Journal: Cureus

Article Title: Role of Adenosine A1 Receptor in Sleep Deprivation-Induced Neuroinflammation: Insights on Rapid Eye Movement Sleep and Fear Extinction Memory Recall in Rats

doi: 10.7759/cureus.75926

Figure Lengend Snippet: The study involved the following experimental timeline: 1) habituation and baseline behavioural recordings, 2) fear conditioning, 3) fear extinction training, 4) animals divided into three groups (a. CC (ad libitum sleep), b. SD (sleep deprivation for 48 hours + vehicle, i.e., 20% DMSO), c. SD + CPT (8-cyclopentyltheophylline, adenosine A1 receptor antagonist was administered for two days during 48-hour SD exposure), 5) fear extinction recall test and depressive behaviour assessment, 6) sleep architecture recording, 6) brain region dissection for molecular analyses. Out of the 44 animals screened for locomotor activity, 39 animals qualified for behavioural experiments. Five animals were excluded due to higher anxiety levels and abnormal locomotor activity. All experimental animals were handled and habituated by the experimenter, twice a day, for three days. Then, the animals were subjected to cued fear conditioning on day 1. After 24 hours, cued fear extinction training was given on day 2. Then, the animals were divided into three groups: CC (ad libitum sleep), SD (sleep deprivation for 48 hours + vehicle, i.e., 20% DMSO), SD + CPT (8-cyclopentyltheophylline, adenosine A1 receptor antagonist was administered for two days during the 48-hour SD exposure). Immediately, after the SD 48-hour exposure, all the experimental animals including control animals with ad libitum sleep were tested for the cued fear extinction recall test, open field test (OFT) on day 4, sucrose preference test for eight hours, forced swim test (FST) on day 5, followed by the dissection and collection of the brain sample. Brain samples were processed further for Immunohistochemistry (IHC), RT-PCR, ELISA, and high-performance liquid chromatography (HPLC) experiments. ‘n’ refers to sample size/number of animals in each group.

Article Snippet: Assessment of Fear Extinction Memory Recall Apparatus: The fear conditioning experiment was carried out in a chamber (Context A) constructed with Plexiglass cages and aluminium walls (Ugo Basile instruments, Italy) on day 1.

Techniques: Dissection, Activity Assay, Control, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, High Performance Liquid Chromatography

Journal: Cureus

Article Title: Role of Adenosine A1 Receptor in Sleep Deprivation-Induced Neuroinflammation: Insights on Rapid Eye Movement Sleep and Fear Extinction Memory Recall in Rats

doi: 10.7759/cureus.75926

Figure Lengend Snippet: A. Schematic representation of fear conditioning and cued extinction recall paradigm. B. Statistical comparison graphs (two-way ANOVA, Tukey’s post-hoc, n = 11) depicting the freezing scores exhibited during fear conditioning (day 1), fear extinction (day 2), and fear extinction recall (day 4) post either 48-hour sleep deprivation with vehicle (DMSO) (F(4, 60) = 15.8; p < 0.001; n = 11) or A1R antagonist CPT (p = 0.01; n = 11), and ad libitum sleep (cage control group). *p < 0.05 represents the comparison between CC and SD; #p < 0.05 represents the comparison between SD and SD + CPT. CC: cage control; SD: 48-hour sleep deprivation; SD + CPT: 48-hour sleep deprivation + 8-cyclopentyltheophylline

Article Snippet: Assessment of Fear Extinction Memory Recall Apparatus: The fear conditioning experiment was carried out in a chamber (Context A) constructed with Plexiglass cages and aluminium walls (Ugo Basile instruments, Italy) on day 1.

Techniques: Comparison, Control

Journal: Nature Communications

Article Title: A pH-sensitive closed-loop nanomachine to control hyperexcitability at the single neuron level

doi: 10.1038/s41467-024-49941-3

Figure Lengend Snippet: A Schematics of the bilateral stereotaxic injection of AAV2/1, encoding either Ctrl or pHIL under the CaMKIIα promoter, in the dorsal hippocampus of 2-month-old wild type C57BL/6 mice followed, one month later, by whole-body bioluminescence imaging and behavioral tests after vehicle or CTZ 400a (CTZ) administration (Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en )). B Left: Representative whole-body bioluminescence images (merged emission of both RLuc8 and E 2 GFP) of Ctrl ( top row ) and pHIL ( bottom row ) transduced mice acquired every 5 min after the intravenous injection of CTZ (0.3 mg/kg). Radiance intensity is shown in pseudocolors. Right: The quantitative evaluation of the mean (± SEM) RLuc8/E 2 GFP live average radiance values (means ± SEM) in Ctrl ( black bars ) and pHIL ( red bars ) transduced mice displays an early emission peak 5 min after CTZ administration, followed by a progressive decrease ( n = 11 mice for both Ctrl and pHIL). C Left: Representative whole-body bioluminescence images acquired 5 min after the administration of increasing doses (0.15, 0.3 and 0.6 mg/kg) of CTZ to pHIL-transduced mice. Right: Corresponding RLuc8/E 2 GFP live average radiance values (means ± SEM) for the three doses as a function of time after CTZ administration. For further details see panel B ( n = 6, 10, 5 mice for 0.15, 0.3 and 0.6 mg/kg). D – F The locomotor activity and hippocampus-dependent behavior were investigated in pHIL-transduced mice upon administration of either CTZ (0.3 and 0.6 mg/kg) or the respective vehicle. The control condition (dose = 0 mg/kg) refers to untreated pHIL-transduced mice. D Open field test. The total distance covered by the mice ( top ) and the time spent in the center or along the border ( bottom ) were comparable under all tested conditions, proving no interference of the pharmaceutical treatment with locomotor activity (means ± SEM of n = 7, 7, 10 for 0, 0.3 and 0.6 mg/kg respectively in both Veh and CTZ groups). E Novel object recognition. No effects of CTZ were observed both in the familiarization phase ( top ) and in the recognition phase ( bottom ) when mice were exposed to one object previously explored and a novel unfamiliar object (means ± SEM of n = 7, 7, 7 for 0, 0.3 and 0.6 mg/kg respectively in both Veh and CTZ groups). F Contextual fear conditioning. No significant differences in freezing times were observed both during the fear conditioning phase ( top ) and in control exposure to a new context ( bottom ) (means ± SEM of n = 7, 7, 7 for 0, 0.3, and 0.6 mg/kg respectively in both Veh and CTZ groups). In E and F, either CTZ or vehicle was administered before the novel recognition phase and the conditioning session, respectively. p > 0.05, two-way repeated measures ANOVA (D-F).

Article Snippet: The contextual fear-conditioning test was conducted in the Video Fear Conditioning apparatus (Med Associates Inc., Fairfax, VT).

Techniques: Injection, Imaging, Activity Assay, Control

Journal: Brain sciences

Article Title: Influence of Stress Severity on Contextual Fear Extinction and Avoidance in a Posttraumatic-like Mouse Model.

doi: 10.3390/brainsci14040311

Figure Lengend Snippet: Figure 1. Timeline of behavioral tests performed by mice from all groups. Stressed mice were conditioned to a specific context by receiving either two footshocks (PTSD2 group, n = 20) or four footshocks (PTSD4 group, n = 20), while mice from the control group (CTRL, n = 18) were only exposed to the context without receiving any electrical shock. Behaviors mimicking PTSD symptoms were evaluated before and after a three-day extinction learning protocol (Days 23–25, 20 min re-exposures to the context without footshocks). Extinction retrieval (Day 28) corresponded to a five-minute-long exposure to the context without footshocks, three days after the extinction learning. The object present in the conditioning context was used as a reminder of the fearful event during the avoidance test (Day 21 and Day 35) and compared to a familiar but neutral object. Objects used during the Novel Object Recognition test (NOR) were never presented to the animals before nor reused for other tests. LDB stands for light and dark box test. CFC stands for contextual fear conditioning.

Article Snippet: Mice were placed i a fear conditioning apparatus (Noldus/Ugo Basile Fear conditioning setup) that is a sound-attenuation chamber containing a cage (26 × 26 × 36 cm(h)cm) with a grid floor allowing footshock delivery.

Techniques: Control

Journal: Brain sciences

Article Title: Influence of Stress Severity on Contextual Fear Extinction and Avoidance in a Posttraumatic-like Mouse Model.

doi: 10.3390/brainsci14040311

Figure Lengend Snippet: Figure 3. The long-term effect of the number of footshocks on PTSD-like behaviors before extinction learning. (A) The distance traveled in the arena during the habituation phase of the NOR was deceased in the conditioned group compared to CTRL, two weeks after fear conditioning. The arena was an unknown circular open field, dimly lit. (B) This distance traveled was negatively correlated to freezing expressed at the first re-exposure only for PTSD4 mice. (C) During the learning phase of the NOR, both conditioned groups took longer to become familiarized with the object compared to CTRL. (D) During the testing phase of the NOR, all groups traveled the same distance within the open field and (E) CTRL mice did not explore a new object more compared to the known object, underlying an absence of learning. However, PTSD4 mice explored this new object less compared to the known object (<50%), the CTRL group (p = 0.05) and the PTSD2 group. (F) Both fear-conditioned groups traveled less distance during the avoidance test compared to CTRL. The arena was divided in three equal chambers and placed under a low red light. (G) The neutral object corresponded to a known object previously displayed in the home cage. The reminder was the object that was present during fear conditioning. CTRL mice explored both object the same amount of time and significantly more than conditioned mice. PTSD2 and PTSD4 mice explored the reminder significantly less compared to the neutral object. (H) The objects were placed in the two opposite chambers and time spent in each chamber was measured. Conditioned mice spent significantly less time in the chamber containing the reminder compared to the other chamber or CTRL no matter the number of footshocks. Results are represented by the mean ± SEM, *** p < 0.001, ** p < 0.01 and * p < 0.05 vs. CTRL; !! p < 0.01 and !!! p < 0.001 neutral object vs. reminder within the same group, ## p < 0.01 vs. 50%, $ p < 0.05 vs. PTSD2. CTRL, control group (n = 18); PTSD2, mice that had received two shocks (n = 20); PTSD4, mice that had received four shocks (n = 20); NOR, Novel Object Recognition test.

Article Snippet: Mice were placed i a fear conditioning apparatus (Noldus/Ugo Basile Fear conditioning setup) that is a sound-attenuation chamber containing a cage (26 × 26 × 36 cm(h)cm) with a grid floor allowing footshock delivery.

Techniques: Control