Home Cage Research Articles

Requisite role of dorsal raphé in contextual cocaine-memory reconsolidation

Memory reconsolidation is a process by which labile drug memories are restabilized in long-term memory stores, permitting their enduring control over drug-seeking behaviors. In the present study, we investigated the involvement of the dorsal raphé nuclei (DRN) in cocaine-memory reconsolidation. Sprague-Dawley rats (male, female) were trained to self-administer cocaine in a distinct environmental context to establish contextual drug memories. They then received extinction training in a different context. Next, the rats were re-exposed to the cocaine-predictive context for 15 min to reactivate their cocaine memories or remained in their home cages (no-reactivation control). Memory reactivation was sufficient to increase c-Fos expression, an index of neuronal activation, in the DRN, but not in the median raphé nuclei, during reconsolidation, compared to no reactivation. To determine whether DRN neuronal activity was necessary for cocaine-memory reconsolidation, rats received intra-DRN baclofen plus muscimol (BM; GABAB/A agonists) or vehicle microinfusions immediately after or 6 h after a memory reactivation session conducted with or without lever access. The effects of DRN functional inactivation on long-term memory strength, as indicated by the magnitude of context-induced cocaine seeking, were assessed 72 h later. Intra-DRN BM treatment immediately after memory reactivation with or without lever access attenuated subsequent context-induced cocaine-seeking behavior, independent of sex. Conversely, BM treatment in the adjacent periaqueductal gray (PAG) immediately after memory reactivation, or BM treatment in the DRN 6 h after memory reactivation, did not alter responding. Together, these findings indicate that the DRN plays a requisite role in maintaining cocaine-memory strength during reconsolidation.

Exercise leads to sex-specific recovery of behavior and pathological AD markers following adolescent ethanol exposure in the TgF344-AD model.

Human epidemiological studies suggest that heavy alcohol consumption may lead to earlier onset of Alzheimer's Disease (AD), especially in individuals with a genetic predisposition for AD. Alcohol-related brain damage (ARBD) during a critical developmental timepoint, such as adolescence, interacts with AD-related pathologies to accelerate disease progression later in life. The current study investigates if voluntary exercise in mid-adulthood can recover memory deficits caused by the interactions between adolescence ethanol exposure and AD-transgenes. Male and female TgF344-AD and wildtype F344 rats were exposed to an intragastric gavage of water (control) or 5 g/kg of 20% ethanol (adolescent intermittent ethanol; AIE) for a 2 day on/off schedule throughout adolescence (PD27-57). At 6 months old, rats either remained in their home cage (stationary) or were placed in a voluntary wheel running apparatus for 4 weeks and then underwent several behavioral tests. The number of cholinergic neurons in the basal forebrain and measure of neurogenesis in the hippocampus were assessed. Voluntary wheel running recovers spatial working memory deficits selectively in female TgF344-AD rats exposed to AIE and improves pattern separation impairment seen in control TgF344-AD female rats. There were sex-dependent effects on brain pathology: Exercise improves the integration of recently born neurons in AIE-exposed TgF344-AD female rats. Exercise led to a decrease in amyloid burden in the hippocampus and entorhinal cortex, but only in male AIE-exposed TgF344-AD rats. Although the number of basal forebrain cholinergic neurons was not affected by AD-transgenes in either sex, AIE did reduce the number of basal forebrain cholinergic neurons in female rats. These data provide support that even after symptom onset, AIE and AD related cognitive decline and associated neuropathologies can be rescued with exercise in unique sex-specific ways.

Antioxidant associated chemoprophylaxis effect of natural spice and green vegetable on hepatotoxicity.

This is a case control and cohort study, conducted at Department of Biochemistry in FUAST, compressing 24 number of albino Wistar rats. Illicium verum (star anise) natural spice and green vegetable broccoli are used for chemo-preventive herbal treatment in relation with vitamin C antioxidant activity. Hepatotoxicity was induced in liver of model rats by giving interperitoneally single injection of 200 mg/kg bod weight (B.W) of N-nitrosodiethylamine and orally giving 1 mg/kg BW phenobarbital for 14 days during disease prorogation period. The vitamin C analysis from Illicium verum (star anise), green broccoli, orange juice, orange peel and orange pulp was done by iodometric quantitative and qualitative titration method. Rats were given compound according to their bodyweight as 1 mg/kg B.W. After 14 days (Disease Promotion and Propagation Period) and 28 days (Disease cure period) of treatment behavioral activity was monitored by locomotors activity in open field experiment, stimulatory activity in home cage and anxiolytic effects observed in light and dark apparatus and also in T maze. Behavioral activity were significantly increased in Illicium verum and green broccoli treated rats. Hematological study including hemoglobin (Hb), white blood cells (WBC), red blood cells (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW) and Platelets was done by abbot laboratory analyzer cell. The value of Hb and RBC was also significantly increased in star anise and broccoli treated rats, showed proper function of RBC in microcirculation. WBC was also in normal range revealed that no disease regards to bone marrow and leukemia's. The biochemical activity of rats including urea, creatinine, total protein, albumin, globulin, A/G ratio, amylase and calcium in blood serum were analyzed while, liver health or performance was also determined by LFT (liver function test). Behavioral and laboratorial variables were analyzed by using SPSS v. 20 and p-value < 0.05 was considered statistically significant. The study report noticeable significant effect of Illicium verum and broccoli on hepatotoxicity of experimental model. Recent experimental study statistical outcomes show reciprocal relationship between the consumption of vegetable diet and natural spice with the risk of Hepatotoxicity. Thus, the aim of the study is to enhance the function of star anise or illicium verum natural spice and green vegetable broccoli as an anticancer or chemoprophylaxis agent.

Tactile stimulation of young WAG/Rij rats prevents development of depression but not absence epilepsy.

Investigations in Wistar Albino Glaxo from Rijswijk (WAG/Rij) rats that are susceptible to genetic absence epilepsy have demonstrated that environmental modifications affect absence seizures. Previously, we showed that neonatal tactile stimulations produce disease-modifying effect on genetically determined absence epilepsy and associated depression in Wag/Rij rats. The study presented here examined the effect of TS during late ontogenesis (adolescence and young adulthood) on epilepsy and depression outcomes in this genetically epileptic rat strain. On postnatal day (PND) 38, male WAG/Rij rats randomly were assigned to either the tactile stimulation (TS), handled or control group (unhandled) with 8 animals in each group. Following a 7-day adaptation period to their new surroundings, the animals were submitted to tactile stimulation from PND 45 to PND 90, five days per week, for 5 min daily. The tactile-stimulated rat was removed from its cage, placed on the experimenter's lap, and had its neck and back gently stroked by the researcher. The handled rats were taken to another cage and left alone for 5 min daily from PND 45 to PND 90. The control rats were left undisturbed in their home cage, except for regular cage cleaning. After PND 90, all rats were left undisturbed until behavioral testing and EEG recording. When the animals were 7 months old, they were subjected to the sucrose consumption test (SCT) and the forced swimming test (FST). Electroencephalogram (EEG) recordings were made at 8 months of age in order to measure electroencephalographic seizure activity, thus, the spike-wave discharges (SWDs). Tactile-stimulated rats showed increased sucrose consumption and number of approaches to the sucrose solution in the SCT when compared with the handled and control rats. In the FST, rats in TS group showed lower immobility time and greater immobility latency, active swimming time and diving frequency than the handled and control rats. The duration and the number of seizures were not different amongst the groups. The data obtained suggest that TS in young rats is able to prevent depression in WAG/Rij rats.

Behavioral and histological analyses of the mouse Bassoon p.P3882A mutation corresponding to the human BSN p.P3866A mutation.

Tauopathy is known to be a major pathognomonic finding in important neurodegenerative diseases such as progressive supranuclear palsy (PSP) and corticobasal degeneration. However, the mechanism by which tauopathy is triggered remains to be elucidated. We previously identified the point mutation c.11596C > G, p.Pro3866Ala in the Bassoon gene (BSN) in a Japanese family with PSP-like syndrome. We showed that mutated BSN may have been involved in its own insolubilization and tau accumulation. Furthermore, BSN mutations have also been related to various neurological diseases. In order to further investigate the pathophysiology of BSN mutation in detail, it is essential to study it in mouse models. We generated a mouse model with the mouse Bassoon p.P3882A mutation, which corresponds to the human BSN p.P3866A mutation, knock-in (KI) and we performed systematic behavioral and histological analyses. Behavioral analyses revealed impaired working memory in a Y-maze test at 3 months of age and decreased locomotor activity in the home cage at 3 and 12 months of age in KI mice compared to those in wild-type mice. Although no obvious structural abnormalities were observed at 3 months of age, immunohistochemical studies showed elevation of Bsn immunoreactivity in the hippocampus and neuronal loss without tau accumulation in the substantia nigra at 12 months of age in KI mice. Although our mice model did not show progressive cognitive dysfunction and locomotor disorder like PSP-like syndrome, dopaminergic neuronal loss was observed in the substantia nigra in 12-month-old KI mice. It is possible that BSN mutation may result in dopaminergic neuronal loss without locomotor symptoms due to the early disease stage. Thus, further clinical course can induce cognitive dysfunction and locomotor symptoms.

Accurate locomotor activity profiles of group-housed mice derived from home cage monitoring data.

Holistic phenotyping of rodent models is increasing, with a growing awareness of the 3Rs and the fact that specialized experimental setups can also impose artificial restrictions. Activity is an important parameter for almost all basic and applied research areas involving laboratory animals. Locomotor activity, the main form of energy expenditure, influences metabolic rate, muscle mass, and body weight and is frequently investigated in metabolic disease research. Additionally, it serves as an indicator of animal welfare in therapeutic, pharmacological, and toxicological studies. Thus, accurate and effective measurement of activity is crucial. However, conventional monitoring systems often alter the housing environment and require handling, which can introduce artificial interference and lead to measurement inaccuracies. Our study focused on evaluating circadian activity profiles derived from the DVC and comparing them with conventional activity measurements to validate them statistically and assess their reproducibility. We utilized data from metabolic studies, an Alzheimer's disease model known for increased activity, and included DVC monitoring in a project investigating treatment effects on activity in a type-1-like diabetes model. The DVC data yielded robust, scientifically accurate, and consistent circadian profiles from group-housed mice, which is particularly advantageous for longitudinal experiments. The activity profiles from both systems were fully comparable, providing matching profiles. Using DVC monitoring, we confirmed the hyperactivity phenotype in an AD model and reproduced a decline in activity in type-1-like diabetes model. In our work, we derived robust circadian activity profiles from the DVC data of group-housed mice, which were scientifically accurate, reproducible and comparable to another activity measurement. This approach can not only improve animal welfare according to the 3R principles but can also be implement in high-throughput longitudinal studies. Furthermore, we discuss the advantages and limitations of DVC activity measurements to highlight its potential and avoid confounders.

Voluntary wheel running access produces opposite effects in male and female rats on both palatable diet consumption and associated ventral striatal opioid- and dopamine-related gene expression.

The relationship between physical activity levels and feeding behaviors has been a focus of preclinical research for decades, yet this interaction has only recently been explored for potential sex differences. The aim of the present study was to isolate sex-dependent effects of voluntary wheel running (RUN) vs. sedentary locked wheel (SED) home cage conditions on palatability-driven feeding behavior using a 2-diet choice task between standard chow and a high-fat diet. The sex-dependent effects of physical activity on feeding behavior were examined following a within-subject novel reversal design of physical activity conditions (i.e., RUN > SED > RUN), to assess temporal sensitivity of the interaction. Following the final 2 weeks of reestablished and sustained RUN vs. SED conditions in separate groups of both males and females, reward-related opioid and dopamine gene expression within the nucleus accumbens (Acb) brain region were analyzed. Results demonstrated that the initial RUN > SED transition led to sex-dependent effects of SED condition, as males increased, and females decreased their high fat consumption, compared to their respective high fat consumption during previous RUN condition phase. Following reintroduction to the RUN condition, males decreased, and females increased their high fat consumption, compared to their separate SED control group. Last, sex-dependent shifts in ventral striatal opioid- and dopamine-related gene expression were observed to parallel the behavioral effects. The major findings of the study reveal that SED and RUN home cage conditions shift palatability-driven feeding in the opposite direction for males and females, these effects are sensitive to reversal, and these sex-dependent feeding behaviors track sex-dependent changes to critical reward-related gene expression patterns in the Acb. Considering the present high rates of sedentary behavior and obesity, furthering our understanding of the interaction between physical activity (or lack thereof) and feeding behavior should be a priority, especially in the context of these divergent sex-dependent outcomes.

Environmental Enrichment during Abstinence Reduces Oxycodone Seeking and c-Fos Expression in a Subpopulation of Medial Prefrontal Cortex Neurons

BackgroundSeveral preclinical studies have demonstrated that environmental enrichment (EE) during abstinence reduces drug seeking for psychostimulant and opioid drugs. Drug seeking is dependent on activity within the dorsomedial prefrontal cortex, and enrichment has been able to reduce drug seeking-associated increases in c-Fos in this region. In this study, we tested the hypothesis that EE during abstinence from oxycodone self-administration would reduce drug seeking and c-Fos immunoreactivity within the prefrontal cortex in a cell-type specific manner. MethodsMale rats self-administered oxycodone in two-hours sessions for three weeks, then underwent an initial drug seeking test under extinction conditions after one week of forced abstinence. Following this test, rats received either EE or remained individually housed in their home cage, then a second drug seeking test, with tissue collection immediately afterward. ResultsCompared to rats in standard housing, environmentally enriched rats had lower oxycodone seeking. In the prelimbic and infralimbic prefrontal cortices, the number of c-Fos+ cells was reduced, and this reduction was predominantly in inhibitory cells neurons, as evidenced by a reduction in the proportion of c-Fos+ cells in GAD+, but not CamKII+ cells. There was also a robust positive relationship between the number of c-Fos+ cells and persistence of oxycodone seeking in both the PrL and IL. ConclusionsThese findings further support the effectiveness of enriched environments to reduce reactivity to drug-associated stimuli and contexts and provide a potential mechanism by which this occurs.

Overexpression of Motopsin, an Extracellular Serine Protease Related to Intellectual Disability, Promotes Adult Neurogenesis and Neuronal Responsiveness in the Dentate Gyrus.

Motopsin, a serine protease encoded by PRSS12, is secreted by neuronal cells into the synaptic clefts in an activity-dependent manner, where it induces synaptogenesis by modulating Na+/K+-ATPase activity. In humans, motopsin deficiency leads to severe intellectual disability and, in mice, it disturbs spatial memory and social behavior. In this study, we investigated mice that overexpressed motopsin in the forebrain using the Tet-Off system (DTG-OE mice). The elevated agrin cleavage or the reduced Na+/K+-ATPase activity was not detected. However, motopsin overexpression led to a reduction in spine density in hippocampal CA1 basal dendrites. While motopsin overexpression decreased the ratio of mature mushroom spines in the DG, it increased the ratio of immature thin spines in CA1 apical dendrites. Female DTG-OE mice showed elevated locomotor activity in their home cages. DTG-OE mice showed aberrant behaviors, such as delayed latency to the target hole in the Barnes maze test and prolonged duration of sniffing objects in the novel object recognition test (NOR), although they retained memory comparable to that of TRE-motopsin littermates, which normally express motopsin. After NOR, c-Fos-positive cells increased in the dentate gyrus (DG) of DTG-OE mice compared with that of DTG-SO littermates, in which motopsin overexpression was suppressed by the administration of doxycycline, and TRE-motopsin littermates. Notably, the numbers of doublecortin- and 5-bromo-2'-deoxyuridine-labeled cells significantly increased in the DG of DTG-OE mice, suggesting increased adult neurogenesis. Importantly, our results revealed a new function in addition to modulating neuronal responsiveness and spine morphology in the DG: the regulation of neurogenesis.

Ethanol’s interaction with BK channel α subunit residue K361 does not mediate behavioral responses to alcohol in mice

Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol’s direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary β subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.

The effect of hippocampal overexpression of dopamine neurotrophic factor (CDNF) on the behavior of mice with genetic predisposition to depressive-like behavior

Cerebral dopamine neurotrophic factor (CDNF) is the promising tool for treatment of Parkinson’s disease. However, its role in the regulation of non-motor behavior, including various psychopathologies, remains unclear. In this regard, the aim of present work was to study the effect of CDNF overexpression in the hippocampus on the behavior of ASC mice (Antidepressant Sensitive Cataleptics) with a genetic predisposition to depression-like behavior. CDNF overexpression in mouse hippocampal neurons was induced using an adeno-associated viral vector. Four weeks after stereotaxic injection of the AAV-CDNF construct into the dorsal hippocampus, home cage behavior, exploratory, anxiety and depressive-like behaviors, as well as spatial and associative learning were assessed. We found significant improvements in the dynamics of spatial learning in the Morris water maze in CDNF-overexpressing animals. At the same time, no effect of CDNF was found on other studied behaviors. The behavior of the experimental animals in the home cage conditions did not differ from that in the control group, except for a decrease in the total amount of food eaten and a slight increase in the number of sleep episodes in the light phase of the day. In the present study we also attempted to determine the molecular basis for the above mentioned changes by genes expression assessment. We did not find significant changes in the mRNA level of key kinases, genes involved in neuroplasticity and neuronal survival as well as genes encoding receptors for the main neurotransmitter systems. However, animals overexpressing CDNF show increased lever of spliced Xbp indicating activation of the Ire1α/Xbp1 pathway traditionally associated with ER stress. Immunohistochemical analysis showed that CDNF was co-localized with the ER marker calreticulin. Thus, the effects of endogenous CDNF on behavior that we have found may be mediated by a specific molecular cascade, which emphasizes its difference from classical neurotrophic factors.

A long-term ketogenic diet in young and aged rats has dissociable effects on prelimbic cortex and CA3 ensemble activity.

Age-related cognitive decline has been linked to distinct patterns of cellular dysfunction in the prelimbic cortex (PL) and the CA3 subregion of the hippocampus. Because higher cognitive functions require both structures, selectively targeting a neurobiological change in one region, at the expense of the other, is not likely to restore normal behavior in older animals. One change with age that both the PL and CA3 share, however, is a reduced ability to utilize glucose, which can produce aberrant neural activity patterns. The current study used a ketogenic diet (KD) intervention, which reduces the brain's reliance on glucose, and has been shown to improve cognition, as a metabolic treatment for restoring neural ensemble dynamics in aged rats. Expression of the immediate-early genes Arc and Homer1a were used to quantify the neural ensembles that were active in the home cage prior to behavior, during a working memory/biconditional association task, and a continuous spatial alternation task. Aged rats on the control diet had increased activity in CA3 and less ensemble overlap in PL between different task conditions than did the young animals. In the PL, the KD was associated with increased activation of neurons in the superficial cortical layers, establishing a clear link between dietary macronutrient content and frontal cortical activity. The KD did not lead to any significant changes in CA3 activity. These observations suggest that the availability of ketone bodies may permit the engagement of compensatory mechanisms in the frontal cortices that produce better cognitive outcomes.

A paradigm for ethanol consumption in head-fixed mice during prefrontal cortical two-photon calcium imaging

The prefrontal cortex (PFC) is a hub for cognitive behaviors and is a key target for neuroadaptations in alcohol use disorders. Recent advances in genetically encoded sensors and functional microscopy allow multimodal in vivo PFC activity recordings at subcellular and cellular scales. While these methods could enable a deeper understanding of the relationship between alcohol and PFC function/dysfunction, they typically require animals to be head-fixed. Here, we present a method in mice for binge-like ethanol consumption during head-fixation. Male and female mice were first acclimated to ethanol by providing home cage access to 20% ethanol (v/v) for 4 or 8 days. After home cage drinking, mice consumed ethanol from a lick spout during head-fixation. We used two-photon calcium imaging during the head-fixed drinking paradigm to record from a large population of PFC neurons (>1000) to explore how acute ethanol affects their activity. Drinking exerted temporally heterogeneous effects on PFC activity at single neuron and population levels. Intoxication modulated the tonic activity of some neurons while others showed phasic responses around ethanol receipt. Population level activity did not show tonic or phasic modulation but tracked ethanol consumption over the minute-timescale. Network level interactions assessed through between-neuron pairwise correlations were largely resilient to intoxication at the population level while neurons with increased tonic activity showed higher synchrony by the end of the drinking period. By establishing a method for binge-like drinking in head-fixed mice, we lay the groundwork for leveraging advanced microscopy technologies to study alcohol-induced neuroadaptations in PFC and other brain circuits.This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".

Assessment of apathy‐like behaviour in transgenic mouse models of neurodegenerative diseases

AbstractBackgroundApathy is the most common behavioural and psychological symptom in neurodegenerative diseases like Alzheimer’s disease (AD), frontotemporal dementia (FTD) or Parkinson’s disease (PD). Apathy typically includes a loss of motivation, initiative and interest, listlessness and indifference, flattening of emotions, absence of drive and passion. Scientists have defined this as a reduction/lack in goal direct behaviour. There are currently no specific treatment options for apathy. In experimental models, selective symptoms of apathy‐like behaviour are measured in goal directed or nesting behavioural tasks, which report on their motivation to perform daily activities. A richer understanding of apathy, however, may be achieved through the development of a battery of tests including goal directed, nesting, anhedonia, activity monitoring, and social interaction. These have been applied here to several animal models of neurodegeneration.MethodMale and female transgenic mice (aged 6 – 8 months) including tau over‐expressors L1 for AD and L66 for FTD (Melis et al. 2015); PLB4 BACE1 mouse model for AD (Plucinska et al. 2014); L62 alpha‐synuclein mouse model of PD (Frahm et al. 2018) and relevant wild type (WT) controls were utilised in this study. Animals were tested in a series of behavioural tests including the nest building task, sucrose preference test, buried cookie test, social interaction, and home cage activity. Performance was recorded and analysed by behavioural tracking software as indicators of apathy‐like behaviour.ResultTwo models showed reproducible results compared with previous studies. Intriguingly, L66 FTD mice presented with deficits in sucrose preference and nest‐building, but not a lowering of general activity. Although this was not found for the sister strain, L1 mice which has AD‐like pathology. Very much unexpected is the observation that neither the amyloid nor the synuclein‐based models showed any significant behavioural anomalies. Further detailed investigations are ongoing.ConclusionInitial observations have confirmed that the transgenic models for neurodegenerative diseases show different levels of apathy‐like behaviour. It appears that the phenotype in L66 is a tau‐related anomaly specific to the tau species expressed, i.e. the mutation at P301S. The lack of deficits in all other models warrants in‐depth confirmation and analysis.

Effect of photobiological modulation therapy on cognitive impairment and cardiovascular structure in an APP/PS1 mouse model of Alzheimer’s Disease

AbstractBackgroundPhotobiological modulation (PBM) therapy, a form of low‐dose light therapy, is beneficial in treating various disease conditions including Alzheimer’s disease (AD). Double‐transgenic mice (APPswe/PS1dE9) develop progressive accumulation of amyloid‐β (Aβ) plaques, cognitive impairment, and alterations in cardiovascular structure and function, and represent a model of AD. Prior studies have shown that PBM therapy reduces the size and number of Aβ plaques in the neocortex and hippocampus in the mouse models of AD. The aim of this study was to investigate the effects of PBM on cardiovascular structure and function and cognitive function in the aged AD mice.MethodsSix‐month old female AD (APPswe/PS1dE9; AD‐PBM, n = 8) and wild type mice (WT‐PBM, n = 8) were exposed to near‐infrared light (wavelength 850 nm, 4.5 J/cm2, 3min/day, 5 days/week) for 6 months. Control mice (AD‐CNT, n = 6; WT‐CNT, n = 6) were placed under the PBM apparatus without turning the light on for 3min/day, 5 days/week. Cardiac parameters were measured by echocardiography. Gait analysis (DigiGait), home cage activity (HCA), and spatial working memory (indicated by alternation rate in cross maze test, CM) were assessed after 6 months of treatment. Group differences were identified by two‐way ANOVA.ResultsIn HCA test, the AD‐CNT mice exhibited circadian locomotor hyperactivity at night‐time relative to WT‐CNT mice (Figure 1A, B), which was reduced by PBM. AD‐CNT mice had lower alternation rates in CM, indicating spatial working memory impairment. PBM had minimal effect on this parameter (Table 1). The AD‐CNT vs. WT‐CNT mice manifested differences in several gait parameters, specifically, they demonstrated a lower fore propel time than WT‐CNT. PBM normalized this parameter in AD‐PBM mice to the level of WT‐CNT (Figure 1C). Both WT‐PBM and AD‐PBM mice had improved cardiovascular structure, which was demonstrated by lower relative wall thickness of left ventricle (LV) and LV mass, and higher LV volume at the end of diastole in PBM‐treated vs. correspondent CNT groups (Table 1, Figure 2).ConclusionPBM improved cardiovascular structure and AD‐associated cognitive impairment in the APP/PS1 mouse model. It is possible that more effective cardiovascular structure and function contributes to Aβ removal from the brain after PBM treatment. Supported by NIA/NIH/IRP

LocoBox: Modular Hardware and Open-Source Software for Circadian Entrainment and Behavioral Monitoring in Home Cages

Day–night locomotor activities are the most readily observed outputs of the circadian (~24-h period) clock in many animals. Temporal patterns of the light–dark schedule serve as input to the clock. While circadian activity patterns under various lighting conditions have been observed and documented, the full extent of circadian locomotor activities by genotype and entrainment remains uncharacterized. To facilitate large-scale, parallel cataloging of circadian input–output patterns, we created the LocoBox, an easy-to-construct and easy-to-operate system that can control environmental light with flexible entrainment scenarios combined with the T-cycle and measure locomotor activities in individual home cages. The LocoBox is made using economical, common components, and normal breeding cages can be used for long-term recording. We provide details of the components and blueprints, along with software programs for Arduino and a Python-based graphical user interface (GUI), so that the system can be easily replicated in other laboratories.

A novel investigation of placebo analgesia through social communication in mice

Background and aimsIn rodents, placebo analgesia is often investigated through direct conditioning of stimuli, but humans can experience placebo analgesia through expectation without experience. In this study, we sought to determine whether placebo analgesia could be elicited through social communication. MethodsMale and female mice were housed in pairs (designated “Active” and “Bystander”) and tested for thermal thresholds on a hot plate (53 °C). Food restriction (1 hr/day) was implemented. The Active mouse was taken to a new cage with food dusted with cocoa (COC) or cinnamon (CINN). The Bystander mice were given regular chow in the home cage. After feeding, the Active mice were given morphine (5 mg/kg, SC) or saline and tested on the hot plate. After 5 pairings of a flavor and treatment (counterbalanced), Active mice were tested following access to a flavored food. Bystander mice were given their first direct exposure to a flavored food and tested on the hot plate. The protocol was repeated with naloxone (10 mg/kg, IP) administered prior to testing. Finally, mice were tested in a two-choice test with both flavored foods available. ResultsActive mice showed a conditioned analgesic response to the morphine-paired flavor that was reduced by naloxone. Bystander mice showed a placebo analgesic response to their cagemate’s morphine-paired flavor that was not significantly impacted by naloxone. Bystander mice spent more time in the chamber associated with their cagemate’s morphine-paired flavor. ConclusionsTo our knowledge, this is the first investigation of placebo analgesia without direct conditioning, instead relying on social communication between mice. The lack of effect with naloxone pretreatment suggests an opioid-independent effect in the Bystander mice. Placebo analgesia in mice may be possible without direct conditioning to better model the effect of expectation of a novel analgesic in humans.

Forced Abstinence from Volitional Ethanol Intake Drives a Vulnerable Period of Hyperexcitability in BNST-Projecting Insular Cortex Neurons.

The insular cortex (IC) integrates sensory and interoceptive cues to inform downstream circuitry executing adaptive behavioral responses. The IC communicates with areas involved canonically in stress and motivation. IC projections govern stress and ethanol recruitment of bed nucleus of the stria terminalis (BNST) activity necessary for the emergence of negative affective behaviors during alcohol abstinence. Here, we assess the impact of the chronic drinking forced abstinence (CDFA) volitional home cage ethanol intake paradigm on synaptic and excitable properties of IC neurons that project to the BNST (IC→BNST). Using whole-cell patch-clamp electrophysiology, we investigated IC→BNST circuitry 24 h or 2 weeks following forced abstinence (FA) in female C57BL6/J mice. We find that IC→BNST cells are transiently more excitable following acute ethanol withdrawal. In contrast, in vivo ethanol exposure via intraperitoneal injection, ex vivo via ethanol wash, and acute FA from a natural reward (sucrose) all failed to alter excitability. In situ hybridization studies revealed that at 24 h post FA BK channel mRNA expression is reduced in IC. Further, pharmacological inhibition of BK channels mimicked the 24 h FA phenotype, while BK activation was able to decrease AP firing in control and 24 h FA subjects. All together these data suggest a novel mechanism of homeostatic plasticity that occurs in the IC→BNST circuitry following chronic drinking.

Refinement of IntelliCage protocols for complex cognitive tasks through replacement of drinking restrictions by incentive-disincentive paradigms.

The IntelliCage allows automated testing of cognitive abilities of mice in a social home cage environment without handling by human experimenters. Restricted water access in combination with protocols in which only correct responses give access to water is a reliable learning motivator for hippocampus-dependent tasks assessing spatial memory and executive function. However, water restriction may negatively impact on animal welfare, especially in poor learners. To better comply with the 3R principles, we previously tested protocols in which water was freely available but additional access to sweetened water could be obtained by learning a task rule. While this purely appetitive motivation worked for simple tasks, too many mice lost interest in the sweet reward during more difficult hippocampus-dependent tasks. In the present study, we tested a battery of increasingly difficult spatial tasks in which water was still available without learning the task rule, but rendered less attractive either by adding bitter tasting quinine or by increasing the amount of work to obtain it. As in previous protocols, learning of the task rule provided access to water sweetened with saccharin. The two approaches of dual motivation were tested in two cohorts of female C57BL/6 N mice. Compared to purely appetitive motivation, both novel protocols strongly improved task engagement and increased task performance. Importantly, neither of the added disincentives had an adverse impact on liquid consumption, health status or body weight of the animals. Our results show that it is possible to refine test protocols in the IntelliCage so that they challenge cognitive functions without restricting access to water.

Homeostatic NREM sleep and salience network function in adult mice exposed to ethanol during development.

Developmental exposure to ethanol is a leading cause of cognitive, emotional and behavioral problems, with fetal alcohol spectrum disorder (FASD) affecting more than 1:100 children. Recently, comorbid sleep deficits have been highlighted in these disorders, with sleep repair a potential therapeutic target. Animal models of FASD have shown non-REM (NREM) sleep fragmentation and slow-wave oscillation impairments that predict cognitive performance. Here we use a mouse model of perinatal ethanol exposure to explore whether reduced sleep pressure may contribute to impaired NREM sleep, and compare the function of a brain network reported to be impacted by insomnia-the Salience network-in developmental ethanol-exposed mice with sleep-deprived, saline controls. Mice were exposed to ethanol or saline on postnatal day 7 (P7) and allowed to mature to adulthood for testing. At P90, telemetered cortical recordings were made for assessment of NREM sleep in home cage before and after 4 h of sleep deprivation to assess basal NREM sleep and homeostatic NREM sleep response. To assess Salience network functional connectivity, mice were exposed to the 4 h sleep deprivation period or left alone, then immediately sacrificed for immunohistochemical analysis of c-Fos expression. The results show that developmental ethanol severely impairs both normal rebound NREM sleep and sleep deprivation induced increases in slow-wave activity, consistent with reduced sleep pressure. Furthermore, the Salience network connectome in rested, ethanol-exposed mice was most similar to that of sleep-deprived, saline control mice, suggesting a sleep deprivation-like state of Salience network function after developmental ethanol even without sleep deprivation.