Neurochemical effects of the endocannabinoid uptake inhibitor UCM707 in various rat brain regions

Life Sciences 80 (2007) 979 – 988 www.elsevier.com/locate/lifescie

Neurochemical effects of the endocannabinoid uptake inhibitor UCM707 in various rat brain regions Eva de Lago a , Silvia Ortega-Gutiérrez b , José A. Ramos a , Maria L. López Rodríguez b , Javier Fernández-Ruiz a,⁎ a

Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, 28040-Madrid, Spain b Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Complutense, 28040-Madrid, Spain Received 28 May 2006; accepted 19 November 2006

Abstract To date, UCM707, (5Z,8Z,11Z,14Z)-N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide, has the highest potency and selectivity in vitro and in vivo as inhibitor of the endocannabinoid uptake. Its biochemical, pharmacological and therapeutic properties have been intensely studied recently, but the information on its capability to modify neurotransmitter activity, which obviously underlies the above properties, is still limited. In the present study, we conducted a time-course experiment in rats aimed at examining the neurochemical effects of UCM707 in several brain regions following a subchronic administration (5 injections during 2.5 days) of this inhibitor in a dose of 5 mg/kg weight. In the hypothalamus, the administration of UCM707 did not modify GABA contents but reduced norepinephrine levels at 5 h after administration, followed by an increase at 12 h. Similar trends were observed for dopamine, whereas serotonin content remained elevated at 1 and, in particular, 5 and 12 h after administration. In the case of the basal ganglia, UCM707 reduced GABA content in the substantia nigra but only at longer (5 or 12 h) times after administration. There were no changes in serotonin content, but a marked reduction in its metabolite 5HIAA was recorded in the substantia nigra. The same pattern was found for dopamine, contents of which were not altered by UCM707 in the caudate–putamen, but its major metabolite DOPAC exhibited a marked decrease at 5 h. In the cerebellum, UCM707 reduced GABA, serotonin and norepinephrine content, but only the reduction found for norepinephrine at 5 h reached statistical significance. The administration of UCM707 did not modify the contents of these neurotransmitters in the hippocampus and the frontal cortex. Lastly, in the case of limbic structures, the administration of UCM707 markedly reduced dopamine content in the nucleus accumbens at 5 h, whereas GABA content remained unchanged in this structure and also in the ventral– tegmental area and the amygdala. By contrast, norepinephrine and serotonin content increased at 5 h in the nucleus accumbens, but not in the other two limbic structures. In summary, UCM707 administered subchronically modified the contents of serotonin, GABA, dopamine and/or norepinephrine with a pattern strongly different in each brain region. So, changes in GABA transmission (decrease) were restricted to the substantia nigra, but did not appear in other regions, whereas dopamine transmission was also altered in the caudate–putamen and the nucleus accumbens. By contrast, norepinephrine and serotonin were altered by UCM707 in the hypothalamus, cerebellum (only norepinephrine), and nucleus accumbens, exhibiting biphasic effects in some cases. © 2006 Elsevier Inc. All rights reserved. Keywords: Endocannabinoid; Endocannabinoid uptake inhibition; UCM707; Neurochemical effects; Neurotransmitters

Introduction Within the present interest in the cannabinoid signaling system as a possible target for novel therapy in several neurological disorders, the mechanism of endocannabinoid uptake has attracted special interest (for a recent review see ⁎ Corresponding author. Tel.: +34 91 3941450; fax: +34 91 3941691. E-mail address: [email protected] (J. Fernández-Ruiz). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.11.029

McFarland and Barker, 2004). This mechanism is part of the process of inactivation of endocannabinoid ligands which also includes the participation of at least two degradative enzymes (Giuffrida et al., 2001; Ortega-Gutierrez, 2005). Several synthetic compounds, such as N-(4-hydroxyphenyl)-arachidonamide (AM404; for details, see Khanolkar et al., 1996; Beltramo et al., 1997), N-(4-hydroxy-2-methylphenyl)-arachidonamide (VDM11; for details, see De Petrocellis et al., 2000), or ( S)- N-oleoyl-(1′-hydroxybenzyl)-2′-ethanolamine

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(OMDM2; for details, see Ortar et al., 2003), have been reported to block the endocannabinoid uptake and have been accordingly evaluated for their therapeutic potential. Other related compounds that have recently attracted interest are: (i) the first covalent inhibitors of anandamide cellular uptake, which might be used for the molecular characterization of the protein involved in the membrane transport of endocannabinoids (Moriello et al., 2006); (ii) the heterocyclic urea inhibitor LY2183240, a compound presented first as a transporter inhibitor (Moore et al., 2005), but that has been recently demonstrated to act as an inhibitor of the fatty acid amide hydrolase (FAAH) enzyme (Alexander and Cravatt, 2006); and (iii) the series of URB compounds, such as URB597, that act as FAAH inhibitors without blocking the transporter (Kathuria et al., 2003). All these compounds, termed “indirect agonists”, act by potentiating the action of endogenous ligands and, hence, they might be particularly recommended for diseases in which an increase in cannabinoid signaling has been postulated to be of therapeutic value (Giuffrida et al., 2001; Pertwee, 2002). The use of these compounds may make it possible to minimize the unwanted effects produced by the “direct activation” of cannabinoid CB1 receptors with classical cannabinoids, through the control of endocannabinoid levels in a concentration range that avoids psychoactive side effects (Felder and Glass, 1998; Ortega-Gutierrez, 2005). However, some of these compounds, such as in the case of AM404, may also behave as agonists of vanilloid TRPV1 receptors (Zygmunt et al., 2000) and, then, exhibit direct effects by themselves (González et al., 1999; Beltramo et al., 2000). We have recently designed and synthesized a series of arachidonic acid derivatives (López-Rodríguez et al., 2001, 2003a) that exhibit a high potency and selectivity in vitro as inhibitors of endocannabinoid uptake, with negligible affinity for cannabinoid, CB1 and CB2, and vanilloid TRPV1 receptors, and the FAAH enzyme. One of these compounds, (5Z,8Z,11Z,14Z)-N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide, so-called UCM707, is the most potent and selective endocannabinoid uptake inhibitor described to date (LópezRodríguez et al., 2001, 2003a,b). When used in vivo at low to moderate doses (0.1–1 mg/kg), this compound does not produce any relevant cannabimimetic effects, such as hypomotility and antinociception (de Lago et al., 2002). However, at these doses, it exhibited an interesting capability to enhance the hypokinetic and antinociceptive actions of subeffective doses of anandamide (de Lago et al., 2002). Higher doses of UCM707 (N1 mg/kg) did produce hypokinetic and analgesic effects by themselves (de Lago et al., 2002), possibly due to the increase of the endogenous cannabinoid tone (de Lago et al., 2005). These observations may enable UCM707 to be used in several neurological disorders, where, as mentioned above, an elevation of the endocannabinoid tone has been postulated to have therapeutic benefits. For instance, we have extensively studied the effects of UCM707 in animal models of Huntington's disease and multiple sclerosis, where we found that this compound has potential as a symptomalleviating agent, producing antihyperkinetic and antispastic effects, respectively (de Lago et al., 2006). It may also attenuate the progression of the disease in a rodent model of multiple

sclerosis (Ortega-Gutiérrez et al., 2005). In addition, UCM707 may be also used as an analgesic agent in chronic or inflammatory pain (de Lago et al., 2002; La Rana et al., 2006). As mentioned above, the neurobiological effects of UCM707 or the other endocannabinoid uptake inhibitors in healthy or pathological conditions would be produced by enhancing the action of endocannabinoids at their receptors (de Lago et al., 2002, 2005). Through this mechanism, UCM707 might influence the activity of several classic neurotransmitters, such as GABA, serotonin or dopamine, which are under the modulatory influence of endocannabinoids (Schlicker and Kathmann, 2001). The literature published so far on UCM707 effects has covered: (i) its in vitro inhibitory action on the endocannabinoid uptake (López-Rodríguez et al., 2001, 2003a), (ii) its capability to elevate endocannabinoid levels (de Lago et al., 2005), (iii) its effects, alone or in combination with anandamide, on several behavioural responses (de Lago et al., 2002), and (iv) its potential therapeutic properties in several neurological disorders (Marsicano et al., 2003; Ortega-Gutiérrez et al., 2005; de Lago et al., 2006). However, there are no relevant data on the effects of UCM707 on the activity of those neurotransmitters, such as GABA, norepinephrine, serotonin or dopamine, that underly the behavioural and therapeutic effects of UCM707 in several brain regions. This has been the main objective of the present study. To address these issues, we conducted a time-course experiment in rats, aimed at examining the effects of UCM707 on these four neurotransmitters in several brain regions following a subchronic administration (five injections during 2.5 days) of this inhibitor at a dose of 5 mg/kg weight. The conditions used for UCM707 treatment (dose, timing, route of administration) were selected from a previous study showing an enhancement by UCM707 of endocannabinoid levels (de Lago et al., 2005). In addition, this dose was revealed to produce motor inhibition and analgesia in previous dose-response analyses (de Lago et al., 2002) and to be beneficial in animal models of some motor disorders (de Lago et al., 2006). Subchronic treatment was chosed because it was expected to have more marked neurochemical effects than those found after a single administration of the same dose in a preliminary study centered in the basal ganglia (de Lago et al., 2006). In the present study, we wanted to also extend the analysis to other brain areas, selecting in each region those neurotransmitters that have been reported to be most affected by the treatment with different cannabinoid compounds. Thus, norepinephrine, serotonin, GABA and dopamine were investigated in the hypothalamus since they have been related to the effects of cannabinoids on several hypothalamic functions, such as regulation of feeding behaviour (Thiébot et al., 2006) and control of anterior pituitary hormone secretion (Fernández-Ruiz et al., 2006). The same neurotransmitters were also investigated in the frontal cortex and several limbic structures because they were involved in the mediation of some cognitive, emotional and motivational effects of cannabinoids (Thiébot et al., 2006). Serotonin, GABA and dopamine were investigated in the basal ganglia (Thiébot et al., 2006; Fernández-Ruiz et al., 2006), whereas GABA, serotonin and norepinephrine were analyzed in relation with the effects of

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Fig. 1. Contents of GABA, norepinephrine (NE), dopamine (DA) and serotonin (5HT) in the hypothalamus of rats subchronically administered (5 injections during 2.5 days) with 5 mg/kg of UCM707 (■) or vehicle (▯) and killed at different times after the last injection. Details in the text. Values are means ± SEM of 5 determinations per group. Data were assessed by two-way (treatment × time) analysis of variance followed by the Student–Newman–Keuls test (⁎p b 0.05 versus the corresponding vehicle; #p b 0.05 versus the other times for the same treatment).

cannabinoids in the cerebellum (Hoffman and Lupica, 2006). These last three neurotransmitters were investigated in the hippocampus because they were involved in memory effects of cannabinoids (Thiébot et al., 2006; Hoffman and Lupica, 2006). Methods Animals, treatments and sampling Male Wistar rats were housed in a room with controlled photoperiod (08:00-20:00 light) and temperature (22 ± 1 °C). They had free access to standard food and water. Animals were used at adult age (N 8 weeks old; 250–300 g weight) for all the experiments, which were performed according to European regulations for experimental work with animals (directive 86/609/ EEC). Rats were administered intraperitoneally with UCM707, synthesized as previously described (López-Rodríguez et al., 2001, 2003a), at a dose of 5 mg/kg. The treatment included 5 injections done every 12 h over a total period of 48 h, following the procedure described in a previous report (de Lago et al., 2005). Rats injected with vehicle (Tween 80-saline solution) were used as controls. The animals were sacrificed after 1, 5 and 12 h from the last administration and their brains were immediately removed and rapidly frozen by immersion in 2-methyl-butane cooled in dry ice. All samples were stored at −80 °C until processed for analysis of concentrations of different neurotransmitters. Determinations of GABA, serotonin, norepinephrine and dopamine contents by HPLC with electrochemical detection Dissection procedure Brains were used to manually obtain coronal slices (around 500 μm thick) at levels containing the different structures to be

analyzed: frontal cortex, caudate–putamen, globus pallidus, substantia nigra, nucleus accumbens, ventral–tegmental area, amygdala, hippocampus, hypothalamus and cerebellum (Palkovits and Brownstein, 1988). Subsequently, the structures were dissected and homogenized in 20–40 vol of cold 150 mM potassium phosphate buffer, pH 6.8, and each homogenate was distributed for the analysis of: (i) GABA contents, (ii) serotonin and its metabolite 5-hydroxyindolacetic acid (5HIAA), and (iii) norepinephrine, dopamine and its metabolite L-3,4-dihydroxyphenylacetic acid (DOPAC), as will be described below. An aliquot of each homogenate was used to analyze protein concentration (Lowry et al., 1951). Analysis of GABA content. GABA content was analyzed by HPLC with electrochemical detection according to Smith and Sharp (1994). Homogenates were diluted with 0.4 N perchloric acid containing 0.4 mM sodium disulfite, 0.90 mM EDTA and 10 μg/ml β-aminobutyrate (BABA) as internal standard. Afterwards, samples were centrifuged for 3 min (15000 g) and 50 μl of each supernatant was removed and neutralized with 100 μl of 0.1 N NaOH. Samples were stored at 4 °C until analysis. This was performed by derivatization of GABA and BABA through the addition of 15 μl of o-phthaldehyde (OPA)-sulfite solution (14.9 mM OPA, 45.4 mM sodium sulfite and 4.5% ethanol in 327 mM borate buffer, pH 10.4). Samples were allowed to react at room temperature for a period of 10 min. After this time, 20 μl of each reaction mixture (including derivatized calibration standards composed of known concentrations of GABA and BABA) were injected into the HPLC system. This consisted of the following elements: the pump was an isocratic Spectra-Physics 8810; the column was a RP-18 (Tracer Excel 120 ODSB; 150 mm, 4.6 mm, 5 μm particle size; Teknokroma, Barcelona, Spain). The mobile phase, previously filtered and degassed, consisted of 0.06 M sodium dihydrogen phosphate, 0.06 mM

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Fig. 2. Contents of GABA, dopamine (DA) and serotonin (5HT) in the basal ganglia of rats subchronically administered (5 injections during 2.5 days) with 5 mg/kg of UCM707 (■) or vehicle (▯) and killed at different times after the last injection. Details in the text. Values are means ± SEM of 5 determinations per group. Data were assessed by two-way (treatment × time) analysis of variance followed by the Student–Newman–Keuls test (⁎p b 0.05 versus the corresponding vehicle; #p b 0.05 versus the other times for the same treatment).

EDTA and 10% methanol (pH 4.4) and the flow rate was 0.8 ml/ min. The effluent was monitored with a Metrohm bioanalytical system amperometric detector using a glassy carbon electrode. The potential was 0.85 V relative to an Ag/AgCl reference electrode with a sensitivity of 50 nA (approximately 2 ng/sample). The signal was recorded on a Spectra-Physics 4290 integrator. The results were obtained from the peaks and calculated by comparison with the area under the corresponding internal standard peak. Values are expressed as μg/mg of protein. Analysis of norepinephrine, dopamine and DOPAC content The contents of norepinephrine, dopamine and DOPAC were analyzed using HPLC with electrochemical detection according to our previously published method (Romero et al., 1995; González et al., 1999). Briefly, homogenates were diluted in icecold 0.4 N perchloric acid containing 0.4 mM sodium disulfite and 0.90 mM EDTA. Dihydroxybenzylamine was added as an internal standard. The diluted homogenates were then centrifuged and the supernatants injected into the HPLC system,

which consisted of a Spectra-Physics 8810 isocratic pump. The column was a RP-18 (Spherisorb ODS-2; 125 mm, 4.6 mm, 5 μm particle size; Waters, Massachusetts, USA). The mobile phase consisted of 100 mM citric acid, 100 mM sodium acetate, 1.2 mM heptane sulphonate, 1 mM EDTA and 7% methanol (pH 3.9) and the flow rate was 0.8 ml/min. The effluent was monitored with a coulochemical detector (Coulochem II, ESA) using a procedure of oxidation/reduction (conditioning cell: + 360 mV; analytical cell #1: +50 mV; analytical cell #2: − 340 mV). The signal was recorded from the analytical cell #2, with a sensitivity of 50 nA (10 pg/sample), on a Spectra-Physics 4290 integrator and the results are given as area under the peaks. Values are expressed as ng/mg of protein. Analysis of serotonin and 5HIAA content The contents of serotonin and its metabolite were analyzed using HPLC with electrochemical detection according to our previously published method (Sagredo et al., 2006). Briefly, homogenates were diluted in ice-cold 0.4 N perchloric acid

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Results Neurochemical effects of UCM707 in the hypothalamus UCM707 reduced dopamine content in the hypothalamus at 1 and 5 hours after administration, followed by an increase at 12 h (Fig. 1), but these effects were only trends and did not reach statistical significance [2-way interaction: F(2,20) = 2.45, p = 0.111]. A similar pattern was followed by norepinephrine (Fig. 1), although, in this case, the effects did reach statistical significance [2-way interaction: F(2,22) = 5.71, p b 0.05]. By contrast, serotonin content remained elevated at 1 and, in particular, 5 and 12 h after administration [treatment: F(1,18) = 10.90, p b 0.05], but GABA content was not affected (Fig. 1). It is also important to note that, in the hypothalamus, GABA [time: F(2,22) = 4.80, p b 0.05], dopamine [time: F(2,20) = 2.07, p = 0.152] and norepinephrine [time: F(2,22) = 3.86, p b 0.05], but not serotonin contents, exhibited time-dependent fluctuations, mainly in vehicle-treated animals, with increased concentrations usually recorded at 5 h (Fig. 1).

Fig. 3. Contents of GABA, norepinephrine (NE) and serotonin (5HT) in the cerebellum of rats subchronically administered (5 injections during 2.5 days) with 5 mg/kg of UCM707 (■) or vehicle (▯) and killed at different times after the last injection. Details in the text. Values are means ± SEM of 5 determinations per group. Data were assessed by two-way (treatment × time) analysis of variance followed by the Student–Newman–Keuls test (⁎p b 0.05 versus the corresponding vehicle; #p b 0.05 versus the other times for the same treatment).

containing 0.4 mM sodium disulfite and 0.90 mM EDTA. Nmethyl-serotonin was added as an internal standard. The diluted homogenates were then centrifuged and the supernatants injected into the HPLC system. This consisted of a SpectraPhysics 8810 isocratic pump and a RP-18 column (Spherisorb ODS-2; 125 mm, 4.6 mm, 5 μm particle size; Waters, Massachusetts, USA). The mobile phase consisted of 100 mM citric acid, 100 mM sodium acetate, 1 mM EDTA and 7% methanol (pH 4.2) and the flow rate was 0.8 ml/min. The effluent was monitored with a Metrohm bioanalytical system amperometric detector using a glassy carbon electrode. The potential was 0.80 V relative to an Ag/AgCl reference electrode with a sensitivity of 50 nA (approximately 2 ng/sample). The signal was recorded on a Spectra-Physics 4290 integrator. Results are given as area under the peaks and calculated by comparison with the area under the corresponding internal standard peak. Values are expressed as ng/mg of protein. Statistics Data were assessed by two-way analysis of variance followed by the Student–Newman–Keuls test.

Fig. 4. Contents of GABA, norepinephrine (NE) and serotonin (5HT) in the hippocampus of rats subchronically administered (5 injections during 2.5 days) with 5 mg/kg of UCM707 (■) or vehicle (▯) and killed at different times after the last injection. Details in the text. Values are means ± SEM of 5 determinations per group. Data were assessed by two-way (treatment × time) analysis of variance followed by the Student–Newman–Keuls test (⁎p b 0.05 versus the corresponding vehicle; #p b 0.05 versus the other times for the same treatment).

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Neurochemical effects of UCM707 in the basal ganglia The administration of UCM707 reduced GABA content in some basal ganglia structures, but the effects reached statistical significance only in the substantia nigra and only at longer (5 or 12 h) times [Fig. 2; treatment: F(1,23) = 7.34, p b 0.05; 2-way interaction: F(2,23) = 3.90, p b 0.05]. The effects were not significant in the caudate–putamen and remained only as a trend in the globus pallidus [Fig. 2; 2-way interaction: F(2,21) = 2.27, p = 0.129]. The administration of UCM707 did not modify serotonin content in the basal ganglia, but a marked reduction in its metabolite 5HIAA was recorded in the substantia nigra at all times tested [Fig. 2; treatment: F(1,20) = 11.50, p b 0.005]. This reduction led to a parallel reduction in the 5HIAA/5HT ratio, which is an index of neurotransmitter turnover. The reduction was particularly significant at 1 h after the administration of UCM707 (vehicle: 1.04 ± 0.31; UCM707: 0.39 ± 0.14, p b 0.005). This was not observed in other basal ganglia structures (data not shown). In a similar way, dopamine content was not altered by the administration of UCM707 in the caudate–putamen but the content of its major metabolite DOPAC decreased albeit significantly only at 5 h [Fig. 2; 2way interaction: F(2,21) = 5.54, p b 0.05]. However, in this case, the changes in DOPAC contents did not represent a change in the DOPAC/DA ratio (data not shown), thus indicating that the administration of UCM707 affected exclusively DA metabolism without altering DA turnover. It is important to note that, also in the case of the basal ganglia, there were some time-dependent fluctuations, observed mainly in vehicle-treated animals, that showed an increased concentration usually recorded at 5 h (Fig. 2): (i) for GABA [time: F(2,23) = 7.26, p b 0.005], serotonin [time: F(2,22) = 7.10, p b 0.005] and 5HIAA [F(2,20) = 10.48, p b 0.0005] in the substantia nigra (Fig. 2), and (ii) for dopamine

[time: F(2,22) = 5.85, p b 0.01], DOPAC [time: F(2,21) = 8.28, p b 0.005] and serotonin [time: F(2,23) = 7.15, p b 0.005] in the caudate–putamen (Fig. 2). Neurochemical effects of UCM707 in the cerebellum GABA and norepinephrine contents were reduced in the cerebellum after the administration of UCM707 (Fig. 3), but only the reduction found for norepinephrine at 5 h after the administration reached statistical significance [2-way interaction: F(2,19) = 4.21, p b 0.05], and the reduction in GABA (treatment: F(1,23) = 3.17, p = 0.088] was merely a trend. No changes were observed in serotonin content, despite the apparent trend towards a decrease found at 12 h (Fig. 3). There were also some time-dependent fluctuations in this region in vehicle-treated rats (Fig. 3) with increased content of norepinephrine at 5 h [time: F(2,19) = 4.07, p b 0.05] and of serotonin at 12 h [time: F(2,21) = 5.29, p b 0.05]. Neurochemical effects of UCM707 in the hippocampus and the frontal cortex There were no changes in the content of GABA, norepinephrine and serotonin in the hippocampus after the administration of UCM707 (Fig. 4) despite a certain trend towards a decrease observed for GABA content at longer times (12 h) [treatment: F(1,24) = 3.03, p = 0.095]. In a similar way, there were no changes in the content of these neurotransmitters or of dopamine in the frontal cortex after the administration of UCM707 (Fig. 5). As it happens with the above regions, there were also some time-dependent fluctuations in both structures in vehicle-treated rats (Figs. 4 and 5) with the highest values found at 5 h for norepinephrine in the hippocampus

Fig. 5. Contents of GABA, norepinephrine (NE), dopamine (DA) and serotonin (5HT) in the frontal cortex of rats subchronically administered (5 injections during 2.5 days) with 5 mg/kg of UCM707 (■) or vehicle (▯) and killed at different times after the last injection. Details in the text. Values are means ± SEM of 5 determinations per group. Data were assessed by two-way (treatment × time) analysis of variance followed by the Student–Newman–Keuls test (⁎p b 0.05 versus the corresponding vehicle; #p b 0.05 versus the other times for the same treatment).

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Fig. 6. Contents of GABA, norepinephrine (NE), dopamine (DA) and serotonin (5HT) in several limbic structures of rats subchronically administered (5 injections during 2.5 days) with 5 mg/kg of UCM707 (■) or vehicle (▯) and killed at different times after the last injection. Details in the text. Values are means ± SEM of 5 determinations per group. Data were assessed by two-way (treatment × time) analysis of variance followed by the Student–Newman–Keuls test (⁎p b 0.05 versus the corresponding vehicle; #p b 0.05 versus the other times for the same treatment).

[time: F(2,22) = 7.14, p b 0.005] and the frontal cortex [time: F (2,22) = 6.66, p b 0.01], and at 5 and 12 h for GABA [time: F (2,23) = 3.04, p = 0.067] and serotonin [time: F(2,21) = 3.64, p b 0.05] only in the frontal cortex. Neurochemical effects of UCM707 in limbic structures UCM707 caused a marked reduction in dopamine content in the nucleus accumbens at 5 hours [see Fig. 6; treatment: F(1,22) = 5.64, p b 0.05; 2-way interaction: F(2,22) = 7.20, p b 0.005], which corresponded with a marked increase in DOPAC/DA ratio (vehicle: 0.15 ± 0.02; UCM707: 0.23 ± 0.02; p b 0.05). By contrast, GABA content remained unchanged in this structure and also in the ventral-tegmental area and the amygdala (Fig. 6). In addition, the contents of norepinephrine [treatment: F(1,22) = 7.62, p b 0.05; 2-way interaction: F(2,22) = 3.09, p = 0.066] and serotonin [treatment: F(1,22) = 4.99, p b 0.05] increased by UCM707 in the nucleus accumbens also at 5 hours after administration, but

not in the other two limbic structures. The increase found in serotonin content at 5 hours was not, however, accompanied by changes in 5HIAA/5HT ratio (data not shown), thus indicating that it mainly reflects an increase in serotonin synthesis rather than changes in turnover. Here, we also found time-dependent fluctuations, in both vehicle-and UCM707-treated rats, and again the maximal values were found at 5 hours (Fig. 6). This was the case for GABA [time: F(2,24) = 10.99, p b 0.0005], norepinephrine [time: F(2,22) = 8,85, p b 0.005], dopamine [time: F(2,22)=27.24, p b 0.0005] and serotonin [time: F(2,22) = 7.34, p b 0.005] in the nucleus accumbens, and for serotonin [time: F (2,19) = 3.83, p b 0.05] in the ventral-tegmental area. This was not seen in the amygdala for GABA, norepinephrine or serotonin (Fig. 6). Discussion As mentioned in the Introduction, the neurobiological effects of UCM707 or other endocannabinoid uptake inhibitors in

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healthy and, in particular, in pathological conditions would be produced by enhancing the action of endocannabinoids at their receptors, as we have recently shown (de Lago et al., 2002, 2005). Through this action, UCM707 might be able to modify the activity of GABA, serotonin, norepinephrine or dopamine, since these and other neurotransmitters are subject to a modulatory action exerted by endocannabinoids (Schlicker and Kathmann, 2001). Therefore, the evaluation of the neurochemical effects of UCM707 has been the main objective of the present study. Collectively, our data may confirm that UCM707 administered subchronically modified the content of serotonin, GABA, dopamine and/or norepinephrine with a pattern that exhibited variations: (i) in each brain region, (ii) for each neurotransmitter, and (iii) with the amount of time that lapsed between the last administration and when the effects were observed, with the time of 5 hours as the most critical for UCM707 effects. These differences might be certainly related to the particular pharmacokinetic properties of UCM707 due to its lipophilic nature, which would result in different bioavailability between the brain and the periphery, and also within the brain. In addition, the fact that endocannabinoid contents present a particular regional distribution may be another key factor that might explain the differences in neurochemical effects of UCM707. On the other hand, one additional aspect to consider is that tissue contents of neurotransmitters do not exactly reflect what is happening at the extracellular synaptic level, so these findings are going to be complemented with additional studies using more dynamic methods to analyse neurotransmitter release in the regions found here to be most affected by the treatment with UCM707. Meanwhile, the ratio between the main metabolite and the corresponding amine may be used as an index to determine whether the potential effects of UCM707 are due to changes in neurotransmitter turnover or merely reflect changes in synthesis or metabolism. With this idea in mind, we have calculated this parameter for serotonin and dopamine in some regions where UCM707 was able to alter neurotransmitter contents. The administration of UCM707 decreased GABA contents, although this effect was restricted only to the substantia nigra and did not appear in other regions. A reduction in GABA content may be interpreted as the consequence of a reduction in GABA synthesis, but it can also reflect a reduction in GABA reuptake, an effect that previous studies have demonstrated that the direct activation of CB1 receptors may produce in the substantia nigra (Romero et al., 1998) and also in the globus pallidus (Maneuf et al., 1996). If this were the case, UCM707, by reducing GABA reuptake in striatal projection neurons that arise in the substantia nigra, the so-called striatonigral pathway, would increase GABA action at their receptors, mainly GABAB receptors (Romero et al., 1996), thus producing a reduction in motor behaviour (see Fernández-Ruiz and González, 2005, for review). In fact, such an effect of UCM707 has been reported (de Lago et al., 2002). This can be also concluded from the observation that UCM707 reduced dopamine metabolism, reflected by a reduction in DOPAC content, in the caudate– putamen, with no effects on dopamine turnover (DOPAC/DA ratio). We have recently found that certain cannabinoids, including anandamide, may directly act on nigrostriatal

dopaminergic neurons by reducing dopamine synthesis and release through an action that would be mediated by the activation of TRPV1 receptors (de Lago et al., 2004). So, it is possible that UCM707, by enhancing the anandamide tone, may facilitate the action of this endocannabinoid at these vanilloid receptors, thus resulting in a reduction of dopamine transmission in the caudate–putamen that would be also compatible with the hypokinetic profile of all cannabimimetic compounds, including UCM707. We also recorded a decrease in serotonin turnover by UCM707 in the basal ganglia reflected by the reduction in 5HIAA levels and in 5HIAA/5HT ratio. This was observed in the substantia nigra and, again, may be associated with a reduction in motor behaviour since serotoninergic terminals coming from the raphe nuclei have been involved in the basal ganglia circuitry with a stimulatory effect on movement (see Nicholson and Brotchie, 2002; Scholtissen et al., 2006, for review), even though the action of serotonin through a large spectrum of possible serotoninergic receptor subtypes allows different types of modulation for a specific process. Norepinephrine is not significantly present in the basal ganglia, but it can be related to the control of movement through its function in the cerebellum, another brain region related to motor control, in particular, to motor coordination. We did not find any changes in GABA and serotonin in the cerebellum, but recorded a reduction in norepinephrine contents that, based on previous evidence (Woodward et al., 1991; Bickford et al., 1999) may be associated with the modulation of several cerebellar functions, including a reduction of motor coordination, an effect that cannabimimetic compounds are able to produce (see Fernández-Ruiz and González, 2005, for review). In addition to the effects found on dopamine transmission in the caudate–putamen, UCM707 also altered this transmission (reduction in dopamine contents but increase in DOPAC/DA ratio) in the nucleus accumbens, a key limbic region involved in the regulation of motivational behaviours and other processes relevant to psychiatry such as anxiety, affect, aggression and drug abuse (for review, see Morgane et al., 2005). This effect was observed at 5 h after the administration of the inhibitor and was paralleled by an increase in norepinephrine and serotonin content, with no changes in serotonin turnover. Other limbic structures, such as the ventral–tegmental area and the amygdala, were not, however, affected by UCM707 treatment. Changes in dopamine, norepinephrine and/or serotonin transmission in limbic structures, relatively similar to those found here after the treatment with UCM707, have been associated with a variety of changes in several limbic-related behaviors (Blum et al., 2000; Comings and Blum, 2000; Gingrich and Hen, 2001; Swanson and Schoepp, 2003). Interestingly, the same types of behavioural responses have been also observed after treatment with different cannabimimetic compounds (see van der Stelt and Di Marzo, 2003, for review), despite the existence of some differences between acute and chronic treatment, among different ranges of doses, and for specific cannabinoid agonists with different potencies (see van der Stelt and Di Marzo, 2003, for review). Therefore, it can be hypothesized that these three neurotransmitters would be key substrates for the limbic effects of UCM707 and for other

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cannabimimetic agents. By contrast, neither dopamine nor the other three neurotransmitters were affected by UCM707 in another important region related to the control of the above processes such as the frontal cortex, and the same is true for the hippocampus. This possibly indicates that other neurotransmitters such as glutamate (which was not analyzed) might be the key neurochemical target for the effects of UCM707 on cognitive processes such as memory and learning. Norepinephrine and serotonin were altered by UCM707 in the hypothalamus, sometimes even exhibiting biphasic effects as in the case of norepinephrine. Other hypothalamic neurotransmitters, such as GABA and dopamine, were, however, not affected by UCM707. Both norepinephrine (whose terminals arise the hypothalamus from the reticular formation of the midbrain and the brainstem), and serotonin (whose terminals come from the raphe nuclei) have been related to several neurobiological functions whose control resides in the hypothalamus. We can mention the control of the release of hypothalamic hypophysiotrophic hormones (Etgen et al., 2001) and processes related to appetite and feeding (Ramos et al., 2005). Cannabimimetic compounds influence anterior pituitary hormone secretion, through effects exerted mainly at the hypothalamic level (see Murphy et al., 1998, for review), and are also orexigenic agents, through effects exerted on several mediators mainly located in the anterior hypothalamus (see Osei-Hyiaman et al., 2006, for review). In both cases, both serotonin and norepinephrine seem to be involved (Arévalo et al., 2001; Tzavara et al., 2001; Avraham et al., 2004), so we can hypothesize that these two transmitters may be the key targets for hypothalamic effects of UCM707. Conclusion In summary, UCM707 administered subchronically modified the contents of serotonin, GABA, dopamine and/or norepinephrine with a pattern strongly different in each brain region and also depending on the time after the last administration at which the effects were observed. So, changes in GABA transmission (decrease) were restricted to the substantia nigra, and did not appear in other regions, whereas dopamine transmission was also altered in the caudate–putamen and the nucleus accumbens. By contrast, norepinephrine and serotonin were altered by UCM707 in the hypothalamus, cerebellum (only norepinephrine), and nucleus accumbens, exhibiting biphasic effects in some cases. Acknowledgements The present work was supported by grants from MEC (SAF2003-08269 and SAF2004-07103-C02-01) and from “Agencia Antidroga de la Comunidad de Madrid”. Authors are indebted to Ana Jurado for her technical support. References Alexander, J.P., Cravatt, B.F., 2006. The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain

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