Neuroscience

Developing preclinical animal models that faithfully mimic the progressive nature of Parkinson’s disease (PD) is crucial for advancing mechanistic insights as well as therapeutic discovery. While recombinant adeno-associated virus (rAAV)-driven α-synuclein overexpression is widely used, its reliance on high viral titers introduces nonspecific toxicity and limits physiological relevance. The SynFib model, which combines modest rAAV-driven α-synuclein expression (Syn) with α-synuclein preformed fibril (PFF) seeding (Fib), has shown promise in reproducing PD-like pathology. However, current implementations of this SynFib model have largely been confined to rats and require sequential surgeries, which increase animal distress and reduce reproducibility. Here, we present a streamlined protocol to generate a SynFib mouse model of PD that integrates rAAV-α-synuclein delivery and PFF injection into a single stereotaxic surgery. Using fine glass capillaries, this method prevents backflow of injected material, reduces injection-induced trauma, minimizes neuroinflammation, and ensures robust lesion development. This streamlined mouse model provides a reproducible and practical system to investigate α-synuclein-associated pathology and serves as a versatile platform for preclinical testing of potential therapeutics for PD.

Zika virus (ZIKV), an arthropod-borne orthoflavivirus, has emerged as a global health concern due to its ability to cause severe fetal neurological disorders, leading to the congenital Zika syndrome (CZS) in neonates. Vertical transmission during pregnancy can alter neural progenitor cell (NPC) proliferation and differentiation and induce apoptosis, leading to microcephaly and other neurodevelopmental abnormalities. While mammalian models have been used to study the impact of ZIKV on NPC behavior, limitations such as high costs, dedicated time, and ethical constraints have fostered the exploration of alternative systems. The zebrafish embryo constitutes an advantageous in vivo model for studying ZIKV neuropathogenesis. Indeed, ZIKV infection phenocopies several features of the CZS while sharing a conserved neuroanatomical layout and offering genetic plasticity and unique accessibility to the infected brain compared to mammals. Here, we describe a protocol for characterizing ZIKV-induced defects of NPCs in this zebrafish model, relying on whole animal flow cytometry.

A basic function of the nervous system is to confer the ability to detect external stimuli and generate appropriate behavioral and physiological responses. These can be modulated when parallel streams of information are provided to the nervous system and neural activity is appropriately altered. The nematode Caenorhabditis elegans utilizes a simple and well characterized neural circuit to mediate avoidance or attraction responses to stimuli, such as the volatile odorant octanol or diacetyl (DA), respectively. Aging and neurodegeneration constitute two important factors altering the ability to detect external signals and, therefore, changing behavior. Here, we present a modified protocol to assess avoidance or attraction responses to diverse stimuli in healthy individuals and Caenorhabditis elegans models associated with neurodegenerative diseases.

Microinflammation enhances the permeability of specific blood vessel sites through an elevation of local inflammatory mediators, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α. By a two-dimensional immunohistochemistry analysis of tissue sections from mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), we previously showed that pathogenic immune cells, including CD4⁺ T cells, specifically accumulate and cause microinflammation at the dorsal vessels of the fifth lumbar cord (L5), resulting in the onset of disease. However, usual pathological analyses by using immunohistochemistry on sections are not effective at identifying the microinflammation sites in organs. Here, we developed a new three-dimensional visualization method of microinflammation using luminescent gold nanoclusters (AuNCs) and the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) tissue-clearing method. Our protocol is based on the detection of leaked AuNCs from the blood vessels due to an enhanced vascular permeability caused by the microinflammation. When we injected ultrasmall coordinated Au₁₃ nanoclusters intravenously (i.v.) to EAE mice, and then subjected the spinal cords to tissue clearing, we detected Au signals leaked from the blood vessels at L5 by light sheet microscopy, which enabled the visualization of complex tissue structures at the whole organ level, consistent with our previous report that microinflammation occurs specifically at this site. Our method will be useful to specify and track the stepwise development of microinflammation in whole organs that is triggered by the recruitment of pathogenic immune cells at specific blood vessels in various inflammatory diseases.

Pavlovian fear conditioning is a widely used procedure to assess learning and memory processes that has also been extensively used as a model of post-traumatic stress disorder (PTSD). Freezing, the absence of movement except for respiratory-related movements, is commonly used as a measure of fear response in non-human animals. However, this measure of fear responses can be affected by a different baseline of locomotor activity between groups and/or conditions. Moreover, fear conditioning procedures are usually restricted to a single conditioned stimulus (e.g., a tone cue, the context, etc.) and thus do not depict the complexity of real-life situations where traumatic memories are composed of a complex set of stimuli associated with the same aversive event. To overcome this issue, we use a conditioned lick suppression paradigm where water-deprived mice are presented with a single conditioned stimulus (CS, a tone cue or the context) previously paired with an unconditioned stimulus (US, a foot shock) while consuming water. We use the ratio of number of licks before and during the CS presentation as a fear measure, thereby neutralizing the potential effect of locomotor activity in fear responses. We further implemented the conditioned lick suppression ratio to assess the effect of cue competition using a compound of contextual and tone cue conditioned stimuli that were extinguished separately. This paradigm should prove useful in assessing potential therapeutics and/or behavioral therapies in PTSD, while neutralizing potential confounding effects between locomotor activity and fear responses on one side, and by considering potential cue-competition effects on the other side.

Graphical abstract

Schematic representation of the compound context-cue condition lick suppression procedure. Illustration reproduced from Bouchekioua et al. (2022).

Late-gestation transient intrauterine hypoxia is a common cause of birth injury. It can lead to long-term neurodevelopmental disabilities even in the absence of gross anatomic injury. Currently, postnatal models of hypoxia–ischemia are most commonly used to study the effect of oxygen deprivation in the fetal brain. These models, however, are unable to take into account placental factors that influence the response to hypoxia, exhibit levels of cell death not seen in many human patients, and are unable to model preterm hypoxia. To address this gap in research, we have developed a protocol to induce transient hypoxia in fetal mice. A pregnant dam at gestational day 17.5 is placed into a hypoxia chamber. Over 30 min, the inspired oxygen is titrated from 21% (ambient air) to 5%. The dam remains in the chamber for up to 8 h, after which fetal brains can be collected or pups delivered for postnatal studies. This protocol recapitulates phenotypes seen in human patients exposed to transient in utero hypoxia and is readily reproducible by researchers.

Graphical abstract:

Repeat expansion diseases, including fragile X syndrome, Huntington’s disease, and C9orf72-related motor neuron disease and frontotemporal dementia, are a group of disorders associated with polymorphic expansions of tandem repeat nucleotide sequences. These expansions are highly repetitive and often hundreds to thousands of repeats in length, making accurate identification and determination of repeat length via PCR or sequencing challenging. Here we describe a protocol for monitoring repeat length in Drosophila models carrying 1,000 repeat C9orf72-related dipeptide repeat transgenes using Southern blotting. This protocol has been used regularly to check the length of these lines for over 100 generations with robust and repeatable results and can be implemented for monitoring any repeat expansion in Drosophila.

Repeated social defeat stress (RSDS) is a model of chronic stress in rodents. There are several variants of social defeat procedures that exert robust effects in mice, but few published detailed protocols to produce a robust stress and altered immunological profile in rats. In this article, we describe the protocol for the induction of RSDS in adult male Sprague-Dawley rats. Using a resident-intruder paradigm, a physical component of stress is induced by direct attack from the resident aggressive retired breeder Long-Evans rats on the intruder experimental rats. A subsequent threat component is induced by the presence of the aggressor in the vicinity of the intruder, but with physical separation between them. The RSDS induced by this protocol produces robust immunological and behavioral changes in the experimental rats, as evidenced by development of anxiety-like behaviors in open field, social interaction, and elevated plus maze tests, as well as by changes in immune parameters (Munshi et al., 2020). This approach has been used as an ethologically relevant model of stressors that are potent enough to impact neural circuits that are similar to the neural circuits impacted in patients with depression and anxiety.

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain’s poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. In this protocol, we describe how to perform recordings to quantify the neuroprotective and regenerative effect of implanted engineered CS-GAG hydrogel (eCS) on brain tissue. This experiment was performed in rats under three conditions: healthy without injury (Sham), controlled cortical impact (CCI) injury on the rostral forelimb area (RFA), and CCI-RFA with eCS implants. This protocol describes the procedure used to perform the craniotomy, the positioning of the cortical recording electrode, the positioning of the stimulation electrode (contralateral paw), and the recording procedure. In addition, a description of the exact electrical setup is provided. This protocol details the recordings in the brain of injured animals while preserving most of the uninjured tissue intact, with additional considerations for intralesional and laminar recordings of multi-unit response.

Graphic abstract:

Sensorimotor response to paw stimulation using cortical laminar recordings.

Translational work in rodents elucidates basic mechanisms that drive complex behaviors relevant to psychiatric and neurological conditions. Nonetheless, numerous promising studies in rodents later fail in clinical trials, highlighting the need for improving the translational utility of preclinical studies in rodents. Imaging of small rodents provides an important strategy to address this challenge, as it enables a whole-brain unbiased search for structural and dynamic changes that can be directly compared to human imaging. The functional significance of structural changes identified using imaging can then be further investigated using molecular and genetic tools available for the mouse. Here, we describe a pipeline for unbiased search and characterization of structural changes and network properties, based on diffusion MRI data covering the entire mouse brain at an isotropic resolution of 100 µm. We first used unbiased whole-brain voxel-based analyses to identify volumetric and microstructural alterations in the brain of adult mice exposed to unpredictable postnatal stress (UPS), which is a mouse model of complex early life stress (ELS). Brain regions showing structural abnormalities were used as nodes to generate a grid for assessing structural connectivity and network properties based on graph theory. The technique described here can be broadly applied to understand brain connectivity in other mouse models of human disorders, as well as in genetically modified mouse strains.

Graphic abstract:

Pipeline for characterizing structural connectome in the mouse brain using diffusion magnetic resonance imaging. Scale bar = 1 mm.