Development of Novel Alaninamide Derivatives with Anticonvulsant Activity and Favorable Safety Profiles in Animal Models.

In our current study, we developed a focused series of original ((benzyloxy)benzyl)propanamide derivatives that demonstrated potent activity across in vivo mouse seizure models, specifically, maximal electroshock (MES) and 6 Hz (32 mA) seizures. Among these derivatives, compound 5 emerged as a lead molecule, exhibiting robust protection following intraperitoneal (i.p.) injection, as follows: ED50 = 48.0 mg/kg in the MES test, ED50 = 45.2 mg/kg in the 6 Hz (32 mA) test, and ED50 = 201.3 mg/kg in the 6 Hz (44 mA) model. Additionally, compound 5 displayed low potential for inducing motor impairment in the rotarod test (TD50 > 300 mg/kg), indicating a potentially favorable therapeutic window. In vitro toxicity assays further supported its promising safety profile. We also attempted to identify a plausible mechanism of action of compound 5 by applying both binding and functional in vitro studies. Overall, the data obtained for this lead molecule justifies the more comprehensive preclinical development of compound 5 as a candidate for a potentially broad-spectrum and safe anticonvulsant.

Looming stimuli reliably drive innate defensive responses in male rats, but not learned defensive responses.

Survival relies on an organism's intrinsic ability to instinctively react to stimuli such as food, water, and threats, ensuring the fundamental ability to feed, drink, and avoid danger even in the absence of prior experience. These natural, unconditioned stimuli can also facilitate associative learning, where pairing them consistently with neutral cues will elicit responses to these cues. Threat conditioning, a well-explored form of associative learning, commonly employs painful electric shocks, mimicking injury, as unconditioned stimuli. It remains elusive whether actual injury or pain is necessary for effective learning, or whether the threat of harm is sufficient. Approaching predators create looming shadows and sounds, triggering strong innate defensive responses like escape and freezing. This study investigates whether visual looming stimuli can induce learned freezing or learned escape responses to a conditioned stimulus in male rats. Surprisingly, pairing a neutral tone with a looming stimulus only weakly evokes learned defensive responses, in contrast to the strong responses observed when the looming stimulus is replaced by a shock. This dissociation sheds light on the boundaries for learned defensive responses thereby impacting our comprehension of learning processes and defensive strategies.

Sex and the facilitation of cued fear by prior contextual fear conditioning in rats.

Previous studies have shown that the formation of new memories can be influenced by prior experience. This includes work using Pavlovian fear conditioning in rodents that has shown that an initial fear conditioning experience can become associated with and facilitate the acquisition of new fear memories, especially when they occur close together in time. However, most of the prior studies used only males as subjects, resulting in questions about the generalizability of the findings from this work. Here we tested whether prior contextual fear conditioning would facilitate later learning of cued fear conditioning in both male and female rats, and if there were differences based on the interval between the two conditioning episodes. Our results showed that levels of cued fear were not influenced by prior contextual fear conditioning or by the interval between training; however, females showed lower levels of cued fear. Freezing behavior in the initial training context differed by sex, with females showing lower levels of contextual fear, and by the type of initial training, with rats given delayed shock showing higher levels of fear than rats given immediate shock during contextual fear conditioning. These results indicate that contextual fear conditioning does not prime subsequent cued fear conditioning and that female rats express lower levels of cued and contextual fear conditioning than males.

Influence of footshock number and intensity on the behavioral and endocrine response to fear conditioning and cognitive fear generalization in male rats.

Foot-shock paradigms have provided valuable insights into the neurobiology of stress and fear conditioning. An extensive body of literature indicates that shock exposure can elicit both conditioned and unconditioned effects, although delineating between the two is a challenging task. This distinction holds crucial implications not only for the theoretical interpretation of fear conditioning, but also for properly evaluating putative preclinical models of post-traumatic stress disorder (PTSD) involving shock exposure. The characteristics of shocks (intensity and number) affect the strength of learning, but how these characteristics interact to influence conditioned and unconditioned consequences of shocks are poorly known. In this study, we aimed to investigate in adult male rats the impact of varying shock number and intensity on the endocrine and behavioral response to contextual fear conditioning and fear generalization to a novel environment markedly distinct from the shock context (i.e., fear generalization). Classical biological markers of stress (i.e., ACTH, corticosterone, and prolactin) were sensitive to manipulations of shock parameters, whereas these parameters had a limited effect on contextual fear conditioning (evaluated by freezing and distance traveled). In contrast, behavior in different novel contexts (fear generalization) was specifically sensitive to shock intensity. Notably, altered behavior in novel contexts markedly improved, but not completely normalized after fear extinction, hypoactivity apparently being the result of both conditioned and unconditioned effects of foot-shock exposure. The present results will contribute to a better understanding of shock exposure as a putative animal model of PTSD.

Role of ventral tegmental area dopamine neurons in fear memory retention in post-traumatic stress disorder model rats.

Ventral tegmental area (VTA) dopamine (DA) neurons have been found to substantially associate with post-traumatic stress disorder (PTSD) pathology, however, whether and how these DA neurons affect fear memory management in PTSD individuals remains largely unknown. In this study, we utilized auditory conditioned foot-shock to evaluate the fear memory retrieval and retention characteristics in a single prolonged stress-induced PTSD rat model. We employed chemogenetic technology to specifically activate VTA DA neurons to examine the freezing behaviors responding to the conditioned stimuli. In vivo extracellular electrophysiological analyses were used to identify VTA DA neuronal firing alterations due to the chemogenetic activation. The results demonstrated that PTSD model rats showed comparable fear memory retrieval (Day 2 after the conditioned foot-shock), but significant enhancements in fear memory retention (Day 8 after the conditioned foot-shock), compared to normal control rats. Chemogenetic activation of VTA DA neurons markedly diminished the retention of fear memory in PTSD model rats, which appeared concomitantly with increases in the firing activities of the DA neurons. These findings revealed that PTSD induced the persistence of fear memory, which could be attenuated by activation of VTA DA neurons. It is presumed that VTA dopaminergic signals may serve as a prospective option for PTSD treatment.

Discovery and Profiling of New Multimodal Phenylglycinamide Derivatives as Potent Antiseizure and Antinociceptive Drug Candidates.

We developed a focused series of original phenyl-glycinamide derivatives which showed potent activity across in vivo mouse seizure models, namely, maximal electroshock (MES) and 6 Hz (using both 32 and 44 mA current intensities) seizure models. Following intraperitoneal (i.p.) administration, compound (R)-32, which was identified as a lead molecule, demonstrated potent protection against all seizure models with ED50 values of 73.9 mg/kg (MES test), 18.8 mg/kg (6 Hz, 32 mA test), and 26.5 mg/kg (6 Hz, 44 mA test). Furthermore, (R)-32 demonstrated efficacy in both the PTZ-induced kindling paradigm and the ivPTZ seizure threshold test. The expression of neurotrophic factors, such as mature brain-derived neurotrophic factor (mBDNF) and nerve growth factor (NGF), in the hippocampus and/or cortex of mice, and the levels of glutamate and GABA were normalized after PTZ-induced kindling by (R)-32. Importantly, besides antiseizure activity, (R)-32 demonstrated potent antinociceptive efficacy in formalin-induced pain, capsaicin-induced pain, as well as oxaliplatin- and streptozotocin-induced peripheral neuropathy in mice (i.p.). No influence on muscular strength and body temperature in mice was observed. Pharmacokinetic studies and in vitro ADME-Tox data (i.e., high metabolic stability in human liver microsomes, a weak influence on CYPs, no hepatotoxicity, satisfactory passive transport, etc.) proved favorable drug-like properties of (R)-32. Thermal stability of (R)-32 shown in thermogravimetry and differential scanning calorimetry gives the opportunity to develop innovative oral solid dosage forms loaded with this compound. The in vitro binding and functional assays indicated its multimodal mechanism of action. (R)-32, beyond TRPV1 antagonism, inhibited calcium and sodium currents at a concentration of 10 µM. Therefore, the data obtained in the current studies justify a more detailed preclinical development of (R)-32 for epilepsy and pain indications.

Discovery of Novel Pyrimidine-Based Derivatives as Nav1.2 Inhibitors with Efficacy in Mouse Models of Epilepsy.

Dysfunction of voltage-gated sodium channel Nav1.2 causes various epileptic disorders, and inhibition of the channel has emerged as an attractive therapeutic strategy. However, currently available Nav1.2 inhibitors exhibit low potency and limited structural diversity. In this study, a novel series of pyrimidine-based derivatives with Nav1.2 inhibitory activity were designed, synthesized, and evaluated. Compounds 14 and 35 exhibited potent activity against Nav1.2, boasting IC50 values of 120 and 65 nM, respectively. Compound 14 displayed favorable pharmacokinetics (F = 43%) following intraperitoneal injection and excellent brain penetration potency (B/P = 3.6). Compounds 14 and 35 exhibited robust antiepileptic activities in the maximal electroshock test, with ED50 values of 3.2 and 11.1 mg/kg, respectively. Compound 35 also demonstrated potent antiepileptic activity in a 6 Hz (32 mA) model, with an ED50 value of 18.5 mg/kg. Overall, compounds 14 and 35 are promising leads for the development of new small-molecule therapeutics for epilepsy.

Contextual fear conditioning in zebrafish: Influence of different shock frequencies, context, and pharmacological modulation on behavior.

Contextual fear conditioning is a protocol used to assess associative learning across species, including fish. Here, our goal was to expand the analysis of behavioral parameters that may reflect aversive behaviors in a contextual fear conditioning protocol using adult zebrafish (Danio rerio) and to verify how such parameters can be modulated. First, we analyzed the influence of an aversive stimulus (3 mild electric shocks for 5 s each at frequencies of 10, 100 or 1000 Hz) on fish behavior, and their ability to elicit fear responses in the absence of shock during a test session. To confirm whether the aversive responses are context-dependent, behaviors were also measured in a different experimental environment in a test session. Furthermore, we investigated the effects of dizocilpine (MK-801, 2 mg/kg, i.p.) on fear-related responses. Zebrafish showed significant changes in baseline activity immediately after shock exposure in the training session, in which 100 Hz induced robust contextual fear responses during the test session. Importantly, when introduced to a different environment, animals exposed to the aversive stimulus did not show any differences in locomotion and immobility-related parameters. MK-801 administered after the training session reduced fear responses during the test, indicating that glutamate NMDA-receptors play a key role in the consolidation of contextual fear-related memory in zebrafish. In conclusion, by further exploring fear-related behaviors in a contextual fear conditioning task, we show the effects of different shock frequencies and confirm the importance of context on aversive responses for associative learning in zebrafish. Additionally, our data support the use of zebrafish in contextual fear conditioning tasks, as well as for advancing pharmacological studies related to associative learning in translational neurobehavioral research.

Comparison of recovery of sheep, goats, and calves from reversible electrical head-only and head-to-body stunning for halal meat production.

AIMS: To compare the recovery of lambs, goats, and calves from head-only (HO) or high-frequency head-to-body stunning and evaluate the complementary use of behaviour and electroencephalography (EEG) to assess return to consciousness after electrical stunning in these species. METHODS: Six-month-old lambs, adult goats and calves (< 7 days old) were subjected to reversible head-only stunning (50 Hz, 1 A, 2 seconds) or reversible high-frequency head-to-body stunning (RHTB: HO followed by 2,000 Hz, 2 A, 4-second stun to body). Following stunning, behavioural recovery was assessed in 21 lambs, 22 goats, and 20 calves. Latencies to first perform behaviours (end of convulsions, head lift, attempt to right, successful righting, attempt to stand, successful standing) after stunning were scored from video recordings. Recovery of electrical brain activity indicative of consciousness was assessed using EEG in a separate cohort of minimally-anaesthetised lambs, goats and calves (n = 20 per species). EEG traces collected before and after stunning were classified as normal, epileptiform, isoelectric, or transitional activity. Following stunning, the duration of epileptiform and isoelectric activity combined (states of brain activity incompatible with conscious awareness) was calculated, as was latency to return of normal (pre-stun) EEG. RESULTS: The RHTB stun was reversible in all three species, although one sheep failed to recover and was euthanised. Both methods caused tonic and clonic convulsions in all species. Behavioural recovery of sheep and calves was similar for both methods while goats took longer to recover from RHTB than HO stunning. There was no evidence of differences between methods in the duration of EEG incompatible with consciousness or the latency to recovery of normal EEG. CONCLUSIONS: Head-to-body stunning as applied here produced a reversible electrical stun in lambs, adult goats and young calves, although the benefits in terms of meat quality and operator safety are uncertain. Goats took longer to recover behaviourally from head-to-body stunning, possibly due to disrupted motor function, but there was no indication that post-stun unconsciousness lasted longer than following head-only stunning in any species. The normal behaviour for the animals' developmental age should be considered when deciding on behavioural indicators of recovery. The minimal anaesthesia model provided excellent quality EEG data that was valuable for interpretation of the behavioural responses. CLINICAL RELEVANCE: For the purposes of pre-slaughter stunning of sheep, goats and young calves, recovery appears comparable between the two methods, with all but 1/63 animals in the behaviour study recovering normal function.

Climbing Fiber Activation Induced by Footshock in the Cerebellar Vermis Lobule IV/V of Freely Moving Mice.

Parallel fibers (PFs) in the cerebellar cortex are involved in a series of coordinated responses in the fear conditioning paradigm induced by footshock. However, whether footshock can activate cerebellar climbing fibers (CFs) remains unclear. In this study, we recorded calcium (Ca2+) activity in CFs by optical fiber photometry in the cerebellar vermis lobule IV/V of freely moving mice with footshock stimulation. We found that the activation of CFs in the lobule IV/V was highly correlated with footshock stimulation but not with the sound stimulation used as a control. This result suggests that afferent information from CFs might be associated with the motor initiation of fear-related behaviors or fear emotion itself. Thus, our results suggest that a characteristic CF signal in the cerebellar cortex might be related to fear processing or footshock-related behaviors (such as startle responses or pain sensation).

Effects of acute administration of 4-allyl-2,6-dimethoxyphenol in mouse models of seizures.

Epilepsy, a chronic neurological disorder characterized by recurrent unprovoked seizures, presents a substantial challenge in approximately one-third of cases exhibiting resistance to conventional pharmacological treatments. This study investigated the effect of 4-allyl-2,6-dimethoxyphenol, a phenolic compound derived from various natural sources, in different models of induced seizures and its impact on animal electroencephalographic (EEG) recordings. Adult male Swiss albino mice were pre-treated (i.p.) with a dose curve of 4-allyl-2,6-dimethoxyphenol (50, 100, or 200 mg/kg), its vehicle (Tween), or standard antiepileptic drug (Diazepam; or Phenytoin). Subsequently, the mice were subjected to different seizure-inducing models - pentylenetetrazole (PTZ), 3-mercaptopropionic acid (3-MPA), pilocarpine (PILO), or maximal electroshock seizure (MES). EEG analysis was performed on other animals surgically implanted with electrodes to evaluate brain activity. Significant results revealed that animals treated with 4-allyl-2,6-dimethoxyphenol exhibited increased latency to the first myoclonic jerk in the PTZ and PILO models; prolonged latency to the first tonic-clonic seizure in the PTZ, 3-MPA, and PILO models; reduced total duration of tonic-clonic seizures in the PTZ and PILO models; decreased intensity of convulsive seizures in the PTZ and 3-MPA models; and diminished mortality in the 3-MPA, PILO, and MES models. EEG analysis indicated an increase in the percentage of total power attributed to beta waves following 4-allyl-2,6-dimethoxyphenol administration. Notably, the substance protected from behavioral and electrographic seizures in the PTZ model, preventing increases in the average amplitude of recording signals while also inducing an increase in the participation of theta and gamma waves. These findings suggest promising outcomes for the tested phenolic compound across diverse pre-clinical seizure models, highlighting the need for further comprehensive studies to elucidate its underlying mechanisms and validate its clinical relevance in epilepsy management.

Synthesis, In vivo, and In silico Evaluation of New Pyrazoline-Benzothiazole Conjugates as Antiepileptic Agents.

New 2-(4-benzothiazol-2-yl-phenoxy)-1-(3,5-diphenyl-4,5-dihydro-pyrazol-1-yl)-ethanones (9a-o) have been designed and synthesized. All the synthesized compounds were characterized by thin layer chromatography and spectral analysis. The antiepileptic potential of the synthesized compounds has been tested by following standard animal screening models, including maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) models. The neurotoxic and antidepression effects of the synthesized compounds were checked by utilizing rotarod apparatus, and motor impairment test (by actophotometer) respectively. The study concluded that compounds 9c, 9d, 9f, 9i, 9n, and 9o possessed good antiepileptic potential compared to standard drugs like carbamazepine and phenytoin. The results of the rotarod performance test also established them without any neurotoxicity. The motor impairment test revealed that the synthesized compounds are also good antidepressants. In-silico studies have been performed for calculation of pharmacophore pattern, prediction of pharmacokinetic properties which determine the eligibility of synthesized compounds as orally administered molecules and interactions with the target proteins. The result of in-silico studies reinforced results obtained by in vivo study of the synthesized compounds and their possible mechanism of antiepileptic action i. e. via inhibiting voltage-gated sodium channels (VGSCs) and gamma-aminobutyric acid-A receptor.

Elemental and configural representation of a conditioned context.

A context can be conceptualized as a stable arrangement of elements or as the sum of single elements. Both configural and elemental representations play a role in associative processes. This study aimed to explore the respective contributions of these two representations of a context in the acquisition of conditioned anxiety in humans. Virtual reality (VR) can be an ecologically valid tool to investigate context-related mechanisms, yet the influence of the sense of presence within the virtual environment remains unclear. Forty-eight healthy individuals participated in a VR-based context conditioning wherein electric shocks (unconditioned stimulus, US) were unpredictably delivered in one virtual office (CTX+), but not in the other (CTX-). During the test phase, nine elements from each context were presented singularly. We found a cluster of participants, who exhibited heightened anticipation of the US for anxiety-related elements as compared to the other group. In contrast to their clear elemental representation, these individuals showed diminished discriminative responses between the two context's configurations. Discriminative responses to the contexts were boosted in those individuals, who had a weaker elemental representation. Importantly, the individual sense of presence significantly influenced the conditioned responses. These findings align with the dual-representation view of context and provide insights into the role of presence in eliciting (conditioned) anxiety responses.

Intense training prevents the amnestic effect of inactivation of dorsomedial striatum and induces high resistance to extinction.

A large body of evidence has shown that treatments that interfere with memory consolidation become ineffective when animals are subjected to an intense learning experience; this effect has been observed after systemic and local administration of amnestic drugs into several brain areas, including the striatum. However, the effects of amnestic treatments on the process of extinction after intense training have not been studied. Previous research demonstrated increased spinogenesis in the dorsomedial striatum, but not in the dorsolateral striatum after intense training, indicating that the dorsomedial striatum is involved in the protective effect of intense training. To investigate this issue, male Wistar rats, previously trained with low, moderate, or high levels of foot shock, were used to study the effect of tetrodotoxin inactivation of dorsomedial striatum on memory consolidation and subsequent extinction of inhibitory avoidance. Performance of the task was evaluated during seven extinction sessions. Tetrodotoxin produced a marked deficit of memory consolidation of inhibitory avoidance trained with low and moderate intensities of foot shock, but normal consolidation occurred when a relatively high foot shock was used. The protective effect of intense training was long-lasting, as evidenced by the high resistance to extinction exhibited throughout the extinction sessions. We discuss the possibility that increased dendritic spinogenesis in dorsomedial striatum may underly this protective effect, and how this mechanism may be related to the resilient memory typical of post-traumatic stress disorder (PTSD).

Chronic Treatment with Oxcarbazepine Attenuates Its Anticonvulsant Effect in the Maximal Electroshock Model in Mice.

The objective of this study was to assess the impact of acute and chronic treatment with oxcarbazepine on its anticonvulsant activity, neurological adverse effects, and protective index in mice. Oxcarbazepine was administered in four protocols: once or twice daily for one week (7 × 1 or 7 × 2) and once or twice daily for two weeks (14 × 1 or 14 × 2). A single dose of the drug was employed as a control. The anticonvulsant effect was evaluated in the maximal electroshock test in mice. Motor and long-term memory impairment were assessed using the chimney test and the passive avoidance task, respectively. The concentrations of oxcarbazepine in the brain and plasma were determined via high-performance liquid chromatography. Two weeks of oxcarbazepine treatment resulted in a significant reduction in the anticonvulsant (in the 14 × 1; 14 × 2 protocols) and neurotoxic (in the 14 × 2 schedule) effects of this drug. In contrast, the protective index for oxcarbazepine in the 14 × 2 protocol was found to be lower than that calculated for the control. No significant deficits in memory or motor coordination were observed following repeated administration of oxcarbazepine. The plasma and brain concentrations of this anticonvulsant were found to be significantly higher in the one-week protocols. Chronic treatment with oxcarbazepine may result in the development of tolerance to its anticonvulsant and neurotoxic effects, which appears to be dependent on pharmacodynamic mechanisms.

The multiple faces of footshock punishment in animal research on addiction.

Continued drug use despite negative consequences is a hallmark of addiction commonly modelled in rodents using punished drug intake. Over the years, addiction research highlighted two subpopulations of punishment sensitive and resistant animals. While helpful to interrogate the neurobiology of drug-related behaviors, these procedures carry some weaknesses that need to be recognized and eventually defused. Mainly focusing on footshock-related work, we will first discuss the criteria used to define punishment-resistant animals and how their relative arbitrariness may impact our findings. With the overarching goal of improving our interpretation of the punishment-resistant phenotype, we will evaluate how tailored punishment protocols may better apprehend resistance to punishment, and how testing the robustness of punishment resistance could yield new results and strengthen interpretations. Second, we will question whether and to what extent punishment sensitivity, as currently defined, is reflective of abstinence and suggest that punishment resistance is, in fact, a prerequisite to model abstinence from addiction. Again, we will examine how challenging the robustness of the punishment-sensitive phenotype may help to better characterize it. Finally, we will evaluate whether diminished relapse-like behavior after repeated punishment-induced abstinence could not only contribute to better understand the mechanisms of abstinence, but also uniquely model progressive recovery (i.e., after repeated failed attempts at recovery) which is the norm in people with addiction. Altogether, by questioning the strengths and weaknesses of our models, we would like to open discussions on the different ways we interpret punishment sensitivity and resistance and the aspects that remain to be explored.

Punishment-induced resurgence.

The phenomenon of extinction-induced resurgence is well established, but there is comparatively little experimental evidence for punishment-induced resurgence. Punishment-induced resurgence can by tested by contingent shocks following the alternative response. The purpose of Experiment 1 was to test whether low-intensity shocks, that do not decrease rate of reinforcement, result in resurgence. Four rats served as subjects. Rats were exposed to three sequential conditions: (a) variable-interval (VI) 30-s food delivery for a lever press (target response); (b) VI 30-s food delivery for a nose poke (alternative response) and extinction of the lever press; (c) VI 30-s reinforcement for a nose poke with superimposed VI 60-s shock delivery. In the final condition, shocks increased gradually from 0.1 to 0.5 mA. Experiment 2 evaluated whether an abrupt introduction of a high-intensity shock would result in resurgence. Three rats served as subjects and were exposed to three sequential conditions: (a) VI 30-s food delivery for a lever press; (b) VI 30-s food delivery for a nose poke and extinction of the lever press; (c) continued VI 30-s reinforcement for a nose poke with superimposed VI 60-s 0.6 mA shock delivery. Resurgence was observed in all subjects, including in situations in which rate of responding, but not rate of reinforcement, decreased. The present study provides additional evidence for punishment-induced resurgence, but future studies are warranted to determine the extent to which punishment can produce resurgence with or without decreases in rates of reinforcement.

Improved Perception of Aggression Under (un)Related Threat of Shock.

Anxiety shifts visual attention and perceptual mechanisms, preparing oneself to detect potentially threatening information more rapidly. Despite being demonstrated for threat-related social stimuli, such as fearful expressions, it remains unexplored if these effects encompass other social cues of danger, such as aggressive gestures/actions. To this end, we recruited a total of 65 participants and asked them to identify, as quickly and accurately as possible, potentially aggressive actions depicted by an agent. By introducing and manipulating the occurrence of electric shocks, we induced safe and threatening conditions. In addition, the association between electric shocks and aggression was also manipulated. Our result showed that participants have improved sensitivity, with no changes to criterion, when detecting aggressive gestures during threat compared to safe conditions. Furthermore, drift diffusion model analysis showed that under threat participants exhibited faster evidence accumulation toward the correct perceptual decision. Lastly, the relationship between threat source and aggression appeared to not impact any of the effects described above. Overall, our results indicate that the benefits gained from states of anxiety, such as increased sensitivity toward threat and greater evidence accumulation, are transposable to social stimuli capable of signaling danger other than facial expressions.