Exercise rejuvenates microglia and reverses T cell accumulation in the aged female mouse brain.

Slowing and/or reversing brain ageing may alleviate cognitive impairments. Previous studies have found that exercise may mitigate cognitive decline, but the mechanisms underlying this remain largely unclear. Here we provide unbiased analyses of single-cell RNA sequencing data, showing the impacts of exercise and ageing on specific cell types in the mouse hippocampus. We demonstrate that exercise has a profound and selective effect on aged microglia, reverting their gene expression signature to that of young microglia. Pharmacologic depletion of microglia further demonstrated that these cells are required for the stimulatory effects of exercise on hippocampal neurogenesis but not cognition. Strikingly, allowing 18-month-old mice access to a running wheel did by and large also prevent and/or revert T cell presence in the ageing hippocampus. Taken together, our data highlight the profound impact of exercise in rejuvenating aged microglia, associated pro-neurogenic effects and on peripheral immune cell presence in the ageing female mouse brain.

ROCK2 regulates microglia proliferation and neuronal survival after traumatic brain injury.

Traumatic brain injury (TBI) results in prolonged and non-resolving activation of microglia. Forced turnover of these cells during the acute phase of TBI aids recovery, but the cell-intrinsic pathways that underpin the pro-repair phenotype of these repopulating microglia remain unclear. Here, we show that selective targeting of ROCK2 with the small molecule inhibitor KD025 impairs the proliferative response of microglia after TBI as well as during genetically induced turnover of microglia. KD025 treatment abolished the substantial neuroprotective and cognitive benefits conferred by repopulating microglia, preventing these cells from replenishing the depleted niche during the early critical time window post-injury. Delaying KD025 treatment to the subacute phase of TBI allowed microglial repopulation to occur, but this did not enhance the benefits conferred by repopulating microglia. Taken together, our data indicate that ROCK2 mediates neuronal survival and microglial population dynamics after TBI, including the emergence of repopulating microglia with a pro-repair phenotype.

Spatial working memory in rats: Crucial role of the hippocampus in the allothetic place avoidance alternation task demanding stimuli segregation.

Working memory is a construct that contains goal maintenance, interference control and memory capacity domains. Spatial working memory in presence of conflicting stimuli requires segregation and maintenance of the relevant information about a goal over a short period of time. Besides the prefrontal cortex, the hippocampus is an anatomical substrate for the working memory. We hypothesized that in a highly challenging task, where spatial stimuli are in a conflict and only some of them describe the goal location, the spatial working memory will be strongly dependant on the hippocampus. To verify this, we used an allothetic place avoidance alternation task (APAAT). Performance of this task demands a small number of entries and a long maximum time avoided between consecutive entries to the shock sector. These parameters reflected both domains of working memory. The experiment was conducted on hippocampal lesioned (HIPP n = 12) and sham-operated (CTRL n = 8) rats trained in four APAAT days, each consisting of four 5-minute stages: habituation, stage1 (st1) and stage2 (st2) of memory training, a 5-minute break followed by a retrieval test. The position of the shock sector was changed each day. The HIPP rats were impaired on both stages of memory training, whereas CTRL rats presented significant memory improvement on stage2. In HIPP rats the cognitive skill learning measured as shock per entrance ratio was compromised. Hippocampal lesions did not impair locomotor activity. In summary, even slight bilateral damage to the hippocampus is blocking working memory formation in a difficult task.

Transient Inactivation of the Medial Prefrontal Cortex and Ventral Hippocampus Impairs Active Place Avoidance Retrieval on a Rotating Arena.

It is well known that communication between the medial prefrontal cortex (mPFC) and the ventral hippocampus (vHPC) is critical for various cognitive and behavioral functions. However, the exact role of these structures in spatial coordination remains to be clarified. Here we sought to determine the involvement of the mPFC and the vHPC in the spatial retrieval of a previously learned active place avoidance task in adult male Long-Evans rats, using a combination of unilateral and bilateral local muscimol inactivations. Moreover, we tested the role of the vHPC-mPFC pathway by performing combined ipsilateral and contralateral inactivations. Our results showed not only bilateral inactivations of either structure, but also the combined inactivations impaired the retrieval of spatial memory, whereas unilateral one-structure inactivations did not yield any effect. Remarkably, muscimol injections in combined groups exerted similar deficits, regardless of whether the inactivations were contralateral or ipsilateral. These findings confirm the importance of these structures in spatial cognition and emphasize the importance of the intact functioning of the vHPC-mPFC pathway.

Effects of adolescent experience of food restriction and exercise on spatial learning and open field exploration of female rats.

The hippocampus carries out multiple functions: spatial cognition dorsally (DH) and regulation of emotionality-driven behavior ventrally (VH). Previously, we showed that dendrites of DH and VH pyramidal neurons of female rats are still developing robustly during adolescence and are altered by the experience of food restriction and voluntary exercise on a wheel. We tested whether such anatomical changes during adolescence impact anxiety-like behavior and spatial cognition. Four groups of female rats were evaluated for these behaviors: those with wheel access in its cage from postnatal day (P) 36-44 (EX); those with food access restricted to 1 hr per day, from P40 to 44 (FR); those with EX from P36 to 44, combined with FR from P40 to 44, which we will refer to as EX + FR; and controls, CON (no EX, no FR). Open field test for anxiety-like behavior and active place avoidance test for spatial cognition were conducted at P47-49, the age when food restricted animals have restored body weight, or at P54-56, to identify more enduring effects. Anxiety-like behavior was elevated for the EX and FR groups at P47-49 but not for the EX + FR group. By P54-56, the EX + FR and EX groups exhibited less anxiety-like behavior, indicating a beneficial delayed main effect of exercise. There was a beneficial main effect of food restriction upon cognition, as the FR group showed cognition superior to CONs' at P44-46 and P54-56, while the EX + FR animals also showed enhanced spatial learning at P54-56. EX + FR animals with best adaptation to the feeding schedule showed the best spatial learning performance but with a delay. The EX group exhibited only a transient improvement. These findings indicate that FR, EX, and EX + FR in mid-adolescence are all beneficial in reducing anxiety-like behavior and improving spatial cognition but with subtle differences in the timing of their manifestation, possibly reflecting the protracted maturation of the hippocampus.

Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner.

Cognitive dysfunction and reactive microglia are hallmarks of traumatic brain injury (TBI), yet whether these cells contribute to cognitive deficits and secondary inflammatory pathology remains poorly understood. Here, we show that removal of microglia from the mouse brain has little effect on the outcome of TBI, but inducing the turnover of these cells through either pharmacologic or genetic approaches can yield a neuroprotective microglial phenotype that profoundly aids recovery. The beneficial effects of these repopulating microglia are critically dependent on interleukin-6 (IL-6) trans-signaling via the soluble IL-6 receptor (IL-6R) and robustly support adult neurogenesis, specifically by augmenting the survival of newborn neurons that directly support cognitive function. We conclude that microglia in the mammalian brain can be manipulated to adopt a neuroprotective and pro-regenerative phenotype that can aid repair and alleviate the cognitive deficits arising from brain injury.

Long-Lasting Input-Specific Experience-Dependent Changes of Hippocampus Synaptic Function Measured in the Anesthetized Rat.

How experience causes long-lasting changes in the brain is a central question in neuroscience. The common view is that synaptic function is altered by experience to change brain circuit functions that underlie conditioned behavior. We examined hippocampus synaptic circuit function in vivo, in three groups of animals, to assess the impact of experience on hippocampus function in rats. The "conditioned" group acquired a shock-conditioned place response during a cognitively-challenging, hippocampus synaptic plasticity-dependent task. The no-shock group had similar exposure to the environmental conditions but no conditioning. The home-cage group was experimentally naive. After the one-week retention test, under anesthesia, we stimulated the perforant path inputs to CA1, which terminate in stratum lacunosum moleculare (slm), and to the dentate gyrus (DG), which terminate in the molecular layer. We find synaptic compartment specific changes that differ amongst the groups. The evoked field EPSP (fEPSP) and pre-spike field response are enhanced only at the DG input layer and only in conditioned animals. The DG responses, measured by the population spiking activity and post-spike responses, are enhanced in both the conditioned and no-shock groups compared to home-cage animals. These changes are pathway specific because no differences are observed in slm of CA1. These findings demonstrate long-term, experience-dependent, pathway-specific alterations to synaptic circuit function of the hippocampus.

Chronic Neurobehavioral Sex Differences in a Murine Model of Repetitive Concussive Brain Injury.

Traumatic brain injury (TBI) resulting from repeated head trauma is frequently characterized by diffuse axonal injury and long-term motor, cognitive and neuropsychiatric symptoms. Given the delay, often decades, between repeated head traumas and the presentation of symptoms in TBI patients, animal models of repeated injuries should be studied longitudinally to properly assess the longer-term effects of multiple concussive injuries on functional outcomes. In this study, male and cycling female C57BL/6J mice underwent repeated (three) concussive brain injuries (rCBI) delivered via a Leica ImpactOne cortical impact device and were assessed chronically on motor (open field and rotarod), cognitive (y-maze and active place avoidance), and neuropsychiatric (marble-burying, elevated zero maze and tail suspension) tests. Motor deficits were significant on the rotarod on the day following the injuries, and slight impairment remained for up to 6 months. All mice that sustained rCBI had significant cognitive deficits on the active place avoidance test and showed greater agitation (less immobility) in the tail suspension test. Only injured male mice were significantly hyperactive in the open field, and had increased time spent in the open quadrants of the elevated zero maze. One year after the injuries, mice of both sexes exhibited persistent pathological changes by the presence of Prussian blue staining (indication of prior microbleeds), primarily in the cortex at the site of the injury, and increased GFAP staining in the perilesional cortex and axonal tracts (corpus callosum and optic tracts). These data demonstrate that a pathological phenotype with motor, cognitive, and neuropsychiatric symptoms can be observed in an animal model of rCBI for at least one year post-injury, providing a pre-clinical setting for the study of the link between multiple brain injuries and neurodegenerative disorders. Furthermore, this is the first study to include both sexes in a pre-clinical long-term rCBI model, and female mice are less impaired functionally than males.

Analysis of hippocampal-dependent learning and memory behaviour in mice lacking Nfix from adult neural stem cells.

OBJECTIVE: The active place avoidance task (APA) is a behavioural task used to assess learning and memory in rodents. This task relies on the hippocampus, a region of the cerebral cortex capable of generating new neurons from neural stem cells. In this study, to gain further insight into the behavioural phenotype of mice deficient in the transcription factor Nfix, a gene expressed by adult neural stem cells, we examined learning and memory parameters from the APA task that were not published in our original investigation. We analysed time to first and second shock, maximum path and time of shock avoidance, number of entries into the shock zone and time spent in the shock zone. We also assessed performance in the APA task based on sex. RESULTS: We found mice deficient in Nfix displayed decreased latency to second shock compared to the control mice. Nfix deficient mice entered the shock zone more frequently and also spent more time in the shock zone. Our data provides further insights into the memory deficits evident in Nfix mutant mice, indicating these mice have a memory retrieval problem and may employ a different navigation strategy in the APA task.

Multimodal analysis of aged wild-type mice exposed to repeated scanning ultrasound treatments demonstrates long-term safety.

The blood-brain barrier presents a major challenge for the delivery of therapeutic agents to the brain; however, it can be transiently opened by combining low intensity ultrasound with microbubble infusion. Studies evaluating this technology have largely been performed in rodents, including models of neurological conditions. However, despite promising outcomes in terms of drug delivery and the amelioration of neurological impairments, the potential for long-term adverse effects presents a major concern in the context of clinical applications. Methods: To fill this gap, we repeatedly treated 12-month-old wild-type mice with ultrasound, followed by a multimodal analysis for up to 18 months of age. Results: We found that spatial memory in these aged mice was not adversely affected as assessed in the active place avoidance test. Sholl analysis of Golgi impregnations in the dentate gyrus of the hippocampus did not reveal any changes to the neuronal cytoarchitecture. Long-term potentiation, a cellular correlate of memory, was still achievable, magnetic resonance spectroscopy revealed no major changes in metabolites, and diffusion tensor imaging revealed normal microstructure and tissue integrity in the hippocampus. More specifically, all measures of diffusion appeared to support a neuroprotective effect of ultrasound treatment on the brain. Conclusion: This multimodal analysis indicates that therapeutic ultrasound for blood-brain barrier opening is safe and potentially protective in the long-term, underscoring its validity as a potential treatment modality for diseases of the brain.

Protocol for Short- and Longer-term Spatial Learning and Memory in Mice.

Studies on the role of the hippocampus in higher cognitive functions such as spatial learning and memory in rodents are reliant upon robust and objective behavioral tests. This protocol describes one such test-the active place avoidance (APA) task. This behavioral task involves the mouse continuously integrating visual cues to orientate itself within a rotating arena in order to actively avoid a shock zone, the location of which remains constant relative to the room. This protocol details the step-by-step procedures for a novel paradigm of the hippocampal-dependent APA task, measuring acquisition of spatial learning during a single 20-min trial (i.e., short-term memory), with spatial memory encoding and retrieval (i.e., long-term memory) assessed by trials conducted over consecutive days. Using the APA task, cognitive flexibility can be assessed using the reversal learning paradigm, as this increases the cognitive load required for efficient performance in the task. In addition to a detailed experimental protocol, this paper also describes the range of its possible applications, the expected key results, as well as the analytical methods to assess the data, and the pitfalls/troubleshooting measures. The protocol described herein is highly robust and produces replicable results, thus presenting an important paradigm that enables the assessment of subtle short-term changes in spatial learning and memory, such as those observed for many experimental interventions.

Persistent modifications of hippocampal synaptic function during remote spatial memory.

A widely accepted notion for a process underlying memory formation is that learning changes the efficacy of synapses by the mechanism of synaptic plasticity. While there is compelling evidence of changes in synaptic efficacy observed after learning, demonstration of persistent synaptic changes accompanying memory has been elusive. We report that acquisition of a hippocampus and long-term potentiation dependent place memory persistently changes the function of CA1 synapses. Using extracellular recordings we measured CA3-CA1 and EC-CA1 synaptic responses and found robust changes in the CA3-CA1 pathway after memory training. Crucially, these changes in synaptic function lasted at least a month and coincided with the persistence of long-term place memories; the changes were only observed in animals that expressed robust memory, and not in animals with poor memory recall. Interestingly, our findings were observed at the level of populations of synapses; suggesting that memory formation recruits widespread synaptic circuits and persistently reorganizes their function to store information.

Dizocilpine (MK-801) impairs learning in the active place avoidance task but has no effect on the performance during task/context alternation.

The prevention of engram interference, pattern separation, flexibility, cognitive coordination and spatial navigation are usually studied separately at the behavioral level. Impairment in executive functions is often observed in patients suffering from schizophrenia. We have designed a protocol for assessing these functions all together as behavioral separation. This protocol is based on alternated or sequential training in two tasks testing different hippocampal functions (the Morris water maze and active place avoidance), and alternated or sequential training in two similar environments of the active place avoidance task. In Experiment 1, we tested, in adult rats, whether the performance in two different spatial tasks was affected by their order in sequential learning, or by their day-to-day alternation. In Experiment 2, rats learned to solve the active place avoidance task in two environments either alternately or sequentially. We found that rats are able to acquire both tasks and to discriminate both similar contexts without obvious problems regardless of the order or the alternation. We used two groups of rats, controls and a rat model of psychosis induced by a subchronic intraperitoneal application of 0.08mg/kg of dizocilpine (MK-801), a non-competitive antagonist of NMDA receptors. Dizocilpine had no selective effect on parallel/sequential learning of tasks/contexts. However, it caused hyperlocomotion and a significant deficit in learning in the active place avoidance task regardless of the task alternation. Cognitive coordination tested by this task is probably more sensitive to dizocilpine than spatial orientation because no hyperactivity or learning impairment was observed in the Morris water maze.

Corrigendum: Acute administration of MK-801 in an animal model of psychosis in rats interferes with cognitively demanding forms of behavioral flexibility on a rotating arena.

[This corrects the article on p. 75 in vol. 9, PMID: 25883558.].