TrkB (Kd = 10 nM)

ANA-12 (0.5 mg/kg, i.p.) reduces anxiety and depression-related behaviors in adult C57BL6/129SveV F1s mice without compromising neuron survival. It also lowers TrKB activity in the brain.[1] Analogous amideamide-12 (0.5 mg/kg, i.p.) reverses the depressive-like effects of lipopolysaccharide-induced behavior in male C57BL/6 mice.[3] The effect of medial nucleus tractus solitarius (mNTS) BDNF on decreasing food intake is blocked in male Sprague-Dawley rats by ANA-12 (3 μg/dose).[4] ANA-12 reverses ethanol intake and causes D3 receptor downregulation in male wild-type mice, but it has no effect on D₃R^-/-mice.[5] The reduced self-administration of cocaine in male CocSired rats is reversed by ANA-12 (0.5 mg/kg, i.p.).[6]

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NMDA receptors in primary afferent terminals can contribute to hyperalgesia by increasing neurotransmitter release. In rats and mice, we found that the ability of intrathecal NMDA to induce neurokinin 1 receptor (NK1R) internalization (a measure of substance P release) required a previous injection of BDNF. Selective knock-down of NMDA receptors in primary afferents decreased NMDA-induced NK1R internalization, confirming the presynaptic location of these receptors. The effect of BDNF was mediated by tropomyosin-related kinase B (trkB) receptors and not p75 neurotrophin receptors (p75(NTR) ), because it was not produced by proBDNF and was inhibited by the trkB antagonist ANA-12 but not by the p75(NTR) inhibitor TAT-Pep5. These effects are probably mediated through the truncated form of the trkB receptor as there is little expression of full-length trkB in dorsal root ganglion (DRG) neurons. Src family kinase inhibitors blocked the effect of BDNF, suggesting that trkB receptors promote the activation of these NMDA receptors by Src family kinase phosphorylation. Western blots of cultured DRG neurons revealed that BDNF increased Tyr(1472) phosphorylation of the NR2B subunit of the NMDA receptor, known to have a potentiating effect. Patch-clamp recordings showed that BDNF, but not proBDNF, increased NMDA receptor currents in cultured DRG neurons. NMDA-induced NK1R internalization was also enabled in a neuropathic pain model or by activating dorsal horn microglia with lipopolysaccharide. These effects were decreased by a BDNF scavenger, a trkB receptor antagonist and a Src family kinase inhibitor, indicating that BDNF released by microglia potentiates NMDA receptors in primary afferents during neuropathic pain. [1]

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LPS caused a reduction of BDNF in the CA3 and dentate gyrus (DG) of the hippocampus and prefrontal cortex (PFC), whereas LPS increased BDNF in the nucleus accumbens (NAc). Dexamethason suppression tests showed hyperactivity of the hypothalamic-pituitary-adrenal axis in LPS-treated mice. Intraperitoneal (i.p.) administration of 7,8-DHF showed antidepressant effects on LPS-induced depression-like behavior, and i.p. pretreatment with ANA-12 blocked its antidepressant effects. Surprisingly, ANA-12 alone showed antidepressant-like effects on LPS-induced depression-like behavior. Furthermore, bilateral infusion of ANA-12 into the NAc showed antidepressant effects. Moreover, LPS caused a reduction of spine density in the CA3, DG, and PFC, whereas LPS increased spine density in the NAc. Interestingly, 7,8-DHF significantly attenuated LPS-induced reduction of p-TrkB and spine densities in the CA3, DG, and PFC, whereas ANA-12 significantly attenuated LPS-induced increases of p-TrkB and spine density in the NAc. Conclusions: The results suggest that LPS-induced inflammation may cause depression-like behavior by altering BDNF and spine density in the CA3, DG, PFC, and NAc, which may be involved in the antidepressant effects of 7,8-DHF and ANA-12, respectively. [2]

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To localize the neurons mediating the energy balance effects of hindbrain ventricle-delivered BDNF, ventricle subthreshold doses were delivered directly to medial nucleus tractus solitarius (mNTS). mNTS BDNF administration reduced food intake significantly, and this effect was blocked by preadministration of a highly selective TrkB receptor antagonist {[N2-2-2-Oxoazepan-3-yl amino]carbonyl phenyl benzo (b)thiophene-2-carboxamide (ANA-12)}, suggesting that TrkB receptor activation mediates hindbrain BDNF's effect on food intake. Because both BDNF and leptin interact with melanocortin signaling to reduce food intake, we also examined whether the intake inhibitory effects of hindbrain leptin involve hindbrain-specific BDNF/TrkB activation. BDNF protein content within the dorsal vagal complex of the hindbrain was increased significantly by hindbrain leptin delivery. To assess if BDNF/TrkB receptor signaling acts downstream of leptin signaling in the control of energy balance, leptin and ANA-12 were coadministered into the mNTS. Administration of the TrkB receptor antagonist attenuated the intake-suppressive effects of leptin, suggesting that mNTS TrkB receptor activation contributes to the mediation of the anorexigenic effects of hindbrain leptin. Collectively, these results indicate that TrkB-mediated signaling in the mNTS negatively regulates food intake and, in part, the intake inhibitory effects of leptin administered into the NTS. [3]

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Indeed, blockade of the BDNF pathway by the TrkB selective antagonist ANA-12 reversed chronic stable ethanol intake and strongly decreased the striatal expression of D3R. Finally, we evaluated buspirone, an approved drug for anxiety disorders endowed with D3R antagonist activity (confirmed by molecular modeling analysis), that resulted effective in inhibiting ethanol intake. Thus, DA signaling via D3R is essential for ethanol-related reward and consumption and may represent a novel therapeutic target for weaning. [4]

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Administration of a BDNF receptor antagonist (the TrkB receptor antagonist ANA-12) reversed the diminished cocaine self-administration in male cocaine-sired rats. In addition, the association of acetylated histone H3 with Bdnf promoters was increased in the sperm of sires that self-administered cocaine. Collectively, these findings indicate that voluntary paternal ingestion of cocaine results in epigenetic reprogramming of the germline, having profound effects on mPFC gene expression and resistance to cocaine reinforcement in male offspring. [5]

Various concentrations of Trk ^BECD -Fc, 20 mg/ml BSA, or 1 mg/mL IgG-Fc (polyclonal anti-TrkB) are coated on Maxisorp ELISA 96-well plates and left overnight at 4°C in a carbonate buffer with a pH of 9.6. After two hours of room temperature solubility in 0.5% BSA in PBS, plates are thoroughly cleaned in PBS-Tween 0.05%. Following an hour of room temperature incubation in 0.5% PBS-BSA, BDNF is added to the solution and incubated for an additional hour. This process is repeated with Bodipy-ANA-12. The amount of bodipy-ANA-12 bound is measured by fluorescence at 520 ± 10 nm following thorough washes in PBS-Tween 0.05%. To determine the detectability range for extrapolation analysis, ELISA plates are coated with bodipy-ANA-12, and the fluorescence at 520 ± 10 nm is measured.

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ANA-12 binding assays [1]
Maxisorp ELISA 96-well plates were coated with various concentrations of TrkBECD-Fc (as indicated in the figures), 20 mg/ml BSA, or 1 mg/ml IgG-Fc (polyclonal anti-TrkB;) in a carbonate buffer (pH 9.6) overnight at 4°C. Plates were saturated with 0.5% BSA in PBS for 2 hours at room temperature and extensively washed in PBS-Tween 0.05%. Bodipy–ANA-12 was then incubated in 0.5% PBS-BSA for 1 hour at room temperature before the addition of BDNF in 0.5% PBS-BSA for another hour, as indicated in the figures. After extensive washes in PBS-Tween 0.05%, the amount of bodipy–ANA-12 bound was quantified by fluorescence at 520 ± 10 nm. Detectability range for extrapolation analysis was assessed by coating ELISA plates with bodipy–ANA-12 and reading fluorescence at 520 ± 10 nm.

In nnr5 PC12-TrkB, -TrkA, and -TrkC cells, the effects of molecules on neurite outgrowth are evaluated following the addition of BDNF (1 nM), NGF (2 nM), and NT-3 (10 nM), respectively. A microscope counts the number of cells in each counting field—three wells per condition, two fields per well—that have neurites longer than two cells in diameter. For three days, the counting is done in the dark every 24 hours.

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KIRA-ELISA [1]
TrkB receptor autophosphorylation was quantified using a modified version of KIRA-ELISA, as described earlier. TetOn-rhTrkB cells were seeded on flat-bottom 96-well culture plates (4 × 104 cells per well) and were incubated overnight with 1000 ng/ml doxycycline to induce the expression of TrkB receptors. Cortical neurons were seeded on polyornithin-coated flat-bottom 96-well culture plates (12 × 104 cells per well) and cultured 7 to 8 days at 37°C in 5% CO2. Fluorescence levels in TetOn-rhTrkB cells were verified before each assay. Cells were carefully washed 4 times with DMEM before being treated with compounds for 20 minutes and stimulated for 20 minutes with BDNF (recombinant cells, 4 nM; neurons, 0.4 nM) in DMEM containing 0.5% BSA and 25 mM HEPES (control medium) at 37°C in 5% CO2. Assay was stopped by removing the medium on ice, and membranes were solubilized by adding a solubilization buffer (150 mM NaCl, 50 mM Hepes, 0.5% Triton X-100, 0.01% thimerosal, 2 mM sodium orthovanadate, supplemented with a cocktail of protease inhibitor) for 1 hour at room temperature. Lysates were transferred to an ELISA microtiter plate precoated with either anti-GFP (1:5000) for rhTrkB or anti-TrkB (1 μg/ml) for neuronal TrkB. Phosphorylation was revealed by incubation with biotinylated anti-phosphotyrosine (0.5 μg/ml) and HRP-conjugated streptavidin (1:4000). After addition of TMB and acidification with 1 N hydrochloric acid, absorbance was read at 450 nm. Total TrkB was also quantified using KIRA-ELISA using either a monoclonal anti-GFP (1:3000) for rhTrkB or a monoclonal anti-TrkB (1:1000) for neuronal TrkB to precipitate the receptor and either a polyclonal anti-GFP (1:5000) for rhTrkB or a polyclonal anti-TrkB (1 μg/ml) for neuronal TrkB to detect the receptor. In cell cultures, the total TrkB signal was found to be unchanged in all treatment conditions. In brain tissues, the amount of total TrkB was variable and was used to normalize the signal obtained for phospho-TrkB.

In a vehicle of 17% dimethyl sulfoxide (DMSO) in phosphate-buffered saline, ketamine (ketamine hydrochloride, 10 mg/kg), 7,8-dihydroxyflavone (7,8-DHF; 10 mg/kg), and ANA-12, N2-(2-{[(2-oxoazepan-3-yl) amino]carbonyl}phenyl)benzo[b]thiophene-2-carboxamide (0.5 mg/kg) are prepared on the day of injection. Ketamine (10 mg/kg), 7,8-DHF (10 mg/kg), and ANA-12 (0.5 mg/kg) are the doses that have been chosen. Mice receive intraperitoneal (i.p.) administration of all compounds.

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Administration of ANA-12 and in vivo KIRA-ELISA analysis [1]
F1 hybrids obtained by crossing C57BL/6 and 129SveV mice were used following the recommendation of the Banbury Conference on genetic background for studying mouse behavior. Adult C57BL6/129SveV F1s (3 months old) were randomly distributed into saline (1% DMSO dissolved in 0.9% NaCl solution) and ANA-12 (dissolved in saline solution) groups. A volume of 10 μl/g body weight was injected i.p. for saline and ANA-12 (0.5 mg/kg body weight) solutions. After 2 or 4 hours, mice were decapitated and brains were rapidly removed on ice. Striatum, cortex, and hippocampus were subsequently dissected when needed. Tissues were rapidly washed in ice-cold PBS, transferred into ice-cold KIRA-ELISA solubilization buffer, and left overnight at 4°C. Protein concentrations were determined, equal amounts of proteins were loaded, and KIRA-ELISA assays were performed as described above.

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Analysis of ANA-12 stability and bioavailability in mouse brains [1]
Analysis of stability in mouse serum and quantification in mouse brain were performed by TechMedILL facilities (École Supérieure de Biotechnologie de Strasbourg, Illkirch, France). Stability was assessed by incubating ANA-12 in mouse serum for 15, 30, 45, and 60 minutes at 37°C. Mixtures were homogenized and proteins precipitated with acetonitrile before being analyzed by liquid chromatography–mass spectrometry (Agilent LC-MS ESI qTOF connected to a C18-1 × 10 × 1.9 column). Bioavailability in mouse brain was assessed by injecting mice i.p. with ANA-12 (0.5 mg/kg) as described above. After 30 minutes, 1, 2, 4, and 6 hours (3 mice/time point), brains were removed and mashed in NaCl 0.9% before treatment with acetonitrile. The mixture was cleared by ultracentrifugation, and the compound was extracted using a series of dehydrations and resuspensions in acetonitrile/water (1/1 v/v) before being subjected to LC-MS for detection and analysis. Reference samples were prepared by adding known amounts of compound to blank mixtures (brains from saline-treated mice). Concentrations of ANA-12 (ng/g brain) were derived from the reference samples by calculating the area under the detection peak.

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In vivo cell death analysis [1]
The effects of subchronic treatments of ANA-12 on cell death in mouse brains were assessed using the TUNEL assay. Mice were randomly distributed into 4 groups and were i.p. injected with saline (containing 1% DMSO) or 0.5, 1.0, or 2.0 mg/kg of ANA-12 once a day for 1 week. Mice were sacrificed 24 hours after the last injection and perfused transcardially with PBS followed by 4% paraformaldehyde. Brains were postfixed overnight in 4% paraformaldehyde, and 50 μm–thick coronal sections were obtained using a vibratome. Free-floating sections were extensively washed in PBS and permeabilized in PBS containing 0.5% Triton X-100 for 90 minutes at room temperature. Sections were then rinsed and mounted on slides. Labeling of 3′ OH-DNA strand breaks was performed using the DeadEnd Fluorometric TUNEL system according to the manufacturer’s instruction. The reaction was terminated by rinsing the slides in SSC 2× and washing extensively with PBS. Sections were mounted with VECTASHIELD plus DAPI, and fluorescein was visualized at 520 nm using fluorescence microscopy. Positive controls were obtained by pretreating sections from saline-treated animals with DNAse (10 U/ml) for 90 minutes at 37°C in PBS containing 0.5% Triton X-100. Negative controls (absence of fluorescent spots) were obtained by processing the sections as described above without the terminal transferase.

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Behavioral testing of ANA-12 [1]
All behavioral testing was started at the same time of the day (1:00 pm) in quiet and separated rooms under bright ambient light conditions (800–900 lux, except for the open field, 300–400 lux). Behavioral tasks were performed 4 hours after injection of 0.5 mg/kg of ANA-12 using the same procedure as described above. To eliminate odor cues, all testing apparatus was thoroughly cleaned after each animal using the disinfectant Roccal.

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Effects of ANA-12 on anxiety-related behaviors [1]
Anxiety-related behaviors were tested using the open field test, elevated plus maze, and novelty-suppressed feeding paradigm.

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Drug Administration [2]
ANA-12, N2-(2-{[(2-oxoazepan-3-yl) amino]carbonyl}phenyl)benzo[b]thiophene-2-carboxamide (0.5mg/kg, i.p.), was dissolved in 1% dimethylsulfoxide in physiological saline. The doses of 7,8-DHF and ANA-12 were also selected as previously reported (Ren et al., 2013 2014; Cazorla et al., 2011).

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Surgery and Bilateral Injection of ANA-12 into NAc [2]
Mice were anesthetized with pentobarbital (5mg/kg), and placed in a stereotaxic frame. Microinjection needles were placed bilaterally into the NAc shell (+1.7 AP, ±0.75 ML, -3.6 DV) (Paxinos and Watson, 1998). Twenty-four hours after surgery, LPS (0.5mg/kg) or saline (10ml/kg) was injected i.p. Twenty-three hours after injection of LPS (or saline), ANA-12 (0.1 nmol/L, 0.1 μL/min for 5min) or vehicle was injected bilaterally. Behavioral evaluation was performed 4 and 6 hours after the final infusion (Figure 3B).

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Experiment 4: BDNF/TrkB receptor signaling within the mNTS. [3]
All rats (n = 9) received two unilateral NTS intraparenchyma injections that were ∼20 min apart, and food intake was determined at 1, 3, 6, and 24 h after the second injection. Conditions were counterbalanced and were as follows: control condition (100 nl of DMSO followed by 100 nl of aCSF), ANA-12 condition (1 or 3 μg of ANA-12 followed by aCSF), BDNF condition (DMSO followed by 0.2 μg of BDNF), and the combination condition (either 1 or 3 μg of ANA-12 followed by 0.2 μg of BDNF). Body weight measurements were made immediately before and 24 h after the injection of the drugs.

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Experiment 6: hindbrain TrkB receptor signaling and leptin. [3]
All rats (n = 9) received two unilateral NTS parenchyma injections that were ∼20 min apart, and food intake was measured at 1, 3, 6, and 24 h after the second injection. The four counterbalanced conditions were as follows: control condition (100 nl of DMSO followed by 100 nl of sodium bicarbonate), ANA-12 condition (3 μg of ANA-12 followed by sodium bicarbonate), leptin condition (DMSO followed by 0.2 μg of leptin), and the combination condition (3 μg of ANA-12 followed by 0.2 μg of leptin). Body weight measurements were made immediately before and 24 h after the injection of drugs.

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Drugs and Treatments [4]
Ethanol, U99194A maleate, SB277011A hydrochloride, buspirone hydrochloride, 8-OH-DPAT and ANA-12 were dissolved in saline and intraperitoneally (i.p.) injected (in a volume of 10 ml/kg), except ANA-12 that was dissolved in 10% dimethyl sulfoxide. U99194A was used at 10 mg/kg (Harrison and Nobrega, 2009), SB277011A was used at 10 mg/kg (Song et al, 2012), buspirone was used in the range 0.1–10 mg/kg (Martin et al, 1992), 8-OH-DPAT was used at 1 mg/kg (Martin et al, 1992), and ANA-12 was used at 0.5 mg/kg (Cazorla et al, 2011).
In the two-bottle choice paradigm, after 30 days of voluntary alcohol-drinking procedure, D3R−/− and WT were randomly allocated to the eight experimental groups (n=6/10 per group): WT/vehicle, WT/U99194A, WT/SB277011A, WT/buspirone, D3R−/−/vehicle, D3R−/−/U99194A, D3R−/−/SB277011A, and D3R−/−/buspirone. Animals were i.p. injected once a day, for 14 consecutive days. On day 14, animals were sacrificed 1 h after the last administration and brain tissues were taken. In another set of experiments, after 30 days of voluntary alcohol-drinking procedure, mice were randomly allocated to five experimental groups (n=5/7 per group): WT naïve, WT/vehicle, WT/ANA-12, D3R−/−/vehicle, and D3R−/−/ANA-12. Animals were i.p. injected once a day, for 4 consecutive days with the selective Trkb antagonist ANA-12 at 0.5 mg/kg (Cazorla et al, 2011; Vassoler et al, 2013). On day 4, animals were sacrificed 1 h after the last administration and brain tissues were taken.

Systemic administration of ANA-12 inhibits TrkB activity in the brain. [1] This study aimed to develop a small molecule that, upon systemic administration, inhibits TrkB activity in the brain of adult mammals. We first examined whether ANA-12 was stable in mouse serum and not degraded into metabolites before reaching brain tissue. To this end, we incubated ANA-12 in mouse serum at 37°C for 15, 30, 45, and 60 minutes. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that no metabolites were detected in the mixture and no degradation of ANA-12 over time was observed (Figure 6A). Subsequently, we examined whether ANA-12 could cross the blood-brain barrier and reach brain tissue after systemic administration. To this end, we injected adult mice intraperitoneally with ANA-12 (0.5 mg/kg). Animals were sacrificed at 0.5, 1, 2, 4, or 6 hours post-injection, and brain tissue was extracted for quantitative analysis of ANA-12 using liquid chromatography-mass spectrometry (LC-MS). Figure 6B shows that after intraperitoneal injection of ANA-12, active concentrations of ANA-12 could be detected in brain tissue as early as 30 minutes (~400 nM) to 6 hours (~10 nM). Subsequently, we examined whether ANA-12 inhibited TrkB activity in adult animal brain tissue, measuring the degree of TrkB inhibition at 2 and 4 hours post-injection of 0.5 mg/kg ANA-12. This time point was chosen based on our previous observations of cyclophosphamide B (cyclotraxin-B), which requires at least 3 hours to inactivate TrkB in brain tissue. KIRA-ELISA quantitative analysis of phosphorylated TrkB showed that 0.5 mg/kg ANA-12 partially inhibited the total activity of endogenous TrkB throughout the brain (8% inhibition at 2 hours and 25% inhibition at 4 hours; Figure 6C). Although TrkB is widely distributed in the brain, the inhibitory effect of ANA-12 on receptors in different brain regions may not be uniform. This may result in an apparent partial inhibition of TrkB activity throughout the brain, with complete inhibition of TrkB activity in some brain regions while TrkB activity in other brain regions is unaffected or only minimally affected. Therefore, we determined the magnitude of inhibition between different brain regions. We again injected adult mice with 0.5 mg/kg ANA-12 and collected samples from different brain regions (striatum, cortex, and hippocampus) at 2 hours or 4 hours (Figure 6D). KIRA-ELISA analysis showed that 2 hours after injection, the inhibitory effect on TrkB in the striatum was better than that in the hippocampus and cortex. Four hours later, the degree of inhibition was comparable across all analyzed structures (25%–30%), but the TrkB inhibition in the striatum appeared to be slightly higher than in the hippocampus and cortex. In summary, these observations suggest that very low doses of ANA-12 are sufficient to uniformly and partially inhibit TrkB activity throughout the brain after four hours.

ANA-12 does not affect neuronal survival. [1]
Since inhibition of the BDNF/TrkB signaling pathway can induce neuronal death in the central nervous system, we verified whether ANA-12 could be administered long-term without causing toxicity to the brain. To this end, we injected adult mice daily with different doses of ANA-12 (0.5, 1.0 and 2.0 mg/kg) or saline for one week. After the last injection, the mice were sacrificed and brain tissue was collected for TUNEL fluorescence staining to detect apoptosis (Figure 9). TUNEL-positive cells were almost undetectable in the brain tissue of mice injected with saline or 0.5 or 1.0 mg/kg ANA-12. Notably, whole-brain examination showed that apoptotic cells were only present in the dentate gyrus of the hippocampus. However, a slight increase in the number of TUNEL-positive cells was observed in the dentate gyrus of mice that received 2.0 mg/kg ANA-12. No staining was detected in any other study areas (cortex, striatum, hippocampal CA1-3 areas, hypothalamus, thalamus, substantia nigra, ventral tegmentum, globus pallidus, and raphe nuclei).

[1]. J Clin Invest. 2011 May 2; 121(5): 1846–1857.

[2]. Eur J Neurosci . 2014 May;39(9):1439-54.

[3]. Int J Neuropsychopharmacol . 2014 Oct 31;18(4):pyu077.

[4]. Am J Physiol Endocrinol Metab . 2012 May 1;302(10):E1252-60.

[5]. Neuropsychopharmacology . 2014 Jul;39(8):2017-28.

[6]. Nat Neurosci . 2013 Jan;16(1):42-7.

ANA-12 is a secondary amide with the structure of anthranilic acid, in which the carboxyl group undergoes a condensation reaction with the primary amino group of α-amino-ε-caprolactam, and the aryl amino group undergoes a condensation reaction with the carboxyl group of 1-benzothiophene-2-carboxylic acid. It is a selective, non-competitive antagonist of tropomyosin receptor kinase B (TrkB, also known as tyrosine receptor kinase B). It functions as a tropomyosin-associated kinase B receptor antagonist, an antidepressant, and an anxiolytic. It is a secondary amide belonging to the caprolactam and 1-benzothiophene classes. Functionally, it is related to 2-aminohexyl-6-lactam and anthranilic acid. Finally, other compounds with the same 3-(acylamino)-ε-caprolactam skeleton as N-T19 and ANA-12 have been described as cognitive enhancers in mice, although their exact mechanisms of action remain unclear. Given the central role of BDNF/TrkB coupling in cognition and memory, it would be meaningful to assess the effects of these compounds on TrkB receptors and compare them with ANA-12 and N-T19. To our knowledge, ANA-12 is the first non-peptide TrkB receptor antagonist to produce a potent and specific effect in vivo. This proteolytically stable small molecule is a valuable pharmacological tool that can help us better study the role of the BDNF/TrkB signaling pathway under pathophysiological conditions and will serve as a lead compound for designing highly effective orally bioavailable TrkB modulators. [1] In summary, our study shows that LPS-induced inflammation leads to depressive-like behavior and causes changes in BDNF protein and dendritic spine density in the hippocampus, prefrontal cortex (PFC), and nucleus accumbens (NAc). In addition, the TrkB agonist 7,8-DHF and the antagonist ANA-12 restored abnormal dendritic spines in the hippocampus and PFC and abnormal dendritic spines in the NAc to normal, thereby showing antidepressant effects against LPS-induced depressive behavior. Therefore, abnormal BDNF-TrkB signaling pathways in the hippocampus, PFC and NAc may play a role in inflammation-induced depression. Finally, in major depressive disorder (MDD), TrkB agonists and TrkB antagonists may serve as potential therapeutic agents for patients with low levels of brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex (PFC) and high levels of BDNF in the nucleus accumbens (NAc). [2]

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Melolimbic dopamine (DA) controls drug and alcohol craving behavior, but the role of specific DA receptor subtypes is unclear. We tested the hypothesis that D3R gene deletion or D3R pharmacological blockade inhibits ethanol preference in mice. We treated D3R-deficient mice (D3R^-/-) and their wild-type (WT) littermates with the D3R antagonists SB277011A and U99194A, with or without treatment, and tested them in a long-term free-choice ethanol drinking experiment (two-bottle selection) and a binge-drinking paradigm (dark drinking, DID). The selectivity of the D3R antagonists was further evaluated by molecular modeling. In both the two-bottle selection and DID paradigms, ethanol intake was negligible in D3R(-/-) mice, while it was high in wild-type (WT) mice. Treatment with the D3R antagonists suppressed ethanol intake in WT mice but had no effect on D3R(-/-) mice. Ethanol intake increased the expression of RACK1 and brain-derived neurotrophic factor (BDNF) in both WT and D3R(-/-) mice; D3R was also significantly overexpressed in WT mice. Therefore, increased D3R expression associated with RACK1/BDNF activation appears to play a reinforcing role in voluntary ethanol intake. Indeed, blocking the BDNF pathway with the TrkB selective antagonist ANA-12 reversed chronic stable ethanol intake and significantly reduced D3R expression in the striatum. Finally, we evaluated buspirone, an approved drug for the treatment of anxiety disorders, which has D3 receptor antagonistic activity (confirmed by molecular modeling analysis) and showed that it effectively inhibited ethanol intake. Therefore, dopamine signaling via the D3 receptor is crucial for ethanol-related reward and intake and may represent a novel target for withdrawal therapy. [4] We describe a heritable phenotype induced by cocaine self-administration in rats. We observed that the learning and maintenance of cocaine self-administration behavior were delayed in male offspring, but not in female offspring. Brain-derived neurotrophic factor (BDNF) mRNA and BDNF protein were increased in the medial prefrontal cortex (mPFC), and acetylated histone H3 binding to the BDNF promoter was increased only in male offspring with cocaine experience. Administration of a BDNF receptor antagonist (TrkB receptor antagonist ANA-12) reversed the reduction in cocaine self-administration behavior in male cocaine-treated offspring rats. Furthermore, acetylated histone H3 binding to the BDNF promoter was increased in the sperm of male rats who self-administered cocaine. In summary, these findings suggest that paternal voluntary cocaine intake leads to epigenetic reprogramming of germ cells, which has profound implications for mPFC gene expression and resistance to cocaine enhancement in male offspring. [5]

C22H21N3O3S

407.49

407.13

C, 64.85; H, 5.19; N, 10.31; O, 11.78; S, 7.87

219766-25-3

2799722

White to off-white solid powder

4.786

3

4

4

29

628

0

S1C2=C([H])C([H])=C([H])C([H])=C2C([H])=C1C(N([H])C1=C([H])C([H])=C([H])C([H])=C1C(N([H])C1([H])C(N([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])=O)=O)=O

TUSCYCAIGRVBMD-UHFFFAOYSA-N

InChI=1S/C22H21N3O3S/c26-20(25-17-10-5-6-12-23-21(17)27)15-8-2-3-9-16(15)24-22(28)19-13-14-7-1-4-11-18(14)29-19/h1-4,7-9,11,13,17H,5-6,10,12H2,(H,23,27)(H,24,28)(H,25,26)

N-[2-[(2-oxoazepan-3-yl)carbamoyl]phenyl]-1-benzothiophene-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 1.43 mg/mL (3.51 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.43 mg/mL (3.51 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 1 mg/mL (2.45 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 0.45 mg/mL (1.10 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: ≥ 0.45 mg/mL (1.10 mM) (saturation unknown) in 5% DMSO + 95% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 6: 2% DMSO+30% PEG 300+2% Tween 80+ddH2O: 2mg/mL .

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 (Please use freshly prepared in vivo formulations for optimal results.)