Bimodal modulation of alpha5 GABAA receptors in an experimental model of Alzheimer’s disease

Ciljevi: Postoje dokazi da je GABAergička modulacija uključena u kognitivne proceseznačajno promenjena kod Alchajmerove bolesti (AD). U široko korišćenom 5xFADmodelu AD, želeli smo da procenimo da li negativni i pozitivni alosterni modulatori α5GABAA receptora (NAM i PAM, respektivno) utiču na socijalnu interakciju, socijalnu,objektnu i prostornu memoriju, senzomotornu funkciju, emocionalnost, motivaciju,ekspresiju subjedinica GABAA receptora i neuroinflamaciju.Metode: Posle produžene primene PAM, NAM ili rastvarača, 6 meseci stari transgeni inetransgeni 5xFAD miševi podvrgnuti su testiranju u bihejvioralnoj bateriji. Ekspresijegena za Gabra2, Gabra3, Gabra5, Il1b, Il-6, Tnfa, Gfap i Iba1 određene su u hipokampusui prefrontalnom korteksu pomoću qPCR metode.Rezultati: PAM tretman narušio je prostorno učenje kod transgenih ženki, socijalnoprepoznavanje kod transgenih i netransgenih mužjaka i motornu funkciju kod transgenihmužjaka. NAM tretman je smanjio socijalnu interakciju kod transgenih i netransgenihmužjaka i emocionalnost kod transgenih mužjaka. NAM je imao povoljan efekat nakognitivnu fleksibilnost kod netransgenih mužjaka. U hipokampusu, oba tretmana su vratilana normalne nivoe recipročne promene u ekspresiji Gabra2 i Gabra3 kod transgenih ženki.U prefrontalnom korteksu, PAM je smanjio Gabra5 kod oba pola, dok je NAM povećaoGabra2 kod transgenih mužjaka. Transgene životinje nisu u potpunosti razvile kognitivnesimptome, ali je potvrđena neuroinflamacija. NAM je smanjio ekspresiju proinflamatornihgena kod transgenih ženki i astroglioze kod transgenih mužjaka.Zaključak: PAM i NAM nisu uspeli da ispolje konzistentno povoljne bihejvioralne efektekod transgenih životinja. Supresija neuroinflamacije dobijena NAM-om zahteva višestudija sa GABAergičkim ligandima u amiloidnim beta- i/ili tau-zavisnim modelima saizraženom neuroinflamacijom.Aims: GABAergic modulation involved in cognitive processing appears to be substantiallychanged in Alzheimer’s disease (AD). In a widely used 5xFAD model of AD, we aimed toassess if negative and positive allosteric modulators of α5 GABAA receptors (NAM andPAM, respectively) would affect social interaction, social, object and spatial memory,sensorimotor function, emotionality, motivation, expression of GABAA receptor subunitsand neuroinflammation.Methods: After protracted treatment with PAM, NAM or solvent, 6-month-old transgenicand non-transgenic 5xFAD mice underwent testing in a behavioral battery. Geneexpressions of Gabra2, Gabra3, Gabra5, Il1b, Il-6, Tnfa, Gfap and Iba1 were determinedin hippocampus and prefrontal cortex by qPCR analysis.Results: PAM treatment impaired spatial learning in transgenic females, and socialrecognition and motor function in both transgenic and non-transgenic males and intransgenic males, respectively. NAM treatment declined social interaction and emotionalityin both transgenic and non-transgenic males and transgenic males, respectively. NAM had abeneficial effect on cognitive flexibility in non-transgenic males. In hippocampus, bothtreatments reversed reciprocal Gabra2 and Gabra3 changes in transgenic females. Inprefrontal cortex, PAM decreased Gabra5 in both genders, while NAM increased Gabra2in transgenic males. Transgenic animals have not fully displayed cognitive symptoms,while neuroinflammation was confirmed. NAM reduced proinflammatory gene expressionsin transgenic females and astrogliosis in transgenic males.Conclusion: PAM and NAM failed to exert consistently favorable behavioral effects intransgenic animals. Suppression of neuroinflammation obtained with NAM calls for morestudies with GABAergic ligands in amyloid beta- and/or tau-dependent models withprominent neuroinflammation

Bimodal modulation of alpha5 GABAA receptors in an experimental model of Alzheimer’s disease

Ciljevi: Postoje dokazi da je GABAergička modulacija uključena u kognitivne procese značajno promenjena kod Alchajmerove bolesti (AD). U široko korišćenom 5xFAD modelu AD, želeli smo da procenimo da li negativni i pozitivni alosterni modulatori α5 GABAA receptora (NAM i PAM, respektivno) utiču na socijalnu interakciju, socijalnu, objektnu i prostornu memoriju, senzomotornu funkciju, emocionalnost, motivaciju, ekspresiju subjedinica GABAA receptora i neuroinflamaciju. Metode: Posle produžene primene PAM, NAM ili rastvarača, 6 meseci stari transgeni i netransgeni 5xFAD miševi podvrgnuti su testiranju u bihejvioralnoj bateriji. Ekspresije gena za Gabra2, Gabra3, Gabra5, Il1b, Il-6, Tnfa, Gfap i Iba1 određene su u hipokampusu i prefrontalnom korteksu pomoću qPCR metode. Rezultati: PAM tretman narušio je prostorno učenje kod transgenih ženki, socijalno prepoznavanje kod transgenih i netransgenih mužjaka i motornu funkciju kod transgenih mužjaka. NAM tretman je smanjio socijalnu interakciju kod transgenih i netransgenih mužjaka i emocionalnost kod transgenih mužjaka. NAM je imao povoljan efekat na kognitivnu fleksibilnost kod netransgenih mužjaka. U hipokampusu, oba tretmana su vratila na normalne nivoe recipročne promene u ekspresiji Gabra2 i Gabra3 kod transgenih ženki. U prefrontalnom korteksu, PAM je smanjio Gabra5 kod oba pola, dok je NAM povećao Gabra2 kod transgenih mužjaka. Transgene životinje nisu u potpunosti razvile kognitivne simptome, ali je potvrđena neuroinflamacija. NAM je smanjio ekspresiju proinflamatornih gena kod transgenih ženki i astroglioze kod transgenih mužjaka. Zaključak: PAM i NAM nisu uspeli da ispolje konzistentno povoljne bihejvioralne efekte kod transgenih životinja. Supresija neuroinflamacije dobijena NAM-om zahteva više studija sa GABAergičkim ligandima u amiloidnim beta- i/ili tau-zavisnim modelima sa izraženom neuroinflamacijom.Aims: GABAergic modulation involved in cognitive processing appears to be substantially changed in Alzheimer’s disease (AD). In a widely used 5xFAD model of AD, we aimed to assess if negative and positive allosteric modulators of α5 GABAA receptors (NAM and PAM, respectively) would affect social interaction, social, object and spatial memory, sensorimotor function, emotionality, motivation, expression of GABAA receptor subunits and neuroinflammation. Methods: After protracted treatment with PAM, NAM or solvent, 6-month-old transgenic and non-transgenic 5xFAD mice underwent testing in a behavioral battery. Gene expressions of Gabra2, Gabra3, Gabra5, Il1b, Il-6, Tnfa, Gfap and Iba1 were determined in hippocampus and prefrontal cortex by qPCR analysis. Results: PAM treatment impaired spatial learning in transgenic females, and social recognition and motor function in both transgenic and non-transgenic males and in transgenic males, respectively. NAM treatment declined social interaction and emotionality in both transgenic and non-transgenic males and transgenic males, respectively. NAM had a beneficial effect on cognitive flexibility in non-transgenic males. In hippocampus, both treatments reversed reciprocal Gabra2 and Gabra3 changes in transgenic females. In prefrontal cortex, PAM decreased Gabra5 in both genders, while NAM increased Gabra2 in transgenic males. Transgenic animals have not fully displayed cognitive symptoms, while neuroinflammation was confirmed. NAM reduced proinflammatory gene expressions in transgenic females and astrogliosis in transgenic males. Conclusion: PAM and NAM failed to exert consistently favorable behavioral effects in transgenic animals. Suppression of neuroinflammation obtained with NAM calls for more studies with GABAergic ligands in amyloid beta- and/or tau-dependent models with prominent neuroinflammation

Deciphering the reward-related impulsivity domains in rats: The big data study of historical control

Impulsivity is a lack of ability to control own impulses, and encompasses many subdomains. The variable-delay-to-signal (VDS) paradigm is behavioral procedure for assessing motor impulsivity and delay intolerance in rats, but it was unclear whether all parameters contributed to these domains. Therefore, the aim of this study was to uncover the relationship between impulsivity parameters in a large cohort. VDS adapted to a touchscreen environment was used to assess impulsivity in adult Sprague-Dawley rats. After 1 week of training, animals were tested in a 3-stage testing protocol. The first stage included 20 trials with 6s inter-trial interval (ITI6si) that suggested motor impulsivity. The second stage, with 60 randomly distributed trials of ITI9s or 15s, was interpreted as delay intolerance, whereas for the last stage (ITI6sf), which is similar to the first stage, it was unclear to which type of impulsivity it was associated. Principal component analysis (PCA) was used to determine the different behavioral domains. The results of 132 controls from 11 independent VDS experiments were analyzed. Based on the cumulative variance explained, scree plot, and eigenvalues, the main components were extracted whereby varimax rotation was used on factor loadings to extract the components. PCA with varimax rotation was performed in R studio. PCA revealed that 96.45% of the variance could be explained by 3 principal components (PCs). After varimax rotation, loadings for ITI9s and ITI15s were 0.8189 and 0.9419, respectively, for rotated PC1 (RC1), loading for ITI6sf was 0.9482 for RC2, and loading for 6si was 0.9183 for RC3. In the VDS paradigm, 3 different impulsivity domains could be determined. In addition to motor impulsivity and delay intolerance, it is suggested that reflection impulsivity can also be assessed as learning-based impulsivity.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Reward-related impulsivity as a possible surrogate marker of motivation in aging Sprague-Dawley rats

Introduction: Impulsivity is an umbrella term that encompasses many subdomains, most of which rely on the decision-making processes. It is reported that in the process of healthy aging, the two dimensions of impulse control, cognition and motivation, are preserved or even improved. On the other hand, the attentive efficiency seems to decrease with age. Therefore, we aimed to investigate the effects of healthy aging on impulsivity in rats and the influence of food deprivation on impulsivity in aged rats as a strategy to enhance motivation. Additionally, we wanted to assess the gene expression for the alpha5 GABAA receptor subunit during aging, which plays a role in cognitive processes. Methodology: The variable-delay-to-signal (VDS) paradigm adapted to a touchscreen environment was used to assess impulsivity and attention in Sprague-Dawley rats at 2, 3, 5, 8, and 14 months of age. After one week of training, animals were tested at different ages in 3-stage testing protocol. Additionally, prior to testing, animals were fed a restricted diet (16 g/animal). The first stage included 20 trials with inter-trial interval of 6s (ITI6si) that reflected motor impulsivity. The second stage, with 60 randomly distributed trials of ITI9s or 15s, was related to delay intolerance, while the final stage (ITI6sf), similar to the first, was related to reflection impulsivity. The strict 3-day restriction diet (24h food deprivation followed by 10g/day/animal and 8g/day/animal) was applied to 14-month-old animals before testing. Gabra5 expression in the hippocampus was determined by qPCR. Results were analyzed by one-way ANOVA with or without repeated measures, followed by Sidak post-hoc test for impulsivity and attention parameters and by t-test for PCR parameters. Results: Animals aged 8 and 14 months had reduced motor impulsivity (p<0.01 for both groups) and delay intolerance (p<0.05 for both groups) and higher number of omissions (p<0.05 for both groups) compared to animals aged 2, 3 and 5 months of age. In addition, half of the animals were unable to successfully complete a task after 14 months. After rigorous food restriction in 14-month-old animals, the level of impulsivity (ITI9s and ITI15s) and attention (number of omissions) returned to the control level (2 and 3 months of age) compared to the performance of 14-month-old animals prior to rigorous food restriction (p<0.05). Further, the peak of reflection impulsivity (ITI6sf) was reached at 5 months compared to all other groups (p<0.01). No changes in Gabra5 expression in hippocampus were detected in 14-month-old compared to 3-month-old animals. Conclusion: From 8 months of age onwards, rats showed reduced impulsivity in the VDS stages where motor impulsivity and delay intolerance were tested, followed by attention deficits. After strict food restriction in 14-month-old animals, delay intolerance and attention were restored, suggesting the prominent role of motivation in controlling these processes, independently of Gabra5 expression levels in the hippocampus. Since the VDS paradigm aims to assess reward-related impulsivity based on cognition and motivation, it is suspected that results related to impaired cognition in older animals in other cognitive tests should be interpreted with caution, and with additional observation of motivation

Maternal deprivation causes CaMKII downregulation and modulates glutamate, norepinephrine and serotonin in limbic brain areas in a rat model of single prolonged stress

Background: Early life stress is a major risk factor for later development of psychiatric disorders, including post-traumatic stress disorder (PTSD). An intricate relationship exists between various neurotransmitters (such as glutamate, norepinephrine or serotonin), calcium/calmodulin-dependent protein kinase II (CaMKII), as an important regulator of glutamatergic synaptic function, and PTSD. Here, we developed a double-hit model to investigate the interaction of maternal deprivation (MD) as an early life stress model and single prolonged stress (SPS) as a PTSD model at the behavioral and molecular levels. Methods: Male Wistar rats exposed to these stress paradigms were subjected to a comprehensive behavioral analysis. In hippocampal synaptosomes we investigated neurotransmitter release and glutamate concentration. The expression of CaMKII and the content of monoamines were determined in selected brain regions. Brain-derived neurotrophic factor (BDNF) mRNA was quantified by radioactive in situ hybridization. Results: We report a distinct behavioral phenotype in the double-hit group. Double-hit and SPS groups had decreased hippocampal presynaptic glutamatergic function. In hippocampus, double-hit stress caused a decrease in autophosphorylation of CaMKII. In prefrontal cortex, both SPS and double-hit stress had a similar effect on CaMKII autophosphorylation. Double-hit stress, rather than SPS, affected the norepinephrine and serotonin levels in prefrontal cortex, and suppressed BDNF gene expression in prefrontal cortex and hippocampus. Limitations: The study was conducted in male rats only. The affected brain regions cannot be restricted to hippocampus, prefrontal cortex and amygdala. Conclusion: Double-hit stress caused more pronounced and distinct behavioral, molecular and functional changes, compared to MD or SPS alone

Deciphering ciprofloxacin-induced neurotoxicity: behavioral and molecular profiling of ciprofloxacin treatment in rats

Introduction: Ciprofloxacin is a fluoroquinolone antibiotic commonly used to treat various bacterial infections, with a potential to induce adverse mood effects in patients. Since the molecular mechanism of ciprofloxacin-induced neurotoxicity is poorly understood, we aimed to identify behavioral changes and corresponding neurotransmitter pattern after its prolonged administration in rats. We screened for untoward effects of ciprofloxacin on locomotor activity, despair, anhedonia, object recognition memory, and anxiety, as behavioral domains affected in various psychiatric diseases. Methodology: Three-month old male Sprague-Dawley rats were orally gavaged with ciprofloxacin (20 or 100 mg/kg) or solvent (0.5% methyl cellulose solution) each day for 4 weeks (n=80). One group of animals (n=40) passed the open field (OF), novel object recognition test (NORT), and forced swimming test (FST). Another group (n=40) underwent elevated plus maze (EPM) and sucrose preference test (SPT). After the completion of behavioral battery, the prefrontal cortex and cerebrospinal fluid (CSF) were collected. The neurotransmitters and metabolites of the kynurenine pathway were determined in the prefrontal cortex (PFC) through HPLC-MS/MS. Additionally, levels of interleukin-2 (IL-2) in CSF were quantified with Luminex. Behavioral and molecular parameters were analyzed by one-way ANOVA followed by Dunnett post hoc test in GraphPad Prism 9. Results: In FST, the treatment with high dose of ciprofloxacin decreased the swim time compared to control, which could be related to induction of despair-like behavior (p<0.05). The ciprofloxacin treatment did not affect object memory in NORT. In OF, the distance travelled and the number of rotations were not changed after treatment with ciprofloxacin compared to the control group. Further, animals treated with ciprofloxacin did not show changes in parameters in EPM and SPT. The acetylcholine levels in PFC were increased after ciprofloxacin treatment (p<0.05) in comparison with controls, which could be associated with depressed mood states. In line with that, high dose of ciprofloxacin treatment showed the tendency to decrease and increase levels of GABA and dopamine, respectively, but without reaching the statistical significance (p=0.07 and p=0.06). No changes in kynurenine pathway were observed after the treatment. The IL-2 concentration in CSF was increased after prolonged administration of low dose of ciprofloxacin treatment compared to the control levels (p<0.05), which could imply immunological stimulation of T lymphocytes and potential neuroinflammation. Conclusion: The despair behavior after treatment with high dose of ciprofloxacin was accompanied by increased levels of acetylcholine in PFC. Furthermore, the high dose of ciprofloxacin treatment showed tendency to decrease GABA levels, and increase dopamine levels in PFC, which could be connected to psychiatric adverse effects. Nonetheless, further studies are essential to confirm these neurotransmitter changes. On the other hand, the low dose of ciprofloxacin treatment elicited the increase of IL-2, which could be a marker of neuroinflammation-related neurotoxicity. In the future, efforts should be made to examine the role of IL-2 in the interaction of the immune system and the central nervous system, as its potential significance as a biomarker

Effects of α5 GABAA receptor modulation on social interaction, memory, and neuroinflammation in a mouse model of Alzheimer's disease

Aims GABAergic modulation involved in cognitive processing appears to be substantially changed in Alzheimer's disease (AD). In a widely used 5xFAD model of AD, we aimed to assess if negative and positive allosteric modulators of α5 GABAA receptors (NAM and PAM, respectively) would affect social interaction, social, object and spatial memory, and neuroinflammation. Methods After 10-day treatment with PAM, NAM, or solvent, 6-month-old transgenic and non-transgenic 5xFAD mice underwent testing in a behavioral battery. Gene expressions of IL-1β, IL-6, TNF-α, GFAP, and IBA-1 were determined in hippocampus and prefrontal cortex by qPCR. Results PAM treatment impaired spatial learning in transgenic females compared to solvent-treated transgenic females, and social recognition in transgenic and non-transgenic males. NAM treatment declined social interaction in transgenic and non-transgenic males, while had beneficial effect on cognitive flexibility in non-transgenic males compared to solvent-treated non-transgenic males. Transgenic animals have not fully displayed cognitive symptoms, but neuroinflammation was confirmed. NAM reduced proinflammatory gene expressions in transgenic females and astrogliosis in transgenic males compared to pathological controls. Conclusion PAM and NAM failed to exert favorable behavioral effects in transgenic animals. Suppression of neuroinflammation obtained with NAM calls for more studies with GABAergic ligands in amyloid beta- and/or tau-dependent models with prominent neuroinflammation

Curcumin loaded pegylated nanoemulsions designed for maintained antioxidant effects and improved bioavailability: A pilot study on rats

The current study describes the experimental design guided development of PEGylated nanoemulsions as parenteral delivery systems for curcumin, a powerful antioxidant, as well as the evaluation of their physicochemical characteristics and antioxidant activity during the two years of storage. Experimental design setup helped development of nanoemulsion templates with critical quality attributes in line with parenteral application route. Curcumin-loaded nanoemulsions showed mean droplet size about 105 nm, polydispersity index <0.15, zeta potential of −40 mV, and acceptable osmolality of about 550 mOsm/kg. After two years of storage at room temperature, all formulations remained stable. Moreover, antioxidant activity remained intact, as demonstrated by DPPH (IC50 values 0.078–0.075 mg/mL after two years) and FRAPS assays. In vitro release testing proved that PEGylated phospholipids slowed down the curcumin release from nanoemulsions. The nanoemulsion carrier has been proven safe by the MTT test conducted with MRC-5 cell line, and effective on LS cell line. Results from the pharmacokinetic pilot study implied the PEGylated nanoemulsions improved plasma residence of curcumin 20 min after intravenous administration, compared to the non-PEGylated nanoemulsion (two-fold higher) or curcumin solution (three-fold higher). Overall, conclusion suggests that developed PEGylated nanoemulsions present an acceptable delivery system for parenteral administration of curcumin, being effective in preserving its stability and antioxidant capacity at the level highly comparable to the initial findings

Bimodal modulation of alpha5 GABAA receptors in an experimental model of Alzheimer’s disease

Ciljevi: Postoje dokazi da je GABAergička modulacija uključena u kognitivne procese značajno promenjena kod Alchajmerove bolesti (AD). U široko korišćenom 5xFAD modelu AD, želeli smo da procenimo da li negativni i pozitivni alosterni modulatori α5 GABAA receptora (NAM i PAM, respektivno) utiču na socijalnu interakciju, socijalnu, objektnu i prostornu memoriju, senzomotornu funkciju, emocionalnost, motivaciju, ekspresiju subjedinica GABAA receptora i neuroinflamaciju. Metode: Posle produžene primene PAM, NAM ili rastvarača, 6 meseci stari transgeni i netransgeni 5xFAD miševi podvrgnuti su testiranju u bihejvioralnoj bateriji. Ekspresije gena za Gabra2, Gabra3, Gabra5, Il1b, Il-6, Tnfa, Gfap i Iba1 određene su u hipokampusu i prefrontalnom korteksu pomoću qPCR metode. Rezultati: PAM tretman narušio je prostorno učenje kod transgenih ženki, socijalno prepoznavanje kod transgenih i netransgenih mužjaka i motornu funkciju kod transgenih mužjaka. NAM tretman je smanjio socijalnu interakciju kod transgenih i netransgenih mužjaka i emocionalnost kod transgenih mužjaka. NAM je imao povoljan efekat na kognitivnu fleksibilnost kod netransgenih mužjaka. U hipokampusu, oba tretmana su vratila na normalne nivoe recipročne promene u ekspresiji Gabra2 i Gabra3 kod transgenih ženki. U prefrontalnom korteksu, PAM je smanjio Gabra5 kod oba pola, dok je NAM povećao Gabra2 kod transgenih mužjaka. Transgene životinje nisu u potpunosti razvile kognitivne simptome, ali je potvrđena neuroinflamacija. NAM je smanjio ekspresiju proinflamatornih gena kod transgenih ženki i astroglioze kod transgenih mužjaka. Zaključak: PAM i NAM nisu uspeli da ispolje konzistentno povoljne bihejvioralne efekte kod transgenih životinja. Supresija neuroinflamacije dobijena NAM-om zahteva više studija sa GABAergičkim ligandima u amiloidnim beta- i/ili tau-zavisnim modelima sa izraženom neuroinflamacijom.Aims: GABAergic modulation involved in cognitive processing appears to be substantially changed in Alzheimer’s disease (AD). In a widely used 5xFAD model of AD, we aimed to assess if negative and positive allosteric modulators of α5 GABAA receptors (NAM and PAM, respectively) would affect social interaction, social, object and spatial memory, sensorimotor function, emotionality, motivation, expression of GABAA receptor subunits and neuroinflammation. Methods: After protracted treatment with PAM, NAM or solvent, 6-month-old transgenic and non-transgenic 5xFAD mice underwent testing in a behavioral battery. Gene expressions of Gabra2, Gabra3, Gabra5, Il1b, Il-6, Tnfa, Gfap and Iba1 were determined in hippocampus and prefrontal cortex by qPCR analysis. Results: PAM treatment impaired spatial learning in transgenic females, and social recognition and motor function in both transgenic and non-transgenic males and in transgenic males, respectively. NAM treatment declined social interaction and emotionality in both transgenic and non-transgenic males and transgenic males, respectively. NAM had a beneficial effect on cognitive flexibility in non-transgenic males. In hippocampus, both treatments reversed reciprocal Gabra2 and Gabra3 changes in transgenic females. In prefrontal cortex, PAM decreased Gabra5 in both genders, while NAM increased Gabra2 in transgenic males. Transgenic animals have not fully displayed cognitive symptoms, while neuroinflammation was confirmed. NAM reduced proinflammatory gene expressions in transgenic females and astrogliosis in transgenic males. Conclusion: PAM and NAM failed to exert consistently favorable behavioral effects in transgenic animals. Suppression of neuroinflammation obtained with NAM calls for more studies with GABAergic ligands in amyloid beta- and/or tau-dependent models with prominent neuroinflammation