Main Research Findings

1) Treatment with CE-123 was found to elicit procognitive effects and impact reward-related dopaminergic areas of the brain.

2) Administration of CE-123 on a daily basis was found to enhance memory retrieval and memory acquisition in animal models.

 

Selected Data

1) The study conducted by the research team of Sagheddu et al aimed to investigate the neurophysiological, neurochemical, and behavioral effects of (S)-CE-123, a novel modafinil analog, and R-modafinil on the mesocorticolimbic dopamine system in adult male rats. The experimental design outlined subject handling, drug synthesis and administration, electrophysiological recordings, in vivo microdialysis, ultrasonic vocalization recordings, and comprehensive data analysis. For subjects, male Sprague-Dawley rats, weighing 250-350 g, were housed under standard conditions including: 22 ± 2 °C temperature, 60% humidity, and a 12-hour light/dark cycle with ad libitum access to food and water [1].

The drugs, (S)-CE-123 and R-modafinil, were synthesized in-house. On the day of experiments, both (S)-CE-123 and R-modafinil were freshly dissolved in a vehicle solution composed of 4% DMSO, 5% TWEEN 80, and 0.9% physiological saline. For electrophysiological experiments, drugs were administered intravenously (i.v.) at cumulative doses of 1.25 to 10 mg/kg/mL. For in vivo microdialysis studies and ultrasonic vocalization (USV) recordings, drugs were injected intraperitoneally (i.p.) at single doses of 1, 5, or 10 mg/kg in 3 mL.

In vivo single-unit extracellular recordings were performed on anesthetized rats. A cannula was inserted into the femoral vein for i.v. drug administration, and rats were placed in a stereotaxic apparatus with body temperature maintained at 37 ± 1 °C. Extracellular activity of neurons was recorded using glass micropipettes, bandpass filtered, isolated and amplified, and displayed on a digital storage oscilloscope. Data was sampled online and offline using Spike2 software and a CED1401 interface. Recordings targeted the infralimbic/prelimbic (IL/PrL) cortex and the ventral tegmental area. Putative pyramidal neurons in IL/PrL were identified by “regular-spiking” or “intrinsically bursting” activity, firing rates below 10 Hz, and action potentials wider than 2 ms. Putative dopamine neurons in the VTA were identified by a firing rate below 10 Hz and action potential duration greater than 2.5 ms, with bursts defined as two spikes with an interspike interval less than 80 ms, terminated by an interval exceeding 160 ms [1].

In vivo brain microdialysis was used to monitor extracellular dopamine levels. Rats were anesthetized with 3 mL/kg Equithesin and probes were implanted in either the nucleus accumbens (NAc) shell or the IL/PrL cortex according to a rat brain atlas. Probes were perfused with Ringer’s solution at 1 µL/min. Dialysate samples were collected and injected into an HPLC system equipped with a reverse-phase column and a coulometric detector for dopamine quantification. The detector’s first electrode was set at +130 mV for oxidation and the second at –175 mV for reduction. The mobile phase consisted of 50 mM NaH2PO4, 0.1 mM Na2-EDTA, 0.5 mM n-octyl sodium sulfate, and 15% methanol, with pH 5.5. The assay sensitivity for dopamine was 5 fmol/sample. After establishing stable basal levels, drugs or vehicle were injected, and dopamine levels were monitored for up to 3 hours. Histological examination confirmed probe localization.

For 50-kHz ultrasonic vocalization (USV) recordings, individual rats were monitored during microdialysis experiments using ultrasonic microphones connected to an ultrasound recording device. Intensity gain was kept constant, and USVs were recorded for 3 hours post-injection. USV recordings were converted into spectrograms using SASLab Pro 4.52, manually processed to remove background noise, and 50-kHz USVs were automatically counted [1].

2) The study conducted by Kristofova et al aimed to characterize the pharmacological properties of a novel modafinil analogue, (CE-123), and evaluate its potential to enhance memory performance in a rat model. The experimental design involved a comprehensive approach, combining in vitro assays, in vivo pharmacokinetic studies, and behavioral assessments, culminating in detailed biochemical analyses of brain tissue [2].

The synthesis of CE-123 was a multi-step chemical process beginning with diphenylmethanol and thiourea to form S-di-phenylmethyl-isothiuronium hydrobromide. This was followed by the synthesis of 5-(chloromethyl)thiazole hydrochloride from 5-(hydroxymethyl)thiazole. These intermediates were then reacted to produce 5-((benzhydrylthio)methyl)thiazole, which was subsequently oxidized to yield the final compound, CE-123. The compound’s purity was determined by C-18 HPLC, and its structure was confirmed using 1H NMR and 13C NMR spectroscopy.

Molecular docking studies were performed using a drosophila Dopamine Transporter (DAT) homology model, derived from a drosophila DAT structure in complex with nortriptyline. Ligands, including R/S-CE-123 and cocaine, were prepared with flexible bonds. The binding site was defined by the center of mass of the nortriptyline, and interaction patterns with amino acids were visualized to generate pharmacophore models [2].

In vitro reuptake inhibition assays utilized HEK293 cells stably expressing human DAT, norepinephrine transporter (NET), and serotonin transporter (SERT). Cells were incubated with various concentrations of CE-123 alongside tritiated substrates including: [3H]dopamine for DAT, [3H]MPP+ for NET, and [3H]5-HT for SERT. Non-specific uptake was determined using mazindole for DAT/NET and paroxetine for SERT. Radioactivity was measured using a liquid scintillation counter to determine inhibitory concentrations (IC50). A DAT-release assay was conducted using HEK-DAT cells pre-incubated with [3H]MPP+. Cells were then superfused, and the release of radioactivity was measured after sequential additions of monensin, D-amphetamine (positive control), or CE-123, to differentiate between substrate and inhibitor activity [2].

To assess blood-brain barrier (BBB) permeability, an in vitro model utilizing mouse cerebEND cells on Transwell inserts was employed. Compound solutions, CE-123 and diazepam as internal standard, were added to the apical compartment, and transport across the cell layer was monitored over time. Transendothelial electrical resistance (TEER) was measured to ensure cell layer integrity, and collected samples were analyzed by HPLC to calculate permeability coefficients (PCall and PCcell). In vivo pharmacokinetic studies were performed on Sprague Dawley rats administered a single intraperitoneal dose of 10 mg/kg CE-123 or R-modafinil. Plasma, cerebrospinal fluid (CSF), and brain tissues were collected at 15 minutes, 1 hour, and 7 hours post-administration. Levels of CE-123 and R-modafinil were quantified using HPLC-MS.

For the behavioral assessment, forty-eight male Sprague Dawley rats were housed under controlled conditions and food-deprived to 80–85% of their free-feeding weight. Rats were trained on a spatial hole-board task, which involved a 1m x 1m board with 16 holes, four of which were consistently baited with food pellets. Olfactory cues were masked, and the apparatus was elevated. After habituation, rats underwent three days of training and testing, involving multiple trials with a 20-minute inter-trial interval. Parameters measured included latency to find pellets, working memory errors defined by revisiting baited holes, and reference memory errors defined by visiting unbaited holes. Memory indices, WMI and RMI, were calculated. Untrained/yoked rats received similar handling but no food reward. CE-123 or vehicle (100% DMSO) was administered daily via intraperitoneal injection of 1 mg/kg and 10 mg/kg, 30 minutes before training sessions [2].

Additionally, a battery of behavioral tests including Open Field, Elevated Plus Maze, Forced Swim Test, Rota Rod, and a neurological observational battery were performed on a separate group of rats treated with 10 mg/kg CE-123 or vehicle to assess general health, motor function, anxiety-like behavior, and antidepressant effects.
Finally, biochemical analyses focused on hippocampal subregions. Synaptosome isolation involves homogenizing CA1, CA3, and dentate gyrus (DG) regions, followed by differential centrifugation to obtain synaptosomal fractions, with protein concentration determined by BCA assay. Immunoblotting was used to quantify protein levels of dopamine receptors (D1R, D2R, D5R) and DAT (total and phosphorylated at Thr53, DATp). Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific rabbit polyclonal antibodies. Immunoreactive bands were visualized and quantified using ImageJ software, normalized to total protein-stained membranes [2].

 

Discussion

1) The study completed by Sagheddu et al examined the neurophysiological, neurochemical, and behavioral effects of (S)-CE-123 and R-modafinil on the rat mesocorticolimbic dopamine system. The results demonstrated distinct patterns of action for the two compounds across different brain regions and experimental paradigms [1].

In the IL/PrL cortex, electrophysiological recordings revealed that intravenous administration of (S)-CE-123 significantly reduced the firing rate of putative pyramidal neurons in a dose-dependent manner. Cumulative doses of (S)-CE-123 (1.25–10 mg/kg i.v.) led to a marked decrease in firing frequency. In contrast, R-modafinil, administered at the same cumulative doses, did not produce any significant effect on the neuron firing rate in the IL/PrL cortex. Vehicle administration similarly showed no effect. These findings highlight a specific inhibitory effect of (S)-CE-123 on IL/PrL pyramidal neurons that is not shared by R-modafinil.

Conversely, in the VTA, neither (S)-CE-123 nor R-modafinil, when administered intravenously at cumulative doses of 1.25–10 mg/kg, significantly affected the firing frequency of putative VTA dopamine neurons. Furthermore, neither compound altered the burst firing activity of these VTA dopamine cells. Vehicle administration was also ineffective for both firing rate and burst firing in the VTA. These results suggest that neither (S)-CE-123 nor R-modafinil directly modulates the activity of VTA dopamine neurons at the tested doses [1].

The study further investigated the neurochemical effects on dopamine transmission using in vivo microdialysis in the IL/PrL cortex and nucleus accumbens (NAc) shell. In the IL/PrL cortex, a three-way ANOVA revealed significant main effects of dose and time, as well as significant treatment × time, dose × time, and treatment × dose × time interactions. Post-hoc analyses showed that (S)-CE-123 at 10 mg/kg i.p. significantly increased dialysate dopamine levels at 40 minutes post-administration, compared to basal values, vehicle, and the lower dose of 1 mg/kg (S)-CE-123. 10 mg/kg R-modafinil also produced significant increases in dopamine at 100, 120, and 160 minutes relative to basal and the 1 mg/kg R-modafinil dose at 80, 100, 120, and 160 minutes. Furthermore, R-modafinil at 5 mg/kg i.p. showed significant increases at 160 and 180 minutes compared to basal and vehicle-treated animals. A significant difference was also noted at 160 minutes between animals treated with 10 mg/kg (S)-CE-123 and 10 mg/kg R-modafinil [1].

Figure 1: Changes in dopamine levels in the NAc shell in response to administration of (S)-CE-123 or R-modafinal.

In the NAc shell, the analysis revealed a main effect of time and a significant dose × time interaction. Tukey’s post-hoc tests indicated that 10 mg/kg (S)-CE-123 caused a significant, but low and transitory, increase in dialysate dopamine at 40 minutes compared to basal values. Notably, at this 40-minute mark, there was also a significant difference between 10 mg/kg (S)-CE-123 and 10 mg/kg R-modafinil. R-modafinil, however, did not induce any significant changes in dopamine levels in the NAc shell. This suggests a preferential impact of (S)-CE-123 on dopamine release in the IL/PrL cortex over the NAc shell, with a distinct temporal profile compared to R-modafinil.

Finally, the behavioral assessment of positive affect and drug-induced reward via 50-kHz USVs showed that neither (S)-CE-123 nor R-modafinil initiated the emission of these vocalizations. Acute administration of (S)-CE-123 (1–10 mg/kg i.p.) did not increase 50-kHz USVs compared to vehicle. Two-way ANOVA revealed no significant effect of treatment or treatment × time interaction, although a significant effect of time was observed. Similarly, R-modafinil (1–10 mg/kg i.p.) did not elevate USV numbers. Two-way ANOVA showed no significant effect of treatment, time, or treatment × time interaction. These findings indicate a lack of reward-related behavioral effects for both compounds at the tested doses, suggesting a minor impact on reward-related dopaminergic areas [1].

2) The results of the study conducted by Kristofova et al characterized CE-123’s pharmacological profile, demonstrating its selective DAT inhibition, favorable pharmacokinetic properties, and significant pro-cognitive effects in a rat model, alongside its modulation of dopamine receptor and transporter levels in hippocampal subregions.

In vitro reuptake inhibition assays revealed that CE-123 is a potent and selective dopamine reuptake inhibitor, exhibiting an IC50 of 4.606 μM for [3H]dopamine uptake in HEK293 cells expressing human DAT. Crucially, CE-123 showed negligible inhibitory effects on SERT and NET activity. Further investigations using a DAT-release assay confirmed that CE-123 specifically blocks DAT without acting as a substrate, differentiating its mechanism from releasers like D-amphetamine, which induced significant substrate efflux. This suggests CE-123 acts as a pure reuptake inhibitor [2].

Molecular docking studies provided insights into CE-123’s binding to the DAT homology model. The binding site for both R- and S-enantiomers of CE-123 overlapped with the substrate-binding pocket of DAT, which is also the binding site for cocaine, modafinil, and benztropine analogues. While the binding modes were similar between enantiomers, subtle differences in interacting amino acids were observed. A notable finding was the absence of ionic interaction with negatively charged ASP79, a characteristic interaction in cocaine binding, suggesting primarily unspecific hydrophobic interactions.

Regarding its ability to cross biological barriers, the in vitro BBB model utilizing cerebEND cells demonstrated that CE-123 possessed a permeability coefficient (PCcell) of 30.31 ± 6.94 µm/min. When normalized to diazepam, a known BBB-permeating compound, CE-123 showed a ratio of 0.87 ± 0.04, indicating significant BBB permeability in vitro. This was supported by in vivo pharmacokinetic studies in rats, which showed rapid brain uptake of CE-123. Brain levels of CE-123 were notably high at 15 minutes post-administration (4.4 ± 0.5 µg/g) and remained detectable at 7 hours, demonstrating rapid brain entry. In comparison to R-modafinil, CE-123 exhibited higher brain levels at 15 minutes and 1 hour ( 2.0 ± 0.4 µg/g vs. 0.1 ± 0.0 µg/g at 1 hour) and a higher elimination rate constant, suggesting more efficient metabolism or clearance [2].

The pro-cognitive effects of CE-123 were evaluated using a spatial hole-board task. The results indicated a significant enhancement in both memory acquisition and retrieval. Reference Memory Indices (RMI) revealed a significant improvement in drug-treated groups compared to vehicle, with CE-123 delivered in doses of 1 mg/kg and 10 mg/kg, leading to significantly higher RMI values on Day 1 (trial 3, 4, 9), Day 2 (trial 7), and Day 3 (retrieval, trial 10). Concurrently, the latency to find all pellets was significantly decreased in the CE-123-treated groups at various points during acquisition (Day 1: trials 2, 3, 4, 5; Day 2: trials 7, 9) and retrieval (Day 3: trial 10), indicating faster task completion. While a significant trial effect and treatment effect were observed for RMI and latency, working memory indices (WMI) did not show significant differences, possibly attributed to a ceiling effect. Importantly, additional behavioral tests, including Open Field, Elevated Plus Maze, Forced Swim Test, and Rota Rod, demonstrated that CE-123 at a dose of 10 mg/kg did not impair motor coordination, locomotion, or induce anxiety-like or antidepressant-like behaviors, suggesting a favorable side-effect profile [2].

Figure 2: Changes in A) RMI, B) Latency measured in seconds, and C) WMI across trials in response to 1 mg/kg CE-123, 10 mg/kg CE-123, or a vehicle of DMSO.

At the molecular level, CE-123 administration modulated dopamine receptor and transporter levels in specific hippocampal subregions. D1R protein levels were significantly increased in both CA1 and CA3 regions in rats that underwent training and received CE-123. Interestingly, direct drug administration without training also increased D1R in CA1 and DG, but led to a decrease in CA3. D2R protein levels showed a significant decrease in CA1 in the trained group but remained unchanged in other regions. D5R protein levels were significantly increased in the DG in both trained and yoked drug-treated groups, suggesting a training-independent effect in this region. Furthermore, total DAT protein levels were significantly increased in the DG of trained drug-treated animals, while not changed in CA1. Phosphorylated DAT (DATp at Thr53) levels were increased in CA1 in the trained group, suggesting a role in DAT surface regulation, and decreased in CA3 in the yoked group. These findings collectively suggest that CE-123’s cognitive benefits are mediated through complex adaptations in hippocampal dopaminergic signaling [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Sagheddu C, Pintori N, Kalaba P, et al. Neurophysiological and Neurochemical Effects of the Putative Cognitive Enhancer (S)-CE-123 on Mesocorticolimbic Dopamine System. Biomolecules. 2020;10(5):779. Published 2020 May 18. doi:10.3390/biom10050779

[2] Kristofova M, Aher YD, Ilic M, et al. A daily single dose of a novel modafinil analogue CE-123 improves memory acquisition and memory retrieval. Behav Brain Res. 2018;343:83-94. doi:10.1016/j.bbr.2018.01.032

What is CE-123?

5-((benzhydrylsulfinyl)methyl) thiazole, commonly referred to as CE-123 is a modafinil analog that has been shown to have enhanced affinity and selectivity for dopamine transporters, allowing for the improvement of cognitive and motivational processes [1]. Modafinil itself belongs to a class of cognitive enhancing nootropic compounds that have a mild psychostimulant effect. That being said, current research focuses on the potential for Modafinil and its analogs, such as CE-123, to treat disorders such as narcolepsy, ADHD, and various other neuropsychiatric conditions related to regulation of dopamine transporters [2].

 

Main Research Findings

1) Data collected during the study supports the hypothesized procognitive effects of the S-enantiomer of CE-123, while also indicating a minor impact on reward-based dopaminergic areas.

2) Administration of CE-123 was found to inhibit hyperactivity and resolve impairments related to the reversal of learning in rats exposed to alcohol, indicating that CE-123 may act as a potential intervention for deficits related to fetal alcohol syndrome.

 

Selected Data

1) The research team of Sagheddu et al conducted various in vivo electrophysiological, neurochemical, and behavioral experiments in adult male rats in order to assess the cognition enhancing abilities of CE-123 and how its mechanism of action relates to the dopaminergic reward based system in the brain. This experiment also focused on the comparison between the S-enantiomer of CE-123 and the R-enantiomer of its parent compound, Modafinil [1].

Male Sprague-Dawley rats with weights ranging from 250-350 grams each were maintained under standard laboratory conditions and provided ad libitum access to food and water until the initiation of the experiment. On the first day of the trial, S-CE-123 and R-modafinil were dissolved in DMSO 4%, TWEEN 80.5%, and a physiological solution of NaCl 0.9% and randomly assigned to the test subjects for treatment. For purposes of electrophysiological experiments, the compounds were intravenously administered in cumulative doses of 1.25 to 10 mg/kg/mL, or the compounds were intraperitoneally injected in doses of 1, 5, or 10 mg/kg in 3 mL for the purpose of ultrasonic vocalization and microdialysis studies [1].

Prior to intravenous administration of the different treatments, rats were anesthetized using 1.3 g/kg or urethane and a cannula was inserted into the femoral vein. The rats were then placed in a stereotaxic apparatus while the extracellular activity of the neurons were recorded with micropipettes containing 2% Pontamine blue dissolved in sodium acetate. Single action potentials were isolated, amplified, and displayed for evaluation with a digital storage oscilloscope while neurons located in layers III-VI in the cortical surface were recorded [1].

That being said, the electrophysiological characteristics of the cells corresponded to pyramidal neurons and were presented as “regular-spiking” or “intrinsically bursting” activity, a firing rate that did not exceed 10 Hz, and an action potential >2 ms wide. Additionally, single unit neuronal activity located in the ventral tegmental area was recorded and putative dopaminergic neurons were isolated and identified based on an observed firing rate of <10 Hz and an action potential of >2.5 ms duration. Bursting was defined by the research team as an occurrence of two spikes at an interspike interval of <80 ms that was terminated when the interspike interval was >160 ms [1].

Performance of brain microdialysis allowed the research team to observe and monitor the extracellular concentration of various endogenous transmitters and how they relate to neurotransmission. Test subjects were anesthetized using a combination of 3 mL/kg chloral hydrate 2.1 g, sodium pentobarbital 0.46 g, MgSO4 1.06 grams, propylene glycol 21.4 mL, ethanol 5.7 mL, and h2O 3 mL, in order for the researcher to properly evaluate extracellular levels of dopamine [1]. Following anaesthetization, microdialysis probes were implanted in the NAc shell or the IL/PrL cortex.

On the first day of the experiment the probes were perfused with Ringer’s solution and dialysate samples were injected into an HPLC equipped with a coulometric detector and reverse-phase column for the purpose of identifying and quantifying dopamine. After basal levels were determined, the test subjects were randomly assigned to receive treatment with S-CE-123, R-modafinil, or a vehicle. Treatment administration was followed by 3 hours of monitoring the animals and observing any changes in extracellular dopamine levels [1]. Additionally, ultrasonic vocalization emissions were recorded from the rats throughout the duration of the microdialysis experiment through the use of ultrasonic microphones connected to a specific recording apparatus. Intensity gain remained consistent during the recordings and emissions were monitored for 3 hours after administration of the assigned treatment.

2) Fetal alcohol spectrum disorder is typically diagnosed in early childhood and characterized by impulsivity, hyperactivity, memory and learning disabilities that are hypothesized to be rooted in the dysfunction of dopaminergic signaling. The research team of Gibula-Tarlowska et al examined the potential of CE-123 to overcome behavioral defects induced by ethanol in an animal model of fetal alcohol spectrum disorder.

163 Wistar rates were used for this study; the test subjects were maintained under standard laboratory conditions and provided ad libitum access to food and water. In order to promote breeding, one male and one female test subject were housed in a cage together for one week; after three weeks monitoring of the female rats began in order to assess for parturition. An equal number of male and female pups from the new litters were retained, however, only male rats were used in the behavioral studies due to their increased vulnerability to fetal and neonatal exposure to ethanol [2].

The S-enantiomer of CE-123 was synthesized in the Lubec laboratory at the University of Vienna, and on the first day of the experiment the compound was dissolved in a solution of 1% DMSO, and 3.3% TWEEN 80 diluted by 0.9% NaCl. CE-123 was administered intraperitoneally in doses of 1, 5, or 10 mg/kg, 30 minutes before the beginning of the behavioral trials. Ethanol was then dissolved in milk solution at a dose of 5 g/kg and given to the animals randomly assigned to the ethanol-exposed treatment group via intragastric intubation. Test subjects not assigned to the ethanol exposed group were assigned to a sham intubated treatment group where no ethanol-milk mixture was provided to the rats [2].

The researchers weighed and examined the rat pups each morning and found that the doses of ethanol produced significant neurotoxicity with the potential for the development of neurobehavioral deficits. All pups were weaned off of milk and randomly assigned to experimental groups for the purpose of completing various behavioral tests such as the locomotor activity test, elevated plus maze test, the Barnes maze task, the probe trial, and reversal learning. The locomotor activity of the rats was measured by placing the rats in transparent, 60 x 60 cm, soundproof rooms with infrared sensors placed at 45 and 100 mm above the floor to assess horizontal activity and distance traveled in meters. 1, 3, and 10 mg/kg doses of CE-123 were administered to the rats 30 minutes before the test to evaluate the effects of ethanol on locomotion [2]. The distance traveled in the horizontal plane was recorded for 30 minutes after the animals were placed in the apparatus.

This behavioral trial was followed by the elevated plus maze (EPM) test performed by placing the rats in the center of a plus-shaped maze with 2 open and 2 closed arms at a height of 50 cm above the floor. The maze apparatus was placed in a quiet, dark room and the rats were handled by the researchers for 5 minutes per day, 3 days before the official trial began. The rats were placed at the center of the EPM facing the open arm while the research team took note of the number of times a rat entered an open arm with all four paws, as well as the amount of time that was spent there over the course of 5 minutes in order to observe anxiety-like behavior in response to ethanol exposure [2].

The next behavioral task was the Barnes maze involving an apparatus formed from a circular gray metal plate that was elevated 100 cm above the floor with 20 holes located in its periphery, each with a diameter of 10 cm. All holes except for one were covered, which led to an escape box made of the same material as the platform apparatus. The open hole was not apparent until the animal was placed right next to it; the researchers placed several visual cues along the walls of the maze in order to assist in evoking the escape response in the test subjects. There are four main phases to the Barnes maze task starting with habituation, where the subjects were allowed to acclimate to the platform and the escape box the day before the acquisition phase began.

The acquisition phase occurred 24 hours after the rats were habituated to the maze. This phase involved one training session per day for five consecutive days, consisting of two 180 second trials and a 5 minute inter-trial interval. Each trial began by placing the rat in the center of the platform and letting it explore for 180 seconds or until it found the escape platform. The hole was then covered for 30 seconds and animals were guided back to their home cages. The following phase included a probe trial to evaluate spatial memory. This phase began 24 hours after the acquisition phase and included the subjects receiving a 180 second probe trial while the primary errors and latency to reach the escape box were recorded by the researchers [2]. In terms of changes elicited in learning processes and spatial memory due to ethanol exposure, a probe trial was carried out after 5 days of learning took place.

The final stage of the Barnes maze test was reversal learning. The reversal learning trials were identical to acquisition trials and took place 24 hours after the probe trial. The position of the escape hole was rotated 180 degrees and the rats were allowed to learn the new location of the hole for 3 days before the trial occurred. Before the first reversal learning trial, the test subjects were randomly assigned to four treatment groups including a vehicle group, 10 mg/kg of CE-123 + vehicle group, ethanol group + vehicle, and ethanol group + 10 mg/kg of CE-123. The treatments were administered once daily, 30 minutes prior to the reversal learning sessions; data obtained from the trials were combined and used to determine primary latency and errors, as well as the effects of CE-123 on cognitive flexibility following exposure to ethanol [2].

 

Discussion

1) When evaluating the electrophysiological effects of S-CE-123 and R-modafinil on pyramidal neurons recorded in the IL/PrL cortex of test subjects the research team of Sagheddu et al found that intravenous administration of doses ranging from 1.25-10 mg/kg of S-CE-123 resulted in a dose dependent decrease in cell firing rate. This was compared to treatment with R-modafinil that did not produce any remarkable effect on the firing rate of the involved neurons. Similar findings were reported for the test subjects administered a vehicle as well [1].

 

Figure 1: Effects of treatment with S-CE-123 and R-modafinil on electrical activity of pyramidal cells from A) rats that received intravenous injection of the vehicle, S-CE-123, or R-modafinil; and B) dose-dependent changes in the the firing rate in response to treatment with the vehicle, S-CE-123, or R-modafinil

Next, when assessing the effects of S-CE-123 and R-modafinil on the firing activity of dopaminergic neurons in the ventral tegmental area, it was found that neither S-CE-123 or R-modafinil elicited significant changes in the firing frequency of neurons in this region. The same findings were noted when interpreting the results gathered from the control group of subjects that were administered a vehicle compound [1].

Figure 2: Effects of treatment with S-CE-123 and R-modafinil on dopaminergic neurons in the ventral tegmental area in A) rats that received intravenous injection of the vehicle, S-CE-123, or R-modafinil; B) changes in the the firing rate in response to treatment with the vehicle, S-CE-123, or R-modafinil; and C) changes in the burst firing activity in response to treatment with the vehicle, S-CE-123, or R-modafinil.

Furthermore, extracellular levels of dopamine in the IL/PrL cortex were studied to determine the effects of doses of 1, 5, and 10 mg/kg of S-CE-123 or R-modafinil. Analysis with three-way ANOVA reveals significant interactions between treatment and time of injection as well as dose and time of injection and treatment, dose, and time of injection. A Tukey’s post hoc analysis found a marked increase in dialysate dopamine only 40 minutes after treatment with 10 mg/kg of S-CE-123 or 10 mg/kg of R-modafinil was administered [1]. These findings were in comparison to base values, the control group administered a vehicle, and the experimental group administered a 1 mg/kg dose of S-CE-123.

Tukey’s post hoc analysis also highlighted a significant increase in dopamine levels measured at 100, 120, and 160 minutes after administration of 10 mg/kg of R-modafinil. These findings were in comparison to the base values, dopamine levels of the control group 100 and 160 minutes after administration of a vehicle compound, and dopamine levels of the experimental group 80, 100, 120, and 160 minutes after administration of a 1 mg/kg dose of R-modafinil. Additionally, notable differences were seen at 160 and 180 minutes after administration of 5 mg/kg of R-modafinil in comparison to base values, test subjects treated with a vehicle, and the differences seen in the animals at 160 minutes after administrations of 10 mg/kg S-CE-123 or 10 mg/kg of R-modafinil [1].

Figure 3: Effects of a vehicle, S-CE-123, and R-modafinil administered in doses of 1, 5, and 10 mg/kg on dopamine levels in relationship to base levels.

Finally, the effects of S-CE-123 and R-modafinil administered in doses of 1, 5, and 10 mg/kg on the emission of 50-kHz ultrasonic vocalizations acting as a drug-induced reward as well as a behavioral marker of positive affect. Administration of all doses of S-CE-123 did not result in an increase in the number of ultrasonic vocalizations emitted in comparison to the vehicle control group. Two-way ANOVA statistical analysis did not indicate a significant interaction effect between treatment and time, however, there was a significant effect of time that was noted by the research team. Similar results were observed with acute administration of R-modafinil as there was no significant increase in ultrasonic vocalization emitted, nor was there a significant effect of treatment, time, or an interaction between treatment and time [1].

Figure 4: Effects of treatment with a vehicle, S-CE-123, and R-modafinil on the emission of 50-kHz ultrasonic vocalizations.

2) When assessing the influence of CE-123 on locomotor activity following ethanol exposure, two-way ANOVA statistical analysis reported a significant difference between the four different experimental treatment groups. In addition to two-way ANOVA, a Bonferroni post-hoc test suggested the exposure to ethanol significantly increased locomotor activity, however, CE-123 administered in doses of 3 or 10 mg/kg had the potential to inhibit this abnormally increased level of locomotor activity. However, the data obtained and analyzed through a Student’s t-test revealed no significant difference between the treatment groups and the number of entries or amount of time they spent with all four paws within the plus shaped apparatus [2].

Figure 5: Changes in locomotor activity in rats exposed to ethanol.

The research team also used the Barnes maze task to observe the effects of CE-123 on reversal learning as it’s related to primary latency and number of errors. In regards to primary latency, mixed effect models were fitted to the dataset assigning animal number as a random effect and concentration of CE-123, presence of ethanol, and day of treatment were defined as fixed effects. Results reported that there was a significant difference between the ethanol and vehicle groups and the time it took for the subjects in each group to reach the target hole.

Overall, the administration of ethanol was found to increase latency in test subjects, proving that ethanol results in memory impairment. When ethanol or CE-123 was administered there were no remarkable changes in the learning slope, however, there was a drastic change in latency as a function of CE-123 when ethanol is administered. These findings allowed the researchers to conclude that CE-123 elicits its effects after administration of ethanol, and the compound is capable of eliciting effects quickly and consistently across treatment days [2].

Figure 6: Changes in primary latency during A) the acquisition phase and C) the probe trial, in response to ethanol exposure

When looking at the number of errors that occurred as the test subjects attempted to find the escape platform, mixed effect models were fitted to an entire datasheet and assigned animal number as a random effect and the concentration of CE-123, ethanol exposure, and treatment day were defined as fixed effects. The results reported that there was a slight difference between rats exposed to ethanol versus exposure to a vehicle, however, the difference was not deemed significant to the research team. However, it is important to note that the researchers were able to observe an overall decrease of 1.58 errors, suggesting the ability of the animals to learn over time. Similar to the primary latency findings, the learning slope demonstrated no significant changes across the experimental groups [2]. However, the amount of errors was found to significantly lower in a dose-dependent manner when CE-123 was administered in cases of ethanol exposure.

Figure 7: Changes in the number of errors during A) the acquisition phase and C) the probe trial, in response to ethanol exposure

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Sagheddu C, Pintori N, Kalaba P, Dragačević V, Piras G, Lubec J, Simola N, De Luca MA, Lubec G, Pistis M. Neurophysiological and Neurochemical Effects of the Putative Cognitive Enhancer (S)-CE-123 on Mesocorticolimbic Dopamine System. Biomolecules. 2020 May 18;10(5):779. doi: 10.3390/biom10050779. PMID: 32443397; PMCID: PMC7277835.

[2] Gibula-Tarlowska E, Korz V, Lopatynska-Mazurek M, Chlopas-Konowalek A, Grochecki P, Kalaba P, Dragacevic V, Kotlinski R, Kujawski R, Szulc M, Czora-Poczwardowska K, Mikolajczak PL, Lubec G, Kotlinska JH. CE-123, a novel dopamine transporter inhibitor, attenuates locomotor hyperactivity and improves cognitive functions in rat model of fetal alcohol spectrum disorders. Behav Brain Res. 2021 Jul 23;410:113326. doi: 10.1016/j.bbr.2021.113326. Epub 2021 Apr 30. PMID: 33940050.

 

CE-123 is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.