whether the D2 or D3 receptor was involved in the observed protection, the potency of D2/D3 agonists at preventing NRHypo neurotoxicity was correlated with their reported in vitro functional activity at these two distinct receptors. Ability to protect against NRHypo neurotoxicity was correlated with D3 activity (p ⬍ 0.01) but not D2 activity

Figure 1. Bromocriptine and n,n-propylnorapomorphine (NPA) protect against NMDA receptor hypofunction (NRHypo) neurotoxicity. (A) Tracing of the dorsal aspect of a coronal section at approximately -5.6 mm from Bregma (Paxinos and Watson 1998), where the severity of NRHypo neurotoxicity is routinely measured. Gray shaded area indicates the approximate location where damage occurs in layer IV–V pyramidal neurons in the granular subdivision of the retrosplenial cortex (RSC). (B) High powered photomicrograph of normal layer IV–V pyramidal neurons taken from a female rat treated 4 hours earlier with saline. (C) High powered photomicrograph of layer IV–V pyramidal neurons taken from a female rat treated 4 hours earlier with CGS-19755 (20 mg/kg, iv). Most neurons are injured (white arrows). They are swollen and have conspicuous vacuoles in the cytoplasm. Occasional normal appearing neurons can be detected (white arrowheads). (D) High powered photomicrograph of layer IV–V pyramidal neurons taken from a female rat treated 4 hours earlier with CGS-19755 (20 mg/kg, iv) and bromocriptine (10 mg/kg, ip). While abnormal neurons (white arrows) are still present, there are less of them. Healthy neurons (white arrowheads) are more numerous. (E–F) Dose response curves demonstrating the protection provided by bromocriptine and N,N-propylnorapomorphine (NPA) against the neurotoxic effects of CGS-19755 in the RSC. The protective ability of the partial agonist, bromocriptine, appeared to peak at approximately 60%. In contrast the full agonist, NPA, was able to provide complete protection. Error bars indicate SEM.

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N.B. Farber et al Table 1. Potency of D2/D3 Agonists in Preventing CGS-19755 Neurotoxicity Agent 7-OH-DPAT Apomorphine Bromocriptine R(⫺)-NPA (⫺)-Quinelorane Quinpirole Talipexole

# Animals

ED25a

ED50a

ED75a

69 47 92 81 47 91 56

.0104 1.2902 .4835 .0072 .0009 .0214 .0319

.1013 6.0950 .5247 .0202 .0128 .1288 .0683

.9184 20.1081 b

.0610 .3464 .6953 .1463

a

In mg/kg. Bromocriptine did not produce ⬎60% protection. Seven D2/D3 agonists were tested for their ability to prevent the neurotoxic effect of CGS-19755 (20 mg/kg, ip). At least 4 doses were tested for each agent. Regression analysis was conducted with the 4-parameter logistic equation model of Prism. ED25, ED50 and ED75 (doses needed to reduce the amount of damage seen in CGS-19755 control animals by 25, 50, and 75% respectively) were calculated from the regression curve. b

(p ⬎ 0.4; Fig. 2). In order to further confirm that dopaminergic agonism was the likely mechanism of action, a fixed dose (20 mg/kg, ip) of U99194A, a 10 –20 fold selective antagonist for the D3 receptor, was administered together with various doses of

Figure 3. Inhibition of quinpirole’s protection against NMDA receptor hypofunction (NRHypo) neurotoxicity by U99194A. Dose response curves for the protection offered by quinpirole and by quinpirole⫹U99194A. Quinpirole, a D2/D3 agonist, prevents the neurotoxic effects of CGS-19755 (20 mg/kg, iv). U99194A (20 mg/kg, ip), a relatively selective D3 antagonist, significantly interferes with quinpirole’s protection (F[1,59] ⫽ 10.0, p ⬍ 0.01). The shifting of the dose-response curve to the right suggests that the relationship is competitive in nature, confirming the involvement of D2/D3 receptors as the likely mediator of the dopaminergic agonists’ protective effect. Error bars indicate SEM.

quinpirole, a weak but somewhat (approximately 3-fold) selective D3 agonist, to CGS-19755 treated rats to see if U99194A would reverse quinpirole’s protection. Consistent with the hypothesis that D3 receptors mediated the observed protection, U99194A significantly (F[1,59] ⫽ 10.0, p ⬍ 0.01) shifted quinpirole’s protection curve to the right in a manner consistent with a competitive interaction (Fig. 3).

Figure 2. Comparison of dopaminergic agonists’ ED50 for preventing NMDA receptor hypofunction (NRHypo) neurotoxicity and their binding affinity for D2 and D3 receptors. Correlation Plot depicting the ED50 values of several D2/D3 agonists in preventing NRHypo neurotoxicity and the in vitro EC50 values of these same agents for the D2 receptor (A) or the D3 receptor (B). In vitro functional activities were obtained from the literature (Sautel et al 1995). There was a significant correlation with D3 activity (Spearman, p ⬍ 0.01) but not D2 activity. Linear regression line in (B) was calculated by Prism.

Potency in Preventing NRHypo Neurotoxicity Induced by MK-801 versus CGS-19755 NPA and bromocriptine also could reverse in a dose dependent fashion NRHypo neurotoxicity induced by the potent and selective non-competitive NMDA antagonist, MK-801, confirming the generalizability of the effect. However, the potency of these agents for reversing MK-801 neurotoxicity appeared to be substantially less than for reversing CGS-19755 neurotoxicity. For example, NPA’s ED50 for preventing CGS-19755 neurotoxicity was 0.020 mg/kg while its ED50 for preventing MK-801 neurotoxicity was 1.8 mg/kg—an almost 100-fold difference in potency. Such a difference could be due to the fact that a 0.5 mg/kg dose of MK-801 produced a greater amount of NMDA receptor blockade than a 20 mg/kg dose of CGS-19755. In order to determine whether the observed difference was due to dose non-equivalence, a fixed dose of NPA (100 ␮g/kg) was administered together with one of several doses of CGS-19755 or MK-801. The resultant curves were strikingly different (Fig. 4). The CGS-19755⫹NPA curve was shifted to the right of the CGS-19755 curve consistent with a competitive interaction. In contrast, the MK-801⫹NPA curve had a maximal effect that was approximately 75% of that seen with MK-801 alone, suggestive of a non-competitive interaction. Thus, the differential potency of NPA in preventing NRHypo neurotoxicity by CGS-19755 and MK-801 is not likely due to different degrees of NMDA receptor blockade but potentially to the different mechanisms via which these two antagonists block the NMDA receptor. www.sobp.org/journal

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N.B. Farber et al

Figure 4. Protective effect of n,n-propylnorapomorphine (NPA) against CGS-19755 and MK-801 neurotoxicity. Dose response curves of the neurotoxic effects of MK-801, MK-801⫹NPA, CGS-19755, and CGS-19755⫹NPA. NPA was administered at a dose of 100 ␮g/kg. MK-801 and CGS-19755 were administered over a range of doses and the severity of the neurotoxicity was judged as described in the methods section. When added to CGS-19755, NPA shifted the dose-response curve to the right in a manner consistent with a competitive interaction. In contrast, when added to MK-801, NPA lowered the maximal amount of damage seen, suggestive of a non-competitive interaction. Error bars indicate SEM.

Chronic Treatment with D2/D3 Agonists is Less Protective than Acute Treatment The second major aim of the study was to determine whether chronic activity at the D2/D3 receptor would have a different effect than that seen acutely. While a single dose (10 mg/kg) of bromocriptine provided significant protection acutely when given on day 1 (p ⬍ 0.05), after seven days of daily administration the same dose of bromocriptine on day eight appeared to worsen the neurotoxicity but this was non-significant (p ⫽ 0.09; Fig. 5) when MK-801 (0.2 mg/kg) was given on day eight. However, the amount of damage seen with MK-801 after eight days of bromocriptine exposure was significantly (p ⬍ 0.01)

Figure 5. Chronic versus acute effects of bromocriptine. When given with MK-801, a single injection of bromocriptine (10 mg/kg, ip) decreases the amount of damage produced by MK-801 (0.2 mg/kg, sc) by 45% (p ⬍ 0.05). In contrast, after bromocriptine is given daily for seven days at a dose of 10 mg/kg, ip, the same dose of bromocriptine on day 8 provides substantially less protection than it did acutely (p ⬍ 0.01). While there is a numerically greater amount of damage seen with the chronically administered bromocriptine (177 vs. 132 injured neurons), the difference is not significant (p ⫽ 0.09), indicating that chronic exposure to bromocriptine results in the inability of the agent to acutely protect against NMDA Receptor Hypofunction (NRHypo) neurotoxicity. Error bars indicate SEM.

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more than that seen with MK-801 after acute exposure to bromocriptine (Fig. 5). A similar effect (i.e., a decrease in the amount of protection) was also seen with chronic administration of NPA compared to acute administration (data not shown). Chronic but not Acute Treatment with Dopaminergic Doses of Haloperidol Protects Against NRHypo Neurotoxicity This loss of protection with chronic dosing of a D2/D3 agonist suggested the possibility that chronic administration of a D2/D3 antagonist, through some mechanism (e.g., receptor upregulation), might have the opposite effect and be protective. To test this possibility haloperidol, a D2/D3 antagonist, was given daily for 0 (i.e., one acute injection), 1, 3, or 5 weeks at a dopaminergic dose (1 mg/kg, ip), which does not have significant acute protective effects against NRHypo neurotoxicity (Farber et al 1993). Control groups received DMSO daily for the same amount of time. On the last day, 15 min after receiving DMSO or haloperidol, MK-801 (0.5 mg/kg) was given and the brain analyzed for NRHypo neurotoxicity. The amount of damage that MK-801 produced was highly dependent on the duration of haloperidol exposure (F [3,68] ⫽ 18.1, p ⬍ 0.0001, Fig. 6). While no protection was seen on the first day of haloperidol treatment (p ⫽ 0.4, compared to placebo), significant neuroprotection was seen at 1, 3, and 5 weeks of treatment (p ⬍ 0.0001 for all comparisons). In addition, the amount of protection increased with longer exposure until 3 weeks of treatment (0 vs. 1 week: p ⫽ 0.0005; 1 vs. 3 weeks: p ⫽ 0.02) with 1 week of exposure providing a 33% decrease in the amount of damage seen and 3 weeks providing a 45% decrease. However, 5 weeks of treatment did not provide any increase in protection over that seen at 3 weeks (p ⬎ 0.4).

Discussion Activity at 8 different receptors/sites—GABAA (Jevtovic-Todorovic et al 1997; Olney et al 1991), m3-muscarinic (Olney et al 1991), non-NMDA glutamatergic (Farber, Kim et al 2002), ␣2-

N.B. Farber et al

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Figure 6. Chronic versus acute effects of haloperidol. The ability of haloperidol or dimethyl sulfoxide (DMSO) to affect the ability of MK-801 to injure neurons depended on the duration of exposure and which agent was given (i.e., a significant duration x agent interaction; F[3,126] ⫽ 8.3, p ⬍ 0.0001). While DMSO’s effect on NMDA Receptor Hypofunction (NRHypo) neurotoxicity did not vary with different durations of exposure (F[3,58] ⫽ 0.8; p ⬎ 0.4), there was a significant main effect of exposure duration on haloperidol’s ability to prevent NRHypo neurotoxicity (F[3,68] ⫽ 18.1, p ⬍ 0.0001). Inspection of the graph indicated that with longer exposure time haloperidol tended to decrease the amount of damage. The severity seen after 1 week of exposure was significantly less than that seen acutely (p ⫽ 0.0005, Fisher’s PLSD). Similarly after 3 weeks of treatment damage was less than that seen at 1 week (p ⫽ 0.02, Fisher’s PLSD). Amount of damage did not appear to change between 3 and 5 weeks of treatment (p ⬎ 0.4, Fisher’s PLSD). Consistent with this analysis, haloperidol treated animals had less damage than DMSO-treated control animals at all exposure durations (p ⬍ 0.0001; denoted by asterisks on graph) except for the acute condition (p ⫽ 0.4). Error bars indicate SEM.

adrenergic (Farber, Foster, Duhan et al 1995), 5HT2A serotonergic (Farber et al 1998), sigma (Farber et al 1993), adenosine A1 (Okamura et al 2004) and group II mGlu receptors (Okamura et al 2003)— can prevent NRHypo neurotoxicity as judged by the vacuole reaction in the RSC. Here we demonstrate that D2/D3 dopaminergic agonists also can prevent NRHypo neurotoxicity most likely by acting at D3 receptors. The conclusion that it is the D3 and not the D2 receptor, which is mediating the effect, rests on a correlational analysis comparing in vivo ED50 values with in vitro functional activity. The results with U99194A, which has a 10 –20 fold relative preference for D3 receptors, provide further support for this conclusion. Further confirmation of this conclusion awaits the development and testing of agents that are even more selective for the D3 receptor. While several antipsychotics with binding affinity for D2/D3 receptors are able to prevent the neurotoxic effects of NRHypo when given acutely, the dose for producing the effect is generally above that needed to antagonize D2/D3 receptors in rodents (Farber, Foster, Olney 1995; Farber et al 1993). For example, haloperidol provides dose dependent protection against NRHypo neurotoxicity with an ED50 of 5.1 mg/kg. In rodents, a dose of 0.5–1.0 mg/kg is the usual dose used to produce blockade of D2/D3 receptors. At 1.0 mg/kg and lower, acute haloperidol administration has minimal effects at best on NRHypo neurotoxicity ((Farber et al 1993) and Fig 6). The results with D2/D3agonists provide additional evidence that the ability of antipsychotics to acutely prevent NRHypo neurotoxicity in rodents cannot be attributed to antago-

nist activity at D2/D3 receptors. Instead, it is likely that these antipsychotic agents acutely prevent NRHypo neurotoxicity through other receptors (e.g., sigma, m3-muscarinic). The finding that lower doses of D2/D3 agonists are needed to protect against the competitive NMDA antagonist, CGS-19755, than the non-competitive NMDA antagonist, MK-801, was unanticipated and interesting. Follow-up experiments suggested that the protective effect is a competitive phenomenon with CGS19755 but a non-competitive one with MK-801. In the future, it will be important to verify that NPA interacts with other competitive and non-competitive antagonists to produce similar dose response curves. Given that CGS-19755 is a competitive NMDA antagonist and MK-801 is a non-competitive NMDA antagonist, one possibility is that agonist action at certain D3 receptors produces, via an unknown mechanism, an increase in the release of glutamate at specific NMDA receptors that need to be blocked in order to produce NRHypo neurotoxicity. The additional glutamate in the synapse or at extrasynaptic receptors (Chen and Diamond 2002; Diamond 2002) would produce a decrease in the amount of CGS-19755 binding, shifting the dose-response curve to the right. In animals treated with MK-801, the additional glutamate would cause activation of the NMDA receptor, allowing a certain amount of MK-801 trapped in the ion channel to dissociate from the channel and produce the observed noncompetitive dose response curves. While D2/D3 agonists do alter the release of glutamate in subcortical regions (e.g., nucleus accumbens and caudate), the effect is to decrease and not www.sobp.org/journal

636 BIOL PSYCHIATRY 2006;60:630 – 638 increase glutamate levels (Kalivas and Duffy 1997; Koga and Momiyama 2000; Yamamoto and Davy 1992). Whether D2/D3 agonists acting in other brain circuits can also produce increases in glutamate release is unknown. Dopaminergic and glutamatergic systems are known to interact in a wide variety of ways (Tzschentke 2001), suggesting that mechanisms other than alteration in glutamate release could be involved. Additional experimentation is required to determine the mechanism via which D2/D3 agonists acutely protect against NRHypo neurotoxicity. Results from such experiments could be important for improving our understanding of how the D2/D3 dopaminergic and NMDA glutamatergic systems interact, especially in the context of psychosis. The finding that daily exposure to bromocriptine for a week reverses its ability to provide protection against NRHypo neurotoxicity suggests that chronic activity at D2/D3 receptors has a different consequence for NRHypo than acute activity. Because the amount of damage seen after one week was not statistically greater than control animals, we have not concluded that chronic bromocriptine worsens NRHypo neurotoxicity. However, given our finding that daily exposure of haloperidol for 3 weeks leads to an effect stronger than that seen after 1 week, it is possible that a 3-week exposure to bromocriptine would prove to worsen NRHypo neurotoxicity. The observed effect seen with chronic exposure to bromocriptine cannot be explained by a withdrawal phenomenon because on the last day the drug was given in the same manner as it was in the acute studies, thus producing similar drug levels. Accumulation of the drug over time is also not a likely explanation since such an effect would be expected to increase protection instead of leading to its loss. Similar arguments and conclusions would hold for the observed chronic effects of haloperidol. The results with haloperidol are consistent with the report that chronic daily injections of haloperidol for 21 days attenuated NRHypo-induced hypermetabolism whereas the acute administration of haloperidol had no effect (Duncan et al 2003). While it is possible that the chronic effects are due to binding with a different receptor from that involved with the acute effect, we doubt that activity at non-dopaminergic receptors is responsible for the chronic effects, given that these agents have their greatest affinity for dopaminergic receptors. However, since their affinities for D2 and D3 receptors are relatively similar, it is possible that the chronic effects could be via D2 receptors and the acute effects via D3 receptors. Future studies with highly selective agents will be needed to address this possibility. There are several parallels between NRHypo neurotoxicity in rodents and NRHypo psychosis in humans. Both phenomena have an onset of vulnerability in adolescence (Farber et al 1995; Noguchi et al 2005; Reich and Silvay 1989; White et al 1982). Furthermore, both can be prevented acutely by ␣2 adrenergic agonists (Farber, Foster, Duhan, et al 1995; Handa et al 2000; Levanen et al 1995; Newcomer et al 1998), GABAmimetics (Farber et al 2003; Farber, Kim et al 2002; Olney et al 1991; Reich and Silvay 1989), clozapine (Farber et al 1993; Malhotra et al 1997) and lamotrigine (Anand et al 2000; Farber, Jiang et al 2002). The data here demonstrating that acute D2/D3 blockade does not prevent NRHypo neurotoxicity are consistent with the finding of Krystal et al (1999) that haloperidol does not acutely ameliorate ketamine-induced psychosis. These parallels are consistent with the proposal that the disinhibition syndrome underlying the neurotoxic effect in rodents is likely also to underlie the psychotomimetic effects in humans. One potential inconsistency between the two phenomena is the report by Lahti et al that chronic www.sobp.org/journal

N.B. Farber et al haloperidol does not prevent ketamine-induced psychosis in schizophrenics (Lahti et al 1995). However, the lack of an effect was based upon results from 6 subjects and, as the authors discuss, their negative finding is likely due to a small sample size or other effects for which they did not control. While NRHypo neurotoxicity in normal rodent brain appears to be a good model for understanding NRHypo psychosis in normal human brain, one should not expect this acute experimental model to be 100% consistent with results obtained in individuals with schizophrenia where mutations in genes and in utero events probably lead to changes in the brain that unfold over the long period between birth and the onset of the disorder. Nonetheless, continued study of this acute drug-induced syndrome in humans and rodents should provide insight into mechanisms underlying psychosis in idiopathologic psychotic disorders such as schizophrenia. The ability of D2/D3 antagonists to treat psychosis has long been recognized. While recent evidence indicates that some ameliorative effect on psychosis can be seen as early the first day of treatment, the improvement in psychosis continues over time with maximal improvement being obtained several weeks later (Agid et al 2003; Kane et al 1994; Kapur et al 2005; Leucht et al 2005; Rabinowitz et al 2001). Thus, the clinical effect of antipsychotics builds over a long period of time and is in contrast to what is seen in other treatments, for example, ␤-adrenergic antagonists for hypertension, and dopamimetics for Parkinson’s disease, where binding to a receptor leads to an immediate and full response. The reason for this delay in maximal clinical response remains unknown, but the delay suggests that some unknown downstream effect must occur over a prolonged basis. In our study we found that a 7-day or longer exposure to haloperidol provides protection against NRHypo neurotoxicity and that no protective effect is seen with this agent acutely. Since we did not study chronic exposures lasting less than 7 days, it is unknown whether a 24-hour exposure would have been protective. However, given the pharmacokinetic and other differences between humans and rodents, the utility of conducting an in-depth duration study aimed at determining exactly when one can first detect a neuroprotective response with chronic haloperidol exposure in rodents is unclear. Our finding that chronically but not acutely administered haloperidol prevents NRHypo neurotoxicity is, we believe, entirely consistent with the clinical time course of the treatment of psychosis by D2/D3 antagonists. Determining in more detail the mechanism(s) by which chronically administered haloperidol attenuates the effects of NRHypo might lead to further insights into the pathophysiological mechanism(s) underlying idiopathologic psychotic disorders as well as the mechanism(s) underlying the observed treatment response. Finally, these studies, by demonstrating that the acute and chronic effects of an antipsychotic can be different, emphasize the importance of treatment duration as a variable when conducting studies seeking to examine possible mechanisms underlying the effects of antipsychotics. NMDA antagonists are of potential therapeutic benefit in a wide variety of clinical conditions where their use would be brief (e.g., hypoxia-ischemia, trauma, and status epilepticus). NMDA antagonists are now beginning to be used or are being evaluated for use chronically or subchronically in conditions like Alzheimer’s disease and neuropathic pain. It had been assumed that the neuroprotective effect seen with a one time dose of a safener agent would be preserved with chronic use of the safener agent. However, the findings here with dopaminergic agents raise concern that the other classes of safener agents might not be

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