Experimental laboratory models as tools for understanding modifiable dementia risk.

Experimental laboratory research has an important role to play in dementia prevention. Mechanisms underlying modifiable risk factors for dementia are promising targets for dementia prevention but are difficult to investigate in human populations due to technological constraints and confounds. Therefore, controlled laboratory experiments in models such as transgenic rodents, invertebrates and in vitro cultured cells are increasingly used to investigate dementia risk factors and test strategies which target them to prevent dementia. This review provides an overview of experimental research into 15 established and putative modifiable dementia risk factors: less early-life education, hearing loss, depression, social isolation, life stress, hypertension, obesity, diabetes, physical inactivity, heavy alcohol use, smoking, air pollution, anesthetic exposure, traumatic brain injury, and disordered sleep. It explores how experimental models have been, and can be, used to address questions about modifiable dementia risk and prevention that cannot readily be addressed in human studies. HIGHLIGHTS: Modifiable dementia risk factors are promising targets for dementia prevention. Interrogation of mechanisms underlying dementia risk is difficult in human populations. Studies using diverse experimental models are revealing modifiable dementia risk mechanisms. We review experimental research into 15 modifiable dementia risk factors. Laboratory science can contribute uniquely to dementia prevention.

Hope for brain health: impacting the life course and society.

Hope is a cognitive process by which an individual can identify their personal goals and develop actionable steps to achieve results. It has the potential to positively impact people's lives by building resilience, and can be meaningfully experienced at both the individual and group level. Despite this significance, there are sizable gaps in our understanding of the neurobiology of hope. In this perspective paper, the authors discuss why further research is needed on hope and its potency to be harnessed in society as a "tool" to promote brain health across healthy and patient populations. Avenues for future research in hope and the brain are proposed. The authors conclude by identifying strategies for the possible applications of hope in brain health promotion within the areas of technology, arts, media, and education.

Repeated Measurement of Microglia-Dendritic Spine Interactions Using Multi-Photon Imaging.

In recent decades, mounting evidence has shown that microglia play a vital role in maintaining synapses throughout life. This maintenance is done via numerous microglial processes, which are long, thin, and highly motile protrusions from the cell body that monitor their environment. However, due to the brevity of the contacts and the potentially transient nature of synaptic structures, establishing the underlying dynamics of this relationship has proven difficult. This article describes a method of using rapidly acquired multiphoton microscopy images to track microglial dynamics and microglia:synapse interactions and the fate of the synaptic structures following those interactions. First, we detail a method for capturing multiphoton images at 1-min intervals for approximately 1 hr and how that process can be done at multiple time points. We then discuss how best to prevent and account for any drifting of the region of interest that can occur during the imaging session and how to remove excessive background noise from those images. Finally, we detail the annotation process for dendritic spines and microglial processes using plugins in MATLAB and Fiji, respectively. These semi-automated plugins allow tracking of individual cell structures, even if both microglia and neurons are imaged in the same fluorescent channel. This protocol presents a method of tracking both microglial dynamics and synaptic structures, in the same animal, at multiple time points, giving the user information on process speed, branching, tip size, location, and dwell time, as well as any dendritic spine gains, losses, and size changes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Rapid multiphoton image capture Basic Protocol 2: Image preparation using MATLAB and Fiji Basic Protocol 3: Dendritic spine and microglial processes annotation using ScanImage and TrackMate.

A profile of students in a university-based distance dementia education program: Implications for policy and practice.

OBJECTIVE: The online program began in 2012 to support aged care workers without a tertiary education or vocational qualification. This paper documents changes in the student profile since initiation of the program, and how the program may support recommendations of the Royal Commission into Aged Care Quality and Safety and engage other educators, providers and policymakers. METHODS: Four hundred and seventy-one commencing undergraduate students completed a 16-item online survey in 2017 to document demographics and reasons for study. Categorical associations were assessed with univariate logistic regression in R v3.6. RESULTS: Most students (71%; 336) were aged between 41 and 60 years but the program now included younger (<41 years) and older (>80 years) people. Unlike the 2012 students, about 41% had tertiary-level qualifications, and 56% were employed in professional positions, including registered nurse, general practitioner and allied health professional. Professional and practice development was the primary reason for study; significantly so for younger (<41 years) participants in aged and dementia care (χ2 (5) = 18.15, p = 0.003) and for those with previous university experience (χ2 (4) = 22.17, p = 0.001). Older (≥61 years) participants enrolled to gain greater knowledge about dementia (χ2 (4) = 17.60, p = 0.002). CONCLUSIONS: Understanding the changed student profile guided program refinement to ensure the provision of effective, evidence-based education in dementia understanding and care. Work now focusses on increasing partnerships with aged care organisations, community and postsecondary training institutions to support a continuum of workforce development options, guided by the recommendations of the Royal Commission.

Microglia directly associate with pericytes in the central nervous system.

Cerebral blood flow (CBF) is important for the maintenance of brain function and its dysregulation has been implicated in Alzheimer's disease (AD). Microglia associations with capillaries suggest they may play a role in the regulation of CBF or the blood-brain-barrier (BBB). We explored the relationship between microglia and pericytes, a vessel-resident cell type that has a major role in the control of CBF and maintenance of the BBB, discovering a spatially distinct subset of microglia that closely associate with pericytes. We termed these pericyte-associated microglia (PEM). PEM are present throughout the brain and spinal cord in NG2DsRed × CX3 CR1+/GFP mice, and in the human frontal cortex. Using in vivo two-photon microscopy, we found microglia residing adjacent to pericytes at all levels of the capillary tree and found they can maintain their position for at least 28 days. PEM can associate with pericytes lacking astroglial endfeet coverage and capillary vessel width is increased beneath pericytes with or without an associated PEM, but capillary width decreases if a pericyte loses a PEM. Deletion of the microglia fractalkine receptor (CX3 CR1) did not disrupt the association between pericytes and PEM. Finally, we found the proportion of microglia that are PEM declines in the superior frontal gyrus in AD. In summary, we identify microglia that specifically associate with pericytes and find these are reduced in number in AD, which may be a novel mechanism contributing to vascular dysfunction in neurodegenerative diseases.

Subthreshold repetitive transcranial magnetic stimulation drives structural synaptic plasticity in the young and aged motor cortex.

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive tool commonly used to drive neural plasticity in the young adult and aged brain. Recent data from mouse models have shown that even at subthreshold intensities (0.12 T), rTMS can drive neuronal and glial plasticity in the motor cortex. However, the physiological mechanisms underlying subthreshold rTMS induced plasticity and whether these are altered with normal ageing are unclear. OBJECTIVE: To assess the effect of subthreshold rTMS, using the intermittent theta burst stimulation (iTBS) protocol on structural synaptic plasticity in the mouse motor cortex of young and aged mice. METHODS: Longitudinal in vivo 2-photon microscopy was used to measure changes to the structural plasticity of pyramidal neuron dendritic spines in the motor cortex following a single train of subthreshold rTMS (in young adult and aged animals) or the same rTMS train administered on 4 consecutive days (in young adult animals only). Data were analysed with Bayesian hierarchical generalized linear regression models and interpreted with the aid of Bayes Factors (BF). RESULTS: We found strong evidence (BF > 10) that subthreshold rTMS altered the rate of dendritic spine losses and gains, dependent on the number of stimulation sessions and that a single session of subthreshold rTMS was effective in driving structural synaptic plasticity in both young adult and aged mice. CONCLUSION: These findings provide further evidence that rTMS drives synaptic plasticity in the brain and uncovers structural synaptic plasticity as a key mechanism of subthreshold rTMS induced plasticity.

Regional differences in beta amyloid plaque deposition and variable response to midlife environmental enrichment in the cortex of APP/PS1 mice.

Environmentally enriched housing conditions can increase performance on cognitive tasks in APP/PS1 mice; however, the potential effects of environmental enrichment (EE) on disease modification in terms of pathological change are inconclusive. We hypothesized that previous contrasting findings may be attributable to regional differences in susceptibility to amyloid beta (Aß) plaque deposition in cortical regions that are functionally associated with EE. We characterized fibrillar plaque deposition in 6, 12, and 18-22 months old APP/PS1 mice in the prefrontal (PFC), somatosensory (SS2), and primary motor cortex (M1). We found a significant increase in plaque load between 6 and 12 months in all regions. In animals over 12 months, only the PFC region continued to significantly accumulate plaques. Additionally, 12 months old animals subjected to 6 months of EE showed improved spatial navigation and had significantly fewer plaques in M1 and SS2, but not in the PFC. These findings suggest that the PFC region is selectively susceptible to Aß deposition and less responsive to the attenuating effects of EE. In contrast, M1 and SS2 regions plateau with respect to Aß deposition by 12 months of age and are susceptible to amyloid pathology modification by midlife EE.

Microtubule-dependent processes precede pathological calcium influx in excitotoxin-induced axon degeneration.

Axon degeneration and axonal loss is a feature of neurodegenerative disease and injury and occurs via programmed pathways that are distinct from cell death pathways. While the pathways of axonal loss following axon severing are well described, less is known about axonal loss following other neurodegenerative insults. Here we use primary mouse cortical neuron cultures grown in compartmentalized chambers to investigate the role of calcium in the degeneration of axons that occurs following a somal insult by the excitotoxin kainic acid. Calcium influx has been implicated in both excitotoxicity and axon degeneration mechanisms, however the link between a somal insult and axonal calcium increase is unclear. Live imaging of axons demonstrated that pharmacologically preventing intracellular calcium increases through the endoplasmic reticulum or mitochondria significantly (p < 0.05) reduced axon degeneration. Live calcium-imaging with the Ca2+ indicator Fluo-4 demonstrated that kainic acid exposure to the soma resulted in a rapid, and transient, increase in calcium in the axon, which occured even at low kainic acid concentrations that do not cause axon degeneration within 24 h. However, this calcium transient was followed by a gradual increase in axonal calcium, which was associated with axonal loss. Furthermore, treatment with a range of doses of the microtubule stabilizing drug taxol, which protects against axon fragmentation in this model, prevented this gradual calcium increase, suggesting that the intra-axonal calcium changes are downstream of microtubule associated events. Biochemical analysis of taxol treated neurons demonstrated a shift in microtubule post-translational modifications, with a significant (p < 0.05) increase in acetylated tubulin and a significant (p < 0.05) decrease in tyrosinated tubulin, suggestive of a more stable microtubule pool. Together our results suggest that axonal degeneration following excitotoxicity is dependent on an increase in axonal calcium, which is downstream of a microtubule-dependent event.

Rod microglia and their role in neurological diseases.

The striking morphology of microglia is one of their most prominent characteristics, with many studies categorising microglial function based on morphology e.g. ramified, hyper-ramified, activated, or amoeboid. Communications regarding rod microglia in neurological disease are scant, and where reported, these cells are rarely the focus of discussion. These factors make it difficult to determine how widespread these cells are not only through the brain but also across diseases. Studies in experimental diffuse brain injury are the first reports of not only significant numbers of rod microglia, but distinct arrangements of these cells, reminiscent of carriages of a train. This review summarises the available reports of rod microglia in vivo and rod-like microglia in vitro and eludes to possible functions and signalling cascades that may evoke this distinct morphology. More investigations are required to fully elucidate the function that rod microglia play in neurological diseases.

Low intensity repetitive transcranial magnetic stimulation modulates skilled motor learning in adult mice.

Repetitive transcranial magnetic stimulation (rTMS) is commonly used to modulate cortical plasticity in clinical and non-clinical populations. Clinically, rTMS is delivered to targeted regions of the cortex at high intensities (>1 T). We have previously shown that even at low intensities, rTMS induces structural and molecular plasticity in the rodent cortex. To determine whether low intensity rTMS (LI-rTMS) alters behavioural performance, daily intermittent theta burst LI-rTMS (120 mT) or sham was delivered as a priming or consolidating stimulus to mice completing 10 consecutive days of skilled reaching training. Relative to sham, priming LI-rTMS (before each training session), increased skill accuracy (~9%) but did not alter the rate of learning over time. In contrast, consolidating LI-rTMS (after each training session), resulted in a small increase in the rate of learning (an additional ~1.6% each day) but did not alter the daily skill accuracy. Changes in behaviour with LI-rTMS were not accompanied with long lasting changes in brain-derived neurotrophic factor (BDNF) expression or in the expression of plasticity markers at excitatory and inhibitory synapses for either priming or consolidation groups. These results suggest that LI-rTMS can alter specific aspects of skilled motor learning in a manner dependent on the timing of intervention.

Construction and Evaluation of Rodent-Specific rTMS Coils.

Rodent models of transcranial magnetic stimulation (TMS) play a crucial role in aiding the understanding of the cellular and molecular mechanisms underlying TMS induced plasticity. Rodent-specific TMS have previously been used to deliver focal stimulation at the cost of stimulus intensity (12 mT). Here we describe two novel TMS coils designed to deliver repetitive TMS (rTMS) at greater stimulation intensities whilst maintaining spatial resolution. Two circular coils (8 mm outer diameter) were constructed with either an air or pure iron-core. Peak magnetic field strength for the air and iron-cores were 90 and 120 mT, respectively, with the iron-core coil exhibiting less focality. Coil temperature and magnetic field stability for the two coils undergoing rTMS, were similar at 1 Hz but varied at 10 Hz. Finite element modeling of 10 Hz rTMS with the iron-core in a simplified rat brain model suggests a peak electric field of 85 and 12.7 V/m, within the skull and the brain, respectively. Delivering 10 Hz rTMS to the motor cortex of anaesthetized rats with the iron-core coil significantly increased motor evoked potential amplitudes immediately after stimulation (n = 4). Our results suggest these novel coils generate modest magnetic and electric fields, capable of altering cortical excitability and provide an alternative method to investigate the mechanisms underlying rTMS-induced plasticity in an experimental setting.

Facilitatory non-invasive brain stimulation in older adults: the effect of stimulation type and duration on the induction of motor cortex plasticity.

Despite holding significant promise for counteracting the deleterious effects of ageing on cognitive and motor function, little is known of the effects of facilitatory non-invasive brain stimulation (NBS) techniques on corticospinal excitability (CSE) in older adults. Thirty-three older adults (≥60 years) participated in four NBS sessions on separate days, receiving 10- and 20-min anodal transcranial direct current stimulation (atDCS), and 300 and 600 pulses of intermittent theta burst stimulation (iTBS) over the left M1. Motor-evoked potentials measured in the contralateral hand served as a measure of CSE before and for 30 min following each NBS intervention. At the group level, generalized post-stimulation CSE increases were observed (p < 0.001) with no significant differences between the two durations of each stimulation type (atDCS: p = 0.5; iTBS: p = 0.9). For individuals exhibiting overall facilitatory change to atDCS ('responders', n = 10), 20-min atDCS resulted in longer lasting CSE facilitation than 10 min. No such difference was observed between the two iTBS protocols. Considerable variability was observed inter-individually, where 52-58 % of the cohort exhibited the expected facilitation after each of the NBS protocols-as well as intra-individually, where 45-48 % of the cohort maintained consistent post-stimulation responses across the varying durations and types of stimulation. In conclusion, as shown previously in young adults, older adults demonstrate substantial variability in response to different facilitatory NBS protocols. Studies to assess the intra-individual reliability of these protocols are critical to progress towards translation of appropriate protocols (i.e. those that elicit the greatest response for each individual) into clinical practice.

Defining the earliest pathological changes of Alzheimer's disease.

The prospects for effectively treating well-established dementia, such as Alzheimer's disease (AD), are slim, due to the destruction of key brain pathways that underlie higher cognitive function. There has been a substantial shift in the field towards detecting conditions such as AD in their earliest stages, which would allow preventative or therapeutic approaches to substantially reduce risk and/or slow the progression of disease. AD is characterized by hallmark pathological changes such as extracellular Aß plaques and intracellular neurofibrillary pathology, which selectively affect specific subclasses of neurons and brain circuits. Current evidence indicates that Aß plaques begin to form many years before overt dementia, a gradual and progressive pathology which offers a potential target for early intervention. Early Aß changes in the brain result in localized damage to dendrites, axonal processes and synapses, to which excitatory synapses and the processes of projection neurons are highly vulnerable. Aß pathology is replicated in a range of transgenic models overexpressing mutant human familial AD genes (e.g. APP and presenilin 1). Studying the development of aberrant regenerative and degenerative changes in neuritic processes associated with Aß plaques may represent the best opportunity to understand the relationship between the pathological hallmarks of AD and neuronal damage, and to develop early interventions to prevent, slow down or mitigate against Aß pathology and/or the neuronal alterations that leads to cognitive impairment.

Laser-Mediated Microlesions in Mouse Neocortex to Investigate Neuronal Degeneration and Regeneration.

In vivo two-photon (2P) imaging enables neural circuitry to be repeatedly visualized in both normal conditions and following trauma. This protocol describes how laser-mediated neuronal microlesions can be created in the cerebral cortex using an ultrafast laser without causing a significant inflammatory reaction or compromising the blood-brain barrier. Furthermore, directives are provided for the acute and chronic in vivo imaging of the lesion site, as well as for post-hoc analysis of the lesion site in fixed tissue, which can be correlated with the live imaging phase.

Inter- and Intra-individual variability following intermittent theta burst stimulation: implications for rehabilitation and recovery.

BACKGROUND: The continued refinement of non-invasive brain stimulation (NBS) techniques is indicative of promising clinical and rehabilitative interventions that are able to modulate cortical excitability. Intermittent theta burst stimulation (iTBS) is one such technique that can increase cortical excitability, purportedly via LTP-like mechanisms. While iTBS may have the capacity to promote recovery after neurological injury, and to combat cognitive and motor decline, recent reports observed highly variable effects across individuals, questioning the efficacy of iTBS as a clinical tool. OBJECTIVE: The aim of this study was to examine intra-individual reliability and inter-individual variability in responses to iTBS. METHODS: Thirty healthy participants completed two experimental sessions of the iTBS protocol 1-3 weeks apart. Motor evoked potentials in response to single pulse TMS were used to assess corticospinal excitability prior to, and up to 36 min following, iTBS. RESULTS: At the group level, iTBS evoked statistically significant increases in motor cortical excitability across both sessions (P < 0.001), with 22 out of 30 participants exhibiting increases in excitability in both sessions. A strong intraclass correlation demonstrated that both the direction, and magnitude of the plastic changes were reliable at the individual level. CONCLUSIONS: Overall, our results suggest that iTBS is capable of inducing relatively robust and consistent effects within and between young individuals. As such, the capacity for iTBS to be exploited in clinical and rehabilitative interventions should continue to be explored.

Cytoskeletal changes during development and aging in the cortex of neurofilament light protein knockout mice.

The neurofilament light (NFL) subunit is considered as an obligate subunit polymer for neuronal intermediate filaments comprising the neurofilament (NF) triplet proteins. We examined cytoskeletal protein levels in the cerebral cortex of NFL knockout (KO) mice at postnatal day 4 (P4), 5 months, and 12 months of age compared with age-matched wild-type (WT) mice of a similar genetic background (C57BL/6). The absence of NFL protein resulted in a significant reduction of phosphorylated and dephosphorylated NFs (NF-P, NF-DP), the medium NF subunit (NFM), and the intermediate filament α-internexin (INT) at P4. At 5 months, NF-DP, NFM, and INT remained significantly lower in knockouts. At 12 months, NF-P was again significantly decreased, and INT significantly increased, in KOs compared with wild type. In addition, protein levels of class III neuron-specific ß-tubulin and microtubule-associated protein 2 were significantly increased in NFL KO mice at P4, 5 months, and 12 months, whereas ß-actin levels were significantly decreased at P4. Immunocytochemical studies demonstrated that NF-DP accumulated abnormally in the perikarya of cortical neurons by 5 months of age in NFL KO mice. Neurons that lacked NF triplet proteins, such as calretinin-immunolabeled nonpyramidal cells, showed no alterations in density or cytoarchitectural distribution in NFL KO mice at 5 months relative to WT mice, although calretinin protein levels were decreased significantly after 12 months in NFL KO mice. These findings suggest that a lack of NFL protein alters the expression of cytoskeletal proteins and disrupts other NF subunits, causing intracellular aggregation but not gross structural changes in cortical neurons or cytoarchitecture. The data also indicate that changes in expression of other cytoskeletal proteins may compensate for decreased NFs.

The DIADEM data sets: representative light microscopy images of neuronal morphology to advance automation of digital reconstructions.

The comprehensive characterization of neuronal morphology requires tracing extensive axonal and dendritic arbors imaged with light microscopy into digital reconstructions. Considerable effort is ongoing to automate this greatly labor-intensive and currently rate-determining process. Experimental data in the form of manually traced digital reconstructions and corresponding image stacks play a vital role in developing increasingly more powerful reconstruction algorithms. The DIADEM challenge (short for DIgital reconstruction of Axonal and DEndritic Morphology) successfully stimulated progress in this area by utilizing six data set collections from different animal species, brain regions, neuron types, and visualization methods. The original research projects that provided these data are representative of the diverse scientific questions addressed in this field. At the same time, these data provide a benchmark for the types of demands automated software must meet to achieve the quality of manual reconstructions while minimizing human involvement. The DIADEM data underwent extensive curation, including quality control, metadata annotation, and format standardization, to focus the challenge on the most substantial technical obstacles. This data set package is now freely released ( http://diademchallenge.org ) to train, test, and aid development of automated reconstruction algorithms.

Regionalized loss of parvalbumin interneurons in the cerebral cortex of mice with deficits in GFRalpha1 signaling.

Inhibitory interneurons are crucially important for cerebral cortex function and behavior. The mechanisms controlling inhibitory interneuron diversification and allocation to distinct cortical areas remain poorly understood. GDNF (glial cell line-derived neurotrophic factor) and its receptor GFRalpha1 have been implicated in the development of GABAergic precursors but, because of the early lethality of null mutants, their roles in postnatal maturation and function of cortical interneurons are unknown. "cis-only" mutant mice lack GFRalpha1 only in cells that do not express the RET signaling receptor subunit and survive to adulthood. At birth, both null mutants and cis-only mice showed a specific loss of GABAergic interneurons in rostro- and caudolateral cortical regions but not in more medial areas. Unexpectedly, the adult cortex of cis-only mice displayed a complete loss of parvalbumin (PV)-expressing GABAergic interneurons in discrete regions (PV holes) interspersed among areas of normal PV cell density. PV holes predominantly occurred in the visual and frontal cortices, and their size could be affected by neuronal activity. Consistent with deficits in cortical inhibitory activity, these mice showed enhanced cortical excitability, increased sensitivity to epileptic seizure, and increased social behavior. We propose that GFRalpha1 signaling guides the development of a subset of PV-expressing GABAergic interneurons populating discrete regions of the cerebral cortex and may thus contribute to the diversification and allocation of specific cortical interneuron subtypes.

Eph tyrosine kinase receptor EphA4 is required for the topographic mapping of the corticospinal tract.

Fine movement in the body is controlled by the motor cortex, which signals in a topographically specific manner to neurons in the spinal cord by means of the corticospinal tract (CST). How the correct topography of the CST is established is unknown. To investigate the possibility that the Eph tyrosine kinase receptor EphA4 is involved in this process, we have traced CST axons in mice in which the EphA4 gene has been deleted. The forelimb subpopulation of CST axons is unaffected in the EphA4-/- mice, but the hindlimb subpopulation branches too early within the cord, both temporally and spatially. EphA4 shows a dynamic expression pattern in the environment of the developing CST in the spinal cord: high at the time of forelimb branching and down-regulated before hindlimb branching. To examine whether the fore- and hindlimb subpopulations of CST axons respond differently to EphA4 in their environment, neurons from fore- and hindlimb motor cortex were cultured on a substrate containing EphA4. Neurons from the hindlimb cortex showed reduced branching on the EphA4 substrate compared with their forelimb counterparts. Neurons from the hindlimb cortex express ephrinA5, a high-affinity ligand for EphA4, at higher levels compared with forelimb cortex neurons, and this expression is down-regulated before hindlimb branching. Together, these findings suggest that EphA4 regulates topographic mapping of the CST by controlling the branching of CST axons in the spinal cord.