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LY354740, a potent group II metabotropic glutamate receptor agonist prevents lactate-induced panic-like response in panic-prone rats
Neuropharmacology 39 (2000) 1139–1146 www.elsevier.com/locate/neuropharm
LY354740, a potent group II metabotropic glutamate receptor agonist prevents lactate-induced panic-like response in panic-prone rats A. Shekhar *, S.R. Keim Departments of Psychiatry, Pharmacology and Toxicology and Program in Medical Neurobiology, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA Accepted 22 October 1999
Abstract LY354740 is a potent and selective agonist at the group II metabotropic glutamate receptors and is shown to be an effective inhibitor of glutamate release with significant anxiolytic and drug withdrawal alleviating properties in certain animal models. Rats with chronic inhibition of GABA synthesis in the dorsomedial hypothalamus (DMH) are highly anxious and exhibit panic-like responses to peripheral lactate infusions similar to patients with panic disorder. Using these panic-prone rats, we tested the efficacy of LY354740 in preventing the lactate-induced panic-like response, comparing it to alprazolam, a clinically effective anti-panic drug. Rats were fitted with femoral arterial and venous catheters and implanted with Alzet pumps infusing the GABA synthesis inhibitor l-allylglycine into the DMH. After four days of recovery, they were confirmed to be panic-prone to lactate infusions as indicated by increases in heart rate, blood pressure, respiratory rate and “anxiety” measured in the social interaction test. Next, they were pretreated with either vehicle, LY354740 (0.3 and 0.6 mg/kg) or alprazolam (0.5 and 1.0 mg/kg) and re-challenged with lactate infusions. LY354740 treatment was equally efficacious as alprazolam in preventing lactate-induced panic attacks in this model. These data suggest that LY354740 could be a novel anti-panic drug, as effective as alprazolam in acute treatment. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Anxiety; Alprazolam; Dorsomedial nucleus; Glutamate; GABA; Hypothalamus; Social interaction; Stress
1. Introduction Metabotropic glutamate receptors (mGlu receptors) are a family of glutamate G-protein-coupled receptors linked to the second messenger systems. They are classified based on molecular structure, function and pharmacology into three major groups, group I (mGlu receptor1 and 5), group II (mGlu receptor2 and 3) and group III (mGlu receptor4,6–8) receptors (Nakanishi, 1992; Schoepp and Conn, 1993). The group II mGlu receptors are implicated generally in negatively modulating glutamate neurotransmission by presynaptic mechanisms (Pin and Duvoisin, 1995). The distribution of group II mGlu receptor
* Corresponding author. Tel.: +1-317-274-3685; fax: +1-317-2741365. E-mail address: [email protected] (A. Shekhar).
subtypes as labeled by 3H-LY354740 are predominant in the cortical and limbic regions suggesting that these receptors may modulate higher CNS functions such as emotions and cognition (Schauffhauser et al., 1998). LY354740 is a conformationally constrained analog of glutamate which is a potent and selective agonist at the mGlu receptor group II receptors (Monn et al., 1997; Schoepp et al., 1997). This compound binds selectively to group II mGlu receptors without significant affinity for the other mGlu receptors or the ionotropic glutamate receptors. It is orally effective and decreases c-AMP formation in a stereoselective manner in the forebrain areas rich in group II mGlu receptors (Schoepp et al., 1998). This compound also inhibits in vivo release of glutamate (Battaglia et al., 1997). Behaviorally, it exhibits potent anxiolytic-like effects in the potentiated startle and elevated plus-maze tests (Helton et al., 1998), as well as amelioration of the symptoms of nicotine withdrawal
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(Helton et al., 1997). It does not have significant effects on motor function and is not sedating (Helton et al., 1998). All of these properties make this compound a potential novel agent useful in the treatment of anxiety disorders, with acute anxiolytic-like effects comparable to the benzodiazepines, but without some of the adverse effects of the latter. Among anxiety disorders, panic disorder is a severe and often debilitating condition affecting about 2% of the population with the characteristic feature of this disorder being panic attacks i.e. sudden onset of severe anxiety and autonomic arousal (DSM-IV, 1990). Another feature of this disorder is that a majority of patients suffering from it reliably experience a panic attack when given intravenous infusions of sodium lactate (Pitts and McClure, 1967; Liebowitz et al., 1986). A variety of antidepressants and some benzodiazepines such as alprazolam and clonazepam are currently used in the treatment of panic disorder. The antidepressants are not acutely effective in treating the disorder and the benzodiazepines have the significant adverse effects such as sedation, motor incoordination and dependence. Thus, there is a need for novel approaches to treating panic disorder, indicating the importance of testing group II mGlu receptor agonists like LY354740 in animal models of panic disorder. Activation of the dorsomedial hypothalamus (DMH) of rats by blocking GABAA receptor-mediated inhibition elicits a physiological and behavioral syndrome similar to certain aspects of human panic attacks (Shekhar, 1994). Inducing chronic dysfunction of GABA in the DMH by infusing a GABA synthesis inhibitor via an Alzet pump not only renders rats to become chronically anxious but also susceptible to panic-like responses following sodium lactate infusions (Shekhar et al., 1996). This procedure has been utilized as an effective model for studying the underlying mechanisms and pathways in the development of panic disorder (Shekhar and Keim, 1997) and has been empirically validated with effective antipanic drugs (Shekhar, 1994). The present study was conducted to test the potential antipanic effects of LY354740 in blocking the lactateinduced panic response in rats with GABA dysfunction in the DMH. Rats were fitted with femoral arterial and venous catheters and implanted with Alzet pumps infusing the GABA synthesis inhibitor l-allylglycine into the DMH. After recovery, the development of panic responses to lactate infusions was confirmed in these rats as indicated by increases in heart rate (HR), mean arterial blood pressure (BP), respiratory rate (RR) and anxiety measured in the social interaction (SI) test. They were then pretreated with either vehicle, LY354740 (0.3 and 0.6 mg/kg) or the effective antipanic drug alprazolam (0.5 and 1.0 mg/kg) and rechallenged with lactate infusions.
2. Methods All experiments were conducted on male Sprague– Dawley rats (Harlan Laboratories, Indianapolis; 300–350 g) in accordance with the National Institutes of Health guide for the care and use of laboratory animals. The rats were housed in individual plastic cages in a temperature controlled room (72°F) kept on a 12 h day/night cycle and given ad lib food and water. 2.1. Surgical procedures Rats were first implanted with femoral arterial and venous catheters as previously described (Shekhar, 1993). The following day, after being tested for baseline lactate response, they were implanted with Alzet minipumps (Alza Corp., CA) in the DMH. Implantation of Alzet minipumps into the DMH has also been described previously (Shekhar et al., 1996). An L-shaped pump cannula with a side arm attached to a small tygon tube was used for pump implantations. Once the cannula was placed at the coordinates of the DMH, 50 pmoles/100 nl of the GABAA receptor antagonist bicuculline methiodide (BMI) was injected through the side tubing to ascertain that the tip was placed at the reactive site (i.e. where BMI elicits ⬎50 beats/min increases in HR) in the DMH. Once the reactive site was found, the side tubing was connected to the metal connector in the Alzet minipump (model 2002) that was previously filled with the GABA synthesis inhibitor l-allylglycine (Orlowski et al., 1977) infusion fluid. The concentration of the allylglycine solutions were such that 3.5 nmoles/0.5 µl/h of the drug was infused into the DMH. The pump was then sutured under the skin in the nape of the neck and the connector and cannula were cemented to the skull as described previously (Shekhar et al., 1996). 2.2. Social interaction test The social interaction (SI) test is a fully validated test of experimental anxiety in rats (File, 1980) and the procedure as used in our laboratory has been previously described (Shekhar and Katner, 1995; Sanders and Shekhar, 1995). The apparatus consists of a solid wooden box with an open roof 36 inches long by 36 inches wide with walls 12 inches high and a video camera fixed above the box. All behavioral tests were videotaped. The “experimental” rat and an unfamiliar “partner” rat were both placed in the center of the box and allowed to freely interact for a period of 5 min. The number of seconds of non-aggressive physical contact (grooming, sniffing, crawling over and under, etc.) initiated by the “experimental” rat was then counted. Sessions were then scored at a later time by two raters of whom at least one was blind to any drug treatment.
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2.3. Intravenous lactate infusions The lactate infusion procedure has been previously described (Shekhar et al., 1996). Briefly, rats were given i.v. infusions of 0.9% saline and 0.5 M sodium lactate (10 ml/kg; 1 ml/5 min), similar to clinical lactate infusions (Liebowitz et al., 1986), in random order with at least 60 min recovery time between infusions. Responses to lactate (HR, BP and RR) reported are the differences between changes elicited by lactate and saline infusions. 2.4. Measurement of GAD activity The radiometric assay for GAD (modified from Bostwick and Le, 1991; Shekhar et al., 1996) was based on supplying the enzyme with glutamate (substrate) with a 14C labelled carboxyl group and measuring the liberated 14CO2. The tissue was homogenized with 20 volumes of a solution containing EDTA, Triton X-100, and aminoethanolthiol in phosphate buffer, pH 7. Each well of tissue culture plate (Falcon 3070) received 10 µl tissue homogenate or blank (in triplicates) and 10 µl buffer substrate after which the plate was covered with a 14×11 cm sheet of gel blot paper, latched shut, incubated in a 37°C water bath for 30 min and then transferred to a 60°C bath for 45 min. The method for measuring the trapped 14CO2 has been previously described (Bostwick and Le, 1991). 2.5. Drugs Alprazolam and bicuculline methiodide were obtained from RBI. The LY354740 compound was obtained from Eli Lilly and Co. (Indianapolis, IN, USA). Alprazolam was prepared as a fine emulsion in 0.9% saline solution with addition of small volume of Tween 80 and DMSO, while LY354740 was dissolved in distilled water and brought up to a pH of 7 with NaOH. Rats were injected with the alprazolam, LY354740 or their appropriate vehicles intraperitoneally (i.p.) 1 h before lactate infusions. The doses of LY354740 was based on previous reports of anxiolytic-like effects with this compound (Helton et al., 1998) and the doses of alprazolam were determined by previous studies with other benzodiazepines in this model (Shekhar, 1994). 2.6. Experimental procedure Rats (n=12) were fitted with femoral arterial catheters for recording BP and HR and venous catheters for i.v. infusions. After recovery, baseline “anxiety” levels were obtained using the SI test. The SI test was used to measure the change in “anxiety” from baseline state (i.e. before Alzet pump implantation into the DMH) to the post-pump state. Following the baseline SI test, baseline
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reactivity to i.v. sodium lactate infusions were determined. The animals were then randomly assigned to two treatment groups (n=6 each) of LY354740 and alprazolam, respectively. Rats in group 1 were challenged with lactate infusions on post-operative days 6, 8 and 10 after being pretreated in a counterbalanced design with vehicle or LY354740 (0.3 or 0.6 mg/kg i.p., given 1 h before i.v. infusions) and the resulting panic-like response was recorded. Similarly, rats in group 2 were tested for lactate-induced panic-like response after being pre-treated with vehicle or alprazolam (0.5 or 1.0 mg/kg, given 1 h before i.v. infusions). At the end of experiments, the site of pump infusion was marked by injecting 250 nl of India ink. Rats were sacrificed, their brains removed, mounted on cryostat at ⫺20°C and 30 µm sections of the implantation sites were obtained. The sections were later stained with Neutral Red and the sites of injection were determined by comparing to a standard atlas (Paxinos and Watson, 1986). The brain section containing the remaining DMH was mounted on a frozen platform. The DMH was microdissected, weighed and stored in a ⫺70°C freezer until assayed. At a later date, tissue levels of the GABA synthetic enzyme glutamic acid decarboxylase (GAD) were determined. 2.7. Data analysis All data were expressed as mean±S.E.M. When comparing two or more means of different groups, one-way ANOVA with Newman–Keul’s test was used. Repeated measures ANOVA was employed to test differences between means when repeated measurements were made. Statistical significance was accepted at P⬍0.05.
3. Results 3.1. Effects of pretreatment with LY354740 When the rats in this group were tested at baseline (i.e. before l-AG Alzet pump implantation in the DMH) with sodium lactate infusions, there were no significant increases in HR, BP, or RR (Baseline, Figs. 1A, 2A, and 4A). After four days of l-AG infusion into the DMH, these rats became reactive to lactate as reported in previous studies (Shekhar et al., 1996; Shekhar and Keim, 1997). Pretreating these rats with vehicle used to dissolve LY354740 and testing with lactate infusions elicited significant increases in HR (F=110.5; d.f.=3,20; P⬍0.0001), BP (F=14.35; d.f.=3,20; P⬍0.001), RR (F=40.96; d.f.=3,20; P⬍0.0001) and anxiety as measured by decreased interaction time in SI (F=15.59; d.f.=3,20; P⬍0.0001), clearly suggesting a panic-like response (Figs. 1A, 2A, 3A and 4A, respectively). However, pre-treatment with LY354740 blocked the lactate-
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Fig. 1. Effect of pre-treatment with either LY354740 or alprazolam on changes in heart rate (HR) following intravenous (i.v.) infusions of sodium lactate (10 ml/kg of 0.5 N solution infused 1 ml/5 min) in rats with GABA dysfunction in the dorsomedial hypothalamus (DMH). (A) Rats (n=6) were first infused with lactate before being implanted with the GABA synthesis inhibitor l-allylglycine Alzet pumps into the DMH (baseline). Following at least four days of recovery, using a balanced design and separated by at least 48 h, they were pretreated i.p. with either vehicle or one of the two doses of LY354740 (0.3 or 0.6 mg/kg) and then rechallenged 1 h later with lactate infusions and the changes in HR noted. (B) Similarly, another group of rats (n=6) were infused with lactate at baseline and then implanted with l-allylglycine pumps into the DMH. Following recovery, they were pretreated i.p. with either vehicle or two doses of the standard antipanic agent alprazolam (0.5 and 1.0 mg/kg) and retested 1 h later with lactate infusions. Both LY354740 and alprazolam blocked the lactate induced increases in HR of panic-prone rats in a dose-related manner. Significantly different from *baseline, #vehicle and $the lower dose group by repeated measures ANOVA coupled with Newman–Keul’s test, P⬍0.001.
induced increases in HR (P⬍0.001), BP (P⬍0.05), RR (P⬍0.001) and anxiety in SI (P⬍0.01) in a dose-dependent manner (Figs. 1B, 2B, and 4B). 3.2. Effects of pretreatment with alprazolam Similarly, when rats in the alprazolam group were tested at baseline with sodium lactate infusions, there
Fig. 2. Effect of pretreatment i.p. with either LY354740 or alprazolam on changes in mean arterial blood pressure (BP) following intravenous (i.v.) infusions of sodium lactate (10 ml/kg of 0.5 N solution; 1 ml/5 min) in rats with GABA dysfunction in the dorsomedial hypothalamus (DMH). Two groups of rats (n=6 each) were infused with lactate at baseline and then implanted with l-allylglycine pumps into the DMH. Following recovery, they were pretreated 1 h earlier with (A) either vehicle or two doses of LY354740 (0.3 and 0.6 mg/kg); (B) either vehicle or two doses of the standard antipanic agent alprazolam (0.5 and 1.0 mg/kg), respectively, and tested with lactate infusions. Both LY354740 and alprazolam blocked the lactate induced increases in BP of panic-prone rats in a dose-related manner. Significantly different from *baseline, #vehicle and $the lower dose group by repeated measures ANOVA coupled with Newman–Keul’s test, P⬍0.05.
were no significant increases in HR, BP, or RR (Baseline, Figs. 1B, 2B, 3B and 4B). After four days of l-AG infusion into the DMH, the rats became reactive to lactate with increases in HR, BP, RR and decreases in SI time. Pretreating these rats with vehicle used to dissolve alprazolam and testing with lactate infusions elicited significant increases in HR (F=57.21; d.f.=3,20; P⬍0.0001), BP (F=15.61; d.f.=3,20; P⬍0.001), RR (F=30.10; d.f.=3,20; P⬍0.0001) and anxiety as measured by decreased interaction time in SI (F=55.05; d.f.=3,20; P⬍0.0001), indicating a panic-like response (Figs. 1B, 2B, 3B, and 4B, respectively). Pretreatment with alprazolam blocked the lactate-induced increases in HR (P⬍0.001), BP (P⬍0.05), RR (P⬍0.01) and anxiety in SI (P⬍0.01) in a dose-dependent manner (Fig. 1B, Fig. 2B, Fig. 3B, Fig. 4B).
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Fig. 3. Effect of pretreatment with either LY354740 or alprazolam on changes in social interaction time (SI) following intravenous (i.v.) infusions of sodium lactate (10 ml/kg of 0.5 N solution; 1 ml/5 min) in rats with GABA dysfunction in the dorsomedial hypothalamus (DMH). Two groups of rats (n=6 each) were infused with lactate at baseline and then implanted with l-allylglycine pumps into the DMH. Following recovery, they were pretreated 1 h earlier with (A) either vehicle or two doses of LY354740 (0.3 and 0.6 mg/kg); (B) either vehicle or two doses of the standard antipanic agent alprazolam (0.5 and 1.0 mg/kg), respectively, and tested with lactate infusions. Both LY354740 and alprazolam blocked the lactate induced decreases in SI of panic-prone rats in a dose-related manner. Significantly different from *baseline; and from #vehicle and the lower dose group by repeated measures ANOVA coupled with Newman–Keul’s test, P⬍0.01.
Histological examination of the pump implantation sites revealed that the cannulae were implanted in the DMH with minimal damage to the surrounding tissue and a representative section is shown in Fig. 5. Measurement of GAD activity in the DMH of these animals also revealed a significant decrease in enzyme activity on the pump side compared to the non-pump side, indicating that GABA synthesis was indeed compromised in the DMH (Table 1).
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Fig. 4. Effect of pretreatment with either LY354740 on changes in respiratory rate (RR) following intravenous (i.v.) infusions of sodium lactate (10 ml/kg of 0.5 N solution) in rats with GABA dysfunction in the dorsomedial hypothalamus (DMH). Rats (n=6) were first infused with lactate before being implanted with the GABA synthesis inhibitor l-allylglycine Alzet pumps into the DMH (baseline). Following at least four days of recovery, using a balanced design and separated by at least 48 h, they were pretreated i.p. with (A) either vehicle or one of the two doses of LY354740 (0.3 or 0.6 mg/kg); (B) either vehicle or two doses of the standard antipanic agent alprazolam (0.5 and 1.0 mg/kg), respectively, and tested 1 h later with lactate infusions. Both LY354740 and alprazolam blocked the changes in RR noted. Significantly different from * baseline, #vehicle and $the lower dose group by repeated measures ANOVA coupled with Newman–Keul’s test, P⬍0.001.
4. Discussion The above data clearly indicate that rats with GABA dysfunction in the DMH develop increases in measures of anxiety in the SI and also exhibit physiological arousal similar to certain symptoms observed in patients with panic disorder following i.v. infusions of sodium lactate. In addition to intense anxiety, lactate-induced panic attacks in patients are particularly characterized by dramatic tachycardia and increases in respiratory rate
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Fig. 5. Schematic drawing of the pump implantation sites and a representative photomicrograph of a histological section showing the site of microinjection cannula implantation (marked by arrow) for l-AG Alzet pump in the DMH. The sites were marked by injecting 50% India ink and only data from the animals with proper cannulae placement were used in the analysis.
Table 1 Effects on implanting Alzet pumps into the DMH infusing l- or dallylglycine on the glutamic acid decarboxylase (GAD) activity in the DMHa Type of Alzet pump in the DMH (n=6 each)
GAD activity on the nonDrug treatment pump side group (pmol/mg protein/30 min)
GAD activity on the pump side (pmol/mg protein/30 min)
l-allylglycine l-allylglycine
LY354740 Alprazolam
872±156* 1146±185*
a
1680±238 1921±339
Rats were implanted with Alzet pumps into the DMH infusing the GABA synthesis inhibitor l-AG and GAD activity was measured after completion of all experiments. In both LY354740 (t=3.434, d.f.=5) and alprazolam (t=4.038, d.f.=5) groups, rats implanted with lAG pumps showed significant decreases in GAD activity within the DMH on the side of pump implantation. *Significantly different from non-pump side by paired t-test, P⬍0.05.
(Liebowitz et al., 1986). This lactate-induced laboratory panic attack has been extensively used in humans to study the pathophysiology of panic disorder as well as to test the effects of antipanic agents (Price et al., 1995). The animal model with inhibition of GABA synthesis in the DMH exhibits a similar pattern of increased “anxiety” and dramatic increases in HR and RR following i.v. lactate infusions (Shekhar et al., 1996; Shekhar and Keim, 1997). Using this animal model of lactate-induced panic-like response, this study tested the efficacy of a novel compound LY354740, a group II mGlu receptor agonist and a putative anxiolytic-like drug, in blocking the lactate effects. Compared to vehicle, LY354740 was able to prevent lactate-induced responses of increased HR, BP, RR and “anxiety” in a dose-related manner, suggesting that according to this model, the LY354740 compound could be an effective antipanic agent in humans. One possibility is that the LY354740 compound may have effects on peripheral cardiovascular mechanisms such that the primary effects may be one of blocking the increases in HR and BP. Panic attacks in patients could possibly represent a response to the vegetative changes such as increases in HR or BP elicited by peripheral infusions like lactate, and if these vegetative symptoms are blocked, then this may result in blocking the panic response. Arguing against such a possibility is the inability of beta-blockers which reduce HR and BP responses following lactate infusions to block the panic responses in patients (Price et al., 1995). Furthermore, we have conducted experiments in an additional group of rats testing the effects of pretreatment with the LY354740 compound on i.v infusions of phenylephrine (a sympathomimetic that increases BP) and nitroprusside (a vasodilator that increases HR). The peripheral cardiovascular effects of these agents were not blocked by the LY354740 compound, suggesting that the anti-panic effects of this agent is not due to peripheral blockade of vegetative changes. Panic disorder is a severely disabling, chronic anxiety syndrome that has a recurrent, life-long course. Currently, antidepressants and benzodiazepines such as alprazolam are the most commonly used drugs to treat panic disorder. Most antidepressants take several weeks to be effective in treating panic disorder and a significant number of patients have difficulty tolerating the initial stimulating effects of the antidepressants. The benzodiazepines are rapidly effective in treating panic attacks but have significant problems such as sedation, interaction with CNS depressants, adverse effects on motor coordination as well as problems of dependence. Thus, there is a great need for rapidly effective antipanic agents without the typical benzodiazepine side effects. Previous studies with the LY354740 compound have shown potent anxiolytic effects in a variety of behavioral paradigms such as the elevated plus-maze, fear-potentiated
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startle (Helton et al., 1998) and nicotine withdrawal (Helton et al., 1997). Unlike the classic anxiolytics like the benzodiazepines, LY354740 does not produce sedation, deficits in neuromuscular coordination, disruption of memory or interact with other central nervous system depressants (Helton et al., 1998). These data suggest that LY354740 could be a novel antipanic agent with considerable advantages over currently available treatments. The panic response elicited by activating the DMH in rats is regulated by a delicate balance between GABA inhibition and glutamatergic excitation. Under normal conditions, this response is under a tonic GABAA receptor mediated inhibition (DiMicco et al., 1992). A full panic response is observed when this GABA inhibition is antagonized and this response can be prevented by blocking glutamate receptors (Soltis and DiMicco, 1991). Chronically disabling the GABA inhibition in this region by blocking its synthesis with the infusion of lAG increases the baseline “anxiety” of these animals and also makes them vulnerable to panic-like response by a variety of so called “panicogenic” substances such as sodium lactate (Shekhar et al., 1996), yohimbine and fenfluramine infusions (Shekhar and Keim, 1998), which induce panic attacks in patients with panic disorder (Price et al., 1995). This panic-prone state that develops in rats following chronic GABA synthesis blockade should be dependent on intact glutamate neurotransmission, which would now presumably be uninhibited. In keeping with this hypothesis, local injection of glutamate receptor antagonists into the DMH of these panic-prone rats blocks the lactate-induced panic-like responses in a dose-dependent manner (Keim and Shekhar, 1998). Thus, antagonizing glutamate neurotransmission in the DMH of rats with chronic local GABA synthesis inhibition would prevent the activation of the panic-like response. The mechanism by which LY354740 blocks the lactate-induced panic in rats with GABA dysfunction in the DMH is most likely by decreasing glutamate neurotransmission in the DMH. Group II mGlu receptors are thought to decrease glutamate neurotransmission by a number of mechanisms (Schoepp, 1994; Pin and Duvoisin, 1995). They may function presynaptically as autoreceptors that inhibit the release of glutamate. Postsynaptically, they are thought to modulate glutamate effects by their actions on potassium and calcium ion channels as well as protein kinase activity. The distribution of group II mGlu receptors is particularly dense in limbic and cortical areas (Onishi et al., 1993) and would be predicted to influence anxiety and fear responses. Supporting such a mechanism of action, LY354740 has been shown to decrease stimulated in vivo release of glutamate in rats (Battaglia et al., 1997). Similarly, LY354740 has also been shown to decrease significantly forskolinstimulated cAMP formation in select brain regions including cerebral cortex, hippocampus and hypothala-
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mus of rats (Schoepp et al., 1998). By such mechanisms, LY354740 could inhibit glutamate neurotransmission in the DMH in the panic-prone rats, thus blocking the panic-like response elicited by lactate infusions. In conclusion, the present study tested the potential antipanic effects of a potent, selective group II mGlu receptor agonist LY354740, in blocking the lactateinduced panic-like response in rats with GABA dysfunction in the DMH, as indicated by increases in HR, BP, RR and “anxiety” as measured in the SI test. When pretreated with either vehicle, LY354740 (0.3 and 0.6 mg/kg) or the effective antipanic drug alprazolam (0.5 and 1.0 mg/kg) and rechallenged with lactate infusions, both LY354740 and alprazolam exhibited dose-dependent inhibition of the lactate-induced panic-like response. These data suggest that LY354740 could be a novel anti-panic drug, as effective as alprazolam, but without some of the adverse effects of benzodiazepines.
Acknowledgements The authors thank Dr Darryle D. Schoepp for the gift of LY354740 and his scientific advice. This study was supported by PHS grant MH 52691 and a research grant from Eli Lilly and Co.
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Johnson, B.G., Howe, T., Alt, C.A., Rhodes, G.A., Robey, R.L., Griffey, K.R., Tizzano, J.P., Kallman, M.J., Helton, D.R., Schoepp, D.D., 1997. Design, synthesis, and pharmacological characterization of (+)-2-aminobicyclo[3.1.0]hexane-2,6-di-carboxylate: a potent, selective, systemically active group 2 metabotropic glutamate receptor agonist possessing anticonvulsant and anxiolytic properties. Journal of Medical Chemistry 40, 528–537. Nakanishi, S., 1992. Molecular diversity of glutamate receptors and implications for brain function. Science 258, 597–603. Onishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993. Distribution for the mRNA for a metabotropic glutamate receptor (mGlu receptor3) in the rat brain: an in situ hybridization study. Journal of Comparative Neurology 335, 252–266. Orlowski, M., Reingold, D.F., Stanley, M.E., 1977. D- and l-stereoisomers of allylglycine: convulsive action and inhibition of brain lglutamate decarboxylase. Journal of Neurochemistry 28, 349–353. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates, second ed. Academic Press, New York. Pin, J.-P., Duvoisin, R., 1995. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34, 1–26. Pitts, F.N., McClure, J.N., 1967. Lactate metabolism in anxiety neurosis. New England Journal of Medicine 227, 1329–1336. Price, L.H., Goddard, A.W., Barr, L.C., Goodman, W.K., 1995. Pharmacological challenges in anxiety disorders. In: Bloom, F.E., Kupfer, D.J. (Eds.) Psychpharmacology: The Fourth Generation of Progression. Raven Press, New York, pp. 1311–1323. Sanders, S.K., Shekhar, A., 1995. GABAA receptors in the basolateral amygdala of rats regulate “anxiety”. Pharmacology, Biochemistry and Behavior 52, 701–706. Schauffhauser, H., Richards, J.G., Cartmell, J., Chaboz, S., Kemp, J.A., Klingschmidt, A., Messer, J., Stadler, H., Woltering, T., Mutel, V., 1998. In vitro binding characteristics of a new selective group II metabotropic glutamate receptor radioligand, [3H]LY354740, in rat brain. Molecular Pharmacology 53, 228–233. Schoepp, D.D., 1994. Novel functions for subtypes of metabotropic glutamate receptors. Neurochemistry International 24, 439–449.
Schoepp, D.D., Conn, P.J., 1993. Metabotropic glutamate receptors in brain function and pathology. Trends in Pharmacological Sciences 14, 13–20. Schoepp, D.D., Johnson, B.G., Wright, R.A., Salhoff, C.R., Mayne, N.G., Wu, S., Cockerham, S.L., Burnett, J.P., Belagaje, R., Bleakman, D., Monn, J.A., 1997. LY374740 is a potent and highly selective group II metabotropic glutamate receptor agonist in cells expressing human glutamate receptor. Neuropharmacology 36, 1– 11. Schoepp, D.D., Johnson, B.G., Wright, R.A., Salhoff, C.R., Monn, J.A., 1998. Potent, stereoselective, and brain region selective modulation of second messengers in the rat brain by (+)LY354740, a novel group II metabotropic glutamate receptor agonist. NaunynSchmiedeberg’s Archives of Pharmacology 358, 175–180. Shekhar, A., 1993. GABA blockade in the region of the dorsomedial hypothalamus regulates “anxiety” in rats in the elevated plus-maze: I. behavioral measures. Brain Research 627, 9–16. Shekhar, A., 1994. Effects of chronic treatment with imipramine and clonazepam in an animal model of panic disorder. Biological Psychiatry 36, 748–758. Shekhar, A., Katner, J.S., 1995. GABAA receptors in the dorsomedial hypothalamus of rats regulate anxiety in the social interaction test. Pharmacology, Biochemistry and Behavior 50, 253–258. Shekhar, A., Keim, S.R., Simon, J.R., McBride, W.J., 1996. Physiological arousal elicited by sodium lactate infusion in rats with dorsomedial hypothalamic GABA dysfunction. Pharmacology, Biochemistry and Behavior 55, 249–256. Shekhar, A., Keim, S.R., 1997. The circumventricular organs form a potential neural pathway for lactate sensitivity: implications for panic disorder. Journal of Neuroscience 17, 9726–9735. Shekhar, A., Keim, S.R., 1998. Panic responses in rats with chronic GABA dysfunction in the dorsomedial hypothalamus (DMH) following i.v. infusions of yohimbine and fenfluramine. Society of Neuroscience Abstracts 24, 1673. Soltis, R.P., DiMicco, J.A., 1991. Interaction of hypothalamic GABAA and excitatory amino acid receptors controlling heart rate in rats. Americal Journal of Physiology 261, R427–R433.
LY354740, a potent group II metabotropic glutamate receptor agonist prevents lactate-induced panic-like response in panic-prone rats A. Shekhar *, S.R. Keim Departments of Psychiatry, Pharmacology and Toxicology and Program in Medical Neurobiology, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA Accepted 22 October 1999
Abstract LY354740 is a potent and selective agonist at the group II metabotropic glutamate receptors and is shown to be an effective inhibitor of glutamate release with significant anxiolytic and drug withdrawal alleviating properties in certain animal models. Rats with chronic inhibition of GABA synthesis in the dorsomedial hypothalamus (DMH) are highly anxious and exhibit panic-like responses to peripheral lactate infusions similar to patients with panic disorder. Using these panic-prone rats, we tested the efficacy of LY354740 in preventing the lactate-induced panic-like response, comparing it to alprazolam, a clinically effective anti-panic drug. Rats were fitted with femoral arterial and venous catheters and implanted with Alzet pumps infusing the GABA synthesis inhibitor l-allylglycine into the DMH. After four days of recovery, they were confirmed to be panic-prone to lactate infusions as indicated by increases in heart rate, blood pressure, respiratory rate and “anxiety” measured in the social interaction test. Next, they were pretreated with either vehicle, LY354740 (0.3 and 0.6 mg/kg) or alprazolam (0.5 and 1.0 mg/kg) and re-challenged with lactate infusions. LY354740 treatment was equally efficacious as alprazolam in preventing lactate-induced panic attacks in this model. These data suggest that LY354740 could be a novel anti-panic drug, as effective as alprazolam in acute treatment. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Anxiety; Alprazolam; Dorsomedial nucleus; Glutamate; GABA; Hypothalamus; Social interaction; Stress
1. Introduction Metabotropic glutamate receptors (mGlu receptors) are a family of glutamate G-protein-coupled receptors linked to the second messenger systems. They are classified based on molecular structure, function and pharmacology into three major groups, group I (mGlu receptor1 and 5), group II (mGlu receptor2 and 3) and group III (mGlu receptor4,6–8) receptors (Nakanishi, 1992; Schoepp and Conn, 1993). The group II mGlu receptors are implicated generally in negatively modulating glutamate neurotransmission by presynaptic mechanisms (Pin and Duvoisin, 1995). The distribution of group II mGlu receptor
* Corresponding author. Tel.: +1-317-274-3685; fax: +1-317-2741365. E-mail address: [email protected] (A. Shekhar).
subtypes as labeled by 3H-LY354740 are predominant in the cortical and limbic regions suggesting that these receptors may modulate higher CNS functions such as emotions and cognition (Schauffhauser et al., 1998). LY354740 is a conformationally constrained analog of glutamate which is a potent and selective agonist at the mGlu receptor group II receptors (Monn et al., 1997; Schoepp et al., 1997). This compound binds selectively to group II mGlu receptors without significant affinity for the other mGlu receptors or the ionotropic glutamate receptors. It is orally effective and decreases c-AMP formation in a stereoselective manner in the forebrain areas rich in group II mGlu receptors (Schoepp et al., 1998). This compound also inhibits in vivo release of glutamate (Battaglia et al., 1997). Behaviorally, it exhibits potent anxiolytic-like effects in the potentiated startle and elevated plus-maze tests (Helton et al., 1998), as well as amelioration of the symptoms of nicotine withdrawal
0028-3908/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 2 1 5 - 4
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(Helton et al., 1997). It does not have significant effects on motor function and is not sedating (Helton et al., 1998). All of these properties make this compound a potential novel agent useful in the treatment of anxiety disorders, with acute anxiolytic-like effects comparable to the benzodiazepines, but without some of the adverse effects of the latter. Among anxiety disorders, panic disorder is a severe and often debilitating condition affecting about 2% of the population with the characteristic feature of this disorder being panic attacks i.e. sudden onset of severe anxiety and autonomic arousal (DSM-IV, 1990). Another feature of this disorder is that a majority of patients suffering from it reliably experience a panic attack when given intravenous infusions of sodium lactate (Pitts and McClure, 1967; Liebowitz et al., 1986). A variety of antidepressants and some benzodiazepines such as alprazolam and clonazepam are currently used in the treatment of panic disorder. The antidepressants are not acutely effective in treating the disorder and the benzodiazepines have the significant adverse effects such as sedation, motor incoordination and dependence. Thus, there is a need for novel approaches to treating panic disorder, indicating the importance of testing group II mGlu receptor agonists like LY354740 in animal models of panic disorder. Activation of the dorsomedial hypothalamus (DMH) of rats by blocking GABAA receptor-mediated inhibition elicits a physiological and behavioral syndrome similar to certain aspects of human panic attacks (Shekhar, 1994). Inducing chronic dysfunction of GABA in the DMH by infusing a GABA synthesis inhibitor via an Alzet pump not only renders rats to become chronically anxious but also susceptible to panic-like responses following sodium lactate infusions (Shekhar et al., 1996). This procedure has been utilized as an effective model for studying the underlying mechanisms and pathways in the development of panic disorder (Shekhar and Keim, 1997) and has been empirically validated with effective antipanic drugs (Shekhar, 1994). The present study was conducted to test the potential antipanic effects of LY354740 in blocking the lactateinduced panic response in rats with GABA dysfunction in the DMH. Rats were fitted with femoral arterial and venous catheters and implanted with Alzet pumps infusing the GABA synthesis inhibitor l-allylglycine into the DMH. After recovery, the development of panic responses to lactate infusions was confirmed in these rats as indicated by increases in heart rate (HR), mean arterial blood pressure (BP), respiratory rate (RR) and anxiety measured in the social interaction (SI) test. They were then pretreated with either vehicle, LY354740 (0.3 and 0.6 mg/kg) or the effective antipanic drug alprazolam (0.5 and 1.0 mg/kg) and rechallenged with lactate infusions.
2. Methods All experiments were conducted on male Sprague– Dawley rats (Harlan Laboratories, Indianapolis; 300–350 g) in accordance with the National Institutes of Health guide for the care and use of laboratory animals. The rats were housed in individual plastic cages in a temperature controlled room (72°F) kept on a 12 h day/night cycle and given ad lib food and water. 2.1. Surgical procedures Rats were first implanted with femoral arterial and venous catheters as previously described (Shekhar, 1993). The following day, after being tested for baseline lactate response, they were implanted with Alzet minipumps (Alza Corp., CA) in the DMH. Implantation of Alzet minipumps into the DMH has also been described previously (Shekhar et al., 1996). An L-shaped pump cannula with a side arm attached to a small tygon tube was used for pump implantations. Once the cannula was placed at the coordinates of the DMH, 50 pmoles/100 nl of the GABAA receptor antagonist bicuculline methiodide (BMI) was injected through the side tubing to ascertain that the tip was placed at the reactive site (i.e. where BMI elicits ⬎50 beats/min increases in HR) in the DMH. Once the reactive site was found, the side tubing was connected to the metal connector in the Alzet minipump (model 2002) that was previously filled with the GABA synthesis inhibitor l-allylglycine (Orlowski et al., 1977) infusion fluid. The concentration of the allylglycine solutions were such that 3.5 nmoles/0.5 µl/h of the drug was infused into the DMH. The pump was then sutured under the skin in the nape of the neck and the connector and cannula were cemented to the skull as described previously (Shekhar et al., 1996). 2.2. Social interaction test The social interaction (SI) test is a fully validated test of experimental anxiety in rats (File, 1980) and the procedure as used in our laboratory has been previously described (Shekhar and Katner, 1995; Sanders and Shekhar, 1995). The apparatus consists of a solid wooden box with an open roof 36 inches long by 36 inches wide with walls 12 inches high and a video camera fixed above the box. All behavioral tests were videotaped. The “experimental” rat and an unfamiliar “partner” rat were both placed in the center of the box and allowed to freely interact for a period of 5 min. The number of seconds of non-aggressive physical contact (grooming, sniffing, crawling over and under, etc.) initiated by the “experimental” rat was then counted. Sessions were then scored at a later time by two raters of whom at least one was blind to any drug treatment.
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2.3. Intravenous lactate infusions The lactate infusion procedure has been previously described (Shekhar et al., 1996). Briefly, rats were given i.v. infusions of 0.9% saline and 0.5 M sodium lactate (10 ml/kg; 1 ml/5 min), similar to clinical lactate infusions (Liebowitz et al., 1986), in random order with at least 60 min recovery time between infusions. Responses to lactate (HR, BP and RR) reported are the differences between changes elicited by lactate and saline infusions. 2.4. Measurement of GAD activity The radiometric assay for GAD (modified from Bostwick and Le, 1991; Shekhar et al., 1996) was based on supplying the enzyme with glutamate (substrate) with a 14C labelled carboxyl group and measuring the liberated 14CO2. The tissue was homogenized with 20 volumes of a solution containing EDTA, Triton X-100, and aminoethanolthiol in phosphate buffer, pH 7. Each well of tissue culture plate (Falcon 3070) received 10 µl tissue homogenate or blank (in triplicates) and 10 µl buffer substrate after which the plate was covered with a 14×11 cm sheet of gel blot paper, latched shut, incubated in a 37°C water bath for 30 min and then transferred to a 60°C bath for 45 min. The method for measuring the trapped 14CO2 has been previously described (Bostwick and Le, 1991). 2.5. Drugs Alprazolam and bicuculline methiodide were obtained from RBI. The LY354740 compound was obtained from Eli Lilly and Co. (Indianapolis, IN, USA). Alprazolam was prepared as a fine emulsion in 0.9% saline solution with addition of small volume of Tween 80 and DMSO, while LY354740 was dissolved in distilled water and brought up to a pH of 7 with NaOH. Rats were injected with the alprazolam, LY354740 or their appropriate vehicles intraperitoneally (i.p.) 1 h before lactate infusions. The doses of LY354740 was based on previous reports of anxiolytic-like effects with this compound (Helton et al., 1998) and the doses of alprazolam were determined by previous studies with other benzodiazepines in this model (Shekhar, 1994). 2.6. Experimental procedure Rats (n=12) were fitted with femoral arterial catheters for recording BP and HR and venous catheters for i.v. infusions. After recovery, baseline “anxiety” levels were obtained using the SI test. The SI test was used to measure the change in “anxiety” from baseline state (i.e. before Alzet pump implantation into the DMH) to the post-pump state. Following the baseline SI test, baseline
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reactivity to i.v. sodium lactate infusions were determined. The animals were then randomly assigned to two treatment groups (n=6 each) of LY354740 and alprazolam, respectively. Rats in group 1 were challenged with lactate infusions on post-operative days 6, 8 and 10 after being pretreated in a counterbalanced design with vehicle or LY354740 (0.3 or 0.6 mg/kg i.p., given 1 h before i.v. infusions) and the resulting panic-like response was recorded. Similarly, rats in group 2 were tested for lactate-induced panic-like response after being pre-treated with vehicle or alprazolam (0.5 or 1.0 mg/kg, given 1 h before i.v. infusions). At the end of experiments, the site of pump infusion was marked by injecting 250 nl of India ink. Rats were sacrificed, their brains removed, mounted on cryostat at ⫺20°C and 30 µm sections of the implantation sites were obtained. The sections were later stained with Neutral Red and the sites of injection were determined by comparing to a standard atlas (Paxinos and Watson, 1986). The brain section containing the remaining DMH was mounted on a frozen platform. The DMH was microdissected, weighed and stored in a ⫺70°C freezer until assayed. At a later date, tissue levels of the GABA synthetic enzyme glutamic acid decarboxylase (GAD) were determined. 2.7. Data analysis All data were expressed as mean±S.E.M. When comparing two or more means of different groups, one-way ANOVA with Newman–Keul’s test was used. Repeated measures ANOVA was employed to test differences between means when repeated measurements were made. Statistical significance was accepted at P⬍0.05.
3. Results 3.1. Effects of pretreatment with LY354740 When the rats in this group were tested at baseline (i.e. before l-AG Alzet pump implantation in the DMH) with sodium lactate infusions, there were no significant increases in HR, BP, or RR (Baseline, Figs. 1A, 2A, and 4A). After four days of l-AG infusion into the DMH, these rats became reactive to lactate as reported in previous studies (Shekhar et al., 1996; Shekhar and Keim, 1997). Pretreating these rats with vehicle used to dissolve LY354740 and testing with lactate infusions elicited significant increases in HR (F=110.5; d.f.=3,20; P⬍0.0001), BP (F=14.35; d.f.=3,20; P⬍0.001), RR (F=40.96; d.f.=3,20; P⬍0.0001) and anxiety as measured by decreased interaction time in SI (F=15.59; d.f.=3,20; P⬍0.0001), clearly suggesting a panic-like response (Figs. 1A, 2A, 3A and 4A, respectively). However, pre-treatment with LY354740 blocked the lactate-
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Fig. 1. Effect of pre-treatment with either LY354740 or alprazolam on changes in heart rate (HR) following intravenous (i.v.) infusions of sodium lactate (10 ml/kg of 0.5 N solution infused 1 ml/5 min) in rats with GABA dysfunction in the dorsomedial hypothalamus (DMH). (A) Rats (n=6) were first infused with lactate before being implanted with the GABA synthesis inhibitor l-allylglycine Alzet pumps into the DMH (baseline). Following at least four days of recovery, using a balanced design and separated by at least 48 h, they were pretreated i.p. with either vehicle or one of the two doses of LY354740 (0.3 or 0.6 mg/kg) and then rechallenged 1 h later with lactate infusions and the changes in HR noted. (B) Similarly, another group of rats (n=6) were infused with lactate at baseline and then implanted with l-allylglycine pumps into the DMH. Following recovery, they were pretreated i.p. with either vehicle or two doses of the standard antipanic agent alprazolam (0.5 and 1.0 mg/kg) and retested 1 h later with lactate infusions. Both LY354740 and alprazolam blocked the lactate induced increases in HR of panic-prone rats in a dose-related manner. Significantly different from *baseline, #vehicle and $the lower dose group by repeated measures ANOVA coupled with Newman–Keul’s test, P⬍0.001.
induced increases in HR (P⬍0.001), BP (P⬍0.05), RR (P⬍0.001) and anxiety in SI (P⬍0.01) in a dose-dependent manner (Figs. 1B, 2B, and 4B). 3.2. Effects of pretreatment with alprazolam Similarly, when rats in the alprazolam group were tested at baseline with sodium lactate infusions, there
Fig. 2. Effect of pretreatment i.p. with either LY354740 or alprazolam on changes in mean arterial blood pressure (BP) following intravenous (i.v.) infusions of sodium lactate (10 ml/kg of 0.5 N solution; 1 ml/5 min) in rats with GABA dysfunction in the dorsomedial hypothalamus (DMH). Two groups of rats (n=6 each) were infused with lactate at baseline and then implanted with l-allylglycine pumps into the DMH. Following recovery, they were pretreated 1 h earlier with (A) either vehicle or two doses of LY354740 (0.3 and 0.6 mg/kg); (B) either vehicle or two doses of the standard antipanic agent alprazolam (0.5 and 1.0 mg/kg), respectively, and tested with lactate infusions. Both LY354740 and alprazolam blocked the lactate induced increases in BP of panic-prone rats in a dose-related manner. Significantly different from *baseline, #vehicle and $the lower dose group by repeated measures ANOVA coupled with Newman–Keul’s test, P⬍0.05.
were no significant increases in HR, BP, or RR (Baseline, Figs. 1B, 2B, 3B and 4B). After four days of l-AG infusion into the DMH, the rats became reactive to lactate with increases in HR, BP, RR and decreases in SI time. Pretreating these rats with vehicle used to dissolve alprazolam and testing with lactate infusions elicited significant increases in HR (F=57.21; d.f.=3,20; P⬍0.0001), BP (F=15.61; d.f.=3,20; P⬍0.001), RR (F=30.10; d.f.=3,20; P⬍0.0001) and anxiety as measured by decreased interaction time in SI (F=55.05; d.f.=3,20; P⬍0.0001), indicating a panic-like response (Figs. 1B, 2B, 3B, and 4B, respectively). Pretreatment with alprazolam blocked the lactate-induced increases in HR (P⬍0.001), BP (P⬍0.05), RR (P⬍0.01) and anxiety in SI (P⬍0.01) in a dose-dependent manner (Fig. 1B, Fig. 2B, Fig. 3B, Fig. 4B).
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Fig. 3. Effect of pretreatment with either LY354740 or alprazolam on changes in social interaction time (SI) following intravenous (i.v.) infusions of sodium lactate (10 ml/kg of 0.5 N solution; 1 ml/5 min) in rats with GABA dysfunction in the dorsomedial hypothalamus (DMH). Two groups of rats (n=6 each) were infused with lactate at baseline and then implanted with l-allylglycine pumps into the DMH. Following recovery, they were pretreated 1 h earlier with (A) either vehicle or two doses of LY354740 (0.3 and 0.6 mg/kg); (B) either vehicle or two doses of the standard antipanic agent alprazolam (0.5 and 1.0 mg/kg), respectively, and tested with lactate infusions. Both LY354740 and alprazolam blocked the lactate induced decreases in SI of panic-prone rats in a dose-related manner. Significantly different from *baseline; and from #vehicle and the lower dose group by repeated measures ANOVA coupled with Newman–Keul’s test, P⬍0.01.
Histological examination of the pump implantation sites revealed that the cannulae were implanted in the DMH with minimal damage to the surrounding tissue and a representative section is shown in Fig. 5. Measurement of GAD activity in the DMH of these animals also revealed a significant decrease in enzyme activity on the pump side compared to the non-pump side, indicating that GABA synthesis was indeed compromised in the DMH (Table 1).
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Fig. 4. Effect of pretreatment with either LY354740 on changes in respiratory rate (RR) following intravenous (i.v.) infusions of sodium lactate (10 ml/kg of 0.5 N solution) in rats with GABA dysfunction in the dorsomedial hypothalamus (DMH). Rats (n=6) were first infused with lactate before being implanted with the GABA synthesis inhibitor l-allylglycine Alzet pumps into the DMH (baseline). Following at least four days of recovery, using a balanced design and separated by at least 48 h, they were pretreated i.p. with (A) either vehicle or one of the two doses of LY354740 (0.3 or 0.6 mg/kg); (B) either vehicle or two doses of the standard antipanic agent alprazolam (0.5 and 1.0 mg/kg), respectively, and tested 1 h later with lactate infusions. Both LY354740 and alprazolam blocked the changes in RR noted. Significantly different from * baseline, #vehicle and $the lower dose group by repeated measures ANOVA coupled with Newman–Keul’s test, P⬍0.001.
4. Discussion The above data clearly indicate that rats with GABA dysfunction in the DMH develop increases in measures of anxiety in the SI and also exhibit physiological arousal similar to certain symptoms observed in patients with panic disorder following i.v. infusions of sodium lactate. In addition to intense anxiety, lactate-induced panic attacks in patients are particularly characterized by dramatic tachycardia and increases in respiratory rate
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Fig. 5. Schematic drawing of the pump implantation sites and a representative photomicrograph of a histological section showing the site of microinjection cannula implantation (marked by arrow) for l-AG Alzet pump in the DMH. The sites were marked by injecting 50% India ink and only data from the animals with proper cannulae placement were used in the analysis.
Table 1 Effects on implanting Alzet pumps into the DMH infusing l- or dallylglycine on the glutamic acid decarboxylase (GAD) activity in the DMHa Type of Alzet pump in the DMH (n=6 each)
GAD activity on the nonDrug treatment pump side group (pmol/mg protein/30 min)
GAD activity on the pump side (pmol/mg protein/30 min)
l-allylglycine l-allylglycine
LY354740 Alprazolam
872±156* 1146±185*
a
1680±238 1921±339
Rats were implanted with Alzet pumps into the DMH infusing the GABA synthesis inhibitor l-AG and GAD activity was measured after completion of all experiments. In both LY354740 (t=3.434, d.f.=5) and alprazolam (t=4.038, d.f.=5) groups, rats implanted with lAG pumps showed significant decreases in GAD activity within the DMH on the side of pump implantation. *Significantly different from non-pump side by paired t-test, P⬍0.05.
(Liebowitz et al., 1986). This lactate-induced laboratory panic attack has been extensively used in humans to study the pathophysiology of panic disorder as well as to test the effects of antipanic agents (Price et al., 1995). The animal model with inhibition of GABA synthesis in the DMH exhibits a similar pattern of increased “anxiety” and dramatic increases in HR and RR following i.v. lactate infusions (Shekhar et al., 1996; Shekhar and Keim, 1997). Using this animal model of lactate-induced panic-like response, this study tested the efficacy of a novel compound LY354740, a group II mGlu receptor agonist and a putative anxiolytic-like drug, in blocking the lactate effects. Compared to vehicle, LY354740 was able to prevent lactate-induced responses of increased HR, BP, RR and “anxiety” in a dose-related manner, suggesting that according to this model, the LY354740 compound could be an effective antipanic agent in humans. One possibility is that the LY354740 compound may have effects on peripheral cardiovascular mechanisms such that the primary effects may be one of blocking the increases in HR and BP. Panic attacks in patients could possibly represent a response to the vegetative changes such as increases in HR or BP elicited by peripheral infusions like lactate, and if these vegetative symptoms are blocked, then this may result in blocking the panic response. Arguing against such a possibility is the inability of beta-blockers which reduce HR and BP responses following lactate infusions to block the panic responses in patients (Price et al., 1995). Furthermore, we have conducted experiments in an additional group of rats testing the effects of pretreatment with the LY354740 compound on i.v infusions of phenylephrine (a sympathomimetic that increases BP) and nitroprusside (a vasodilator that increases HR). The peripheral cardiovascular effects of these agents were not blocked by the LY354740 compound, suggesting that the anti-panic effects of this agent is not due to peripheral blockade of vegetative changes. Panic disorder is a severely disabling, chronic anxiety syndrome that has a recurrent, life-long course. Currently, antidepressants and benzodiazepines such as alprazolam are the most commonly used drugs to treat panic disorder. Most antidepressants take several weeks to be effective in treating panic disorder and a significant number of patients have difficulty tolerating the initial stimulating effects of the antidepressants. The benzodiazepines are rapidly effective in treating panic attacks but have significant problems such as sedation, interaction with CNS depressants, adverse effects on motor coordination as well as problems of dependence. Thus, there is a great need for rapidly effective antipanic agents without the typical benzodiazepine side effects. Previous studies with the LY354740 compound have shown potent anxiolytic effects in a variety of behavioral paradigms such as the elevated plus-maze, fear-potentiated
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startle (Helton et al., 1998) and nicotine withdrawal (Helton et al., 1997). Unlike the classic anxiolytics like the benzodiazepines, LY354740 does not produce sedation, deficits in neuromuscular coordination, disruption of memory or interact with other central nervous system depressants (Helton et al., 1998). These data suggest that LY354740 could be a novel antipanic agent with considerable advantages over currently available treatments. The panic response elicited by activating the DMH in rats is regulated by a delicate balance between GABA inhibition and glutamatergic excitation. Under normal conditions, this response is under a tonic GABAA receptor mediated inhibition (DiMicco et al., 1992). A full panic response is observed when this GABA inhibition is antagonized and this response can be prevented by blocking glutamate receptors (Soltis and DiMicco, 1991). Chronically disabling the GABA inhibition in this region by blocking its synthesis with the infusion of lAG increases the baseline “anxiety” of these animals and also makes them vulnerable to panic-like response by a variety of so called “panicogenic” substances such as sodium lactate (Shekhar et al., 1996), yohimbine and fenfluramine infusions (Shekhar and Keim, 1998), which induce panic attacks in patients with panic disorder (Price et al., 1995). This panic-prone state that develops in rats following chronic GABA synthesis blockade should be dependent on intact glutamate neurotransmission, which would now presumably be uninhibited. In keeping with this hypothesis, local injection of glutamate receptor antagonists into the DMH of these panic-prone rats blocks the lactate-induced panic-like responses in a dose-dependent manner (Keim and Shekhar, 1998). Thus, antagonizing glutamate neurotransmission in the DMH of rats with chronic local GABA synthesis inhibition would prevent the activation of the panic-like response. The mechanism by which LY354740 blocks the lactate-induced panic in rats with GABA dysfunction in the DMH is most likely by decreasing glutamate neurotransmission in the DMH. Group II mGlu receptors are thought to decrease glutamate neurotransmission by a number of mechanisms (Schoepp, 1994; Pin and Duvoisin, 1995). They may function presynaptically as autoreceptors that inhibit the release of glutamate. Postsynaptically, they are thought to modulate glutamate effects by their actions on potassium and calcium ion channels as well as protein kinase activity. The distribution of group II mGlu receptors is particularly dense in limbic and cortical areas (Onishi et al., 1993) and would be predicted to influence anxiety and fear responses. Supporting such a mechanism of action, LY354740 has been shown to decrease stimulated in vivo release of glutamate in rats (Battaglia et al., 1997). Similarly, LY354740 has also been shown to decrease significantly forskolinstimulated cAMP formation in select brain regions including cerebral cortex, hippocampus and hypothala-
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mus of rats (Schoepp et al., 1998). By such mechanisms, LY354740 could inhibit glutamate neurotransmission in the DMH in the panic-prone rats, thus blocking the panic-like response elicited by lactate infusions. In conclusion, the present study tested the potential antipanic effects of a potent, selective group II mGlu receptor agonist LY354740, in blocking the lactateinduced panic-like response in rats with GABA dysfunction in the DMH, as indicated by increases in HR, BP, RR and “anxiety” as measured in the SI test. When pretreated with either vehicle, LY354740 (0.3 and 0.6 mg/kg) or the effective antipanic drug alprazolam (0.5 and 1.0 mg/kg) and rechallenged with lactate infusions, both LY354740 and alprazolam exhibited dose-dependent inhibition of the lactate-induced panic-like response. These data suggest that LY354740 could be a novel anti-panic drug, as effective as alprazolam, but without some of the adverse effects of benzodiazepines.
Acknowledgements The authors thank Dr Darryle D. Schoepp for the gift of LY354740 and his scientific advice. This study was supported by PHS grant MH 52691 and a research grant from Eli Lilly and Co.
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