Neuroscience

Stroke is a worldwide leading cause of death and long-term disability, with ischemic strokes making up approximately 85% of all cases. There is a significant need for an ideal animal model that accurately replicates the disease’s pathology to study the molecular mechanisms of brain injury. Various experimental models have been created to induce middle cerebral artery occlusion (MCAO), including intraluminal MCAO, photothrombotic models, endothelin-1 injections, and electrocoagulation. However, these often result in large infarct or lesion volumes accompanied by considerable variability. In this study, we present a ministroke model that specifically targets the mouse barrel cortex, making it suitable for investigating the mechanisms of minor strokes and stroke recurrence. In our model, the distal branch of the right middle cerebral artery (MCA), which supplies the sensorimotor cortex, is permanently ligated using 10-0 sutures. This is followed by a 7-min occlusion of the bilateral common carotid arteries (CCAs) and subsequent reperfusion. This approach produces a mild stroke characterized by small and consistent lesion volumes and very low mortality rates. A well-trained experimenter can achieve nearly zero mortality with this technique. Furthermore, this model of localized ischemia induces lesions in the functionally defined barrel cortex, allowing the use of the vibrissae-evoked forelimb placing test to assess functional outcomes.

Drosophila larvae exhibit rolling motor behavior as an escape response to avoid predators and painful stimuli. We introduce an accessible method for applying optogenetics to study the motor circuits driving rolling behavior. For this, we simultaneously implement the Gal4-UAS and LexA-Aop binary systems to express two distinct optogenetic channels, GtACR and Chrimson, in motor neuron (MN) subsets and rolling command neurons (Goro), respectively. Upon exposure to white LED light, Chrimson permits the influx of positive ions into Goro neurons, leading to depolarization, whereas GtACR mediates chloride influx into MNs, resulting in hyperpolarization. This method allows researchers to selectively activate certain neurons while simultaneously inhibiting others within a circuit of interest, offering a unique advantage over current optogenetic approaches, which often utilize a single type of optogenetic actuator. Here, we provide a detailed protocol for the dual silencing-activation approach using GtACR and Chrimson optogenetic channels and present a robust methodological framework for investigating the neuromuscular basis of rolling in larvae. Our cost-effective and scalable approach utilizes readily accessible equipment and can be applied to study other locomotor behaviors in Drosophila larvae, thereby enhancing our understanding of the neural circuit mechanisms underlying sensorimotor transformation.

Adult mammals lack the ability to regenerate retinal neurons after injury. However, in previous studies from this lab, topical application of the selective alpha7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, has been associated with an increase in the number of retinal neurons in adult murine models both in the presence and absence of injury to the retina. Additionally, studies assaying mitotic markers have shown a substantial increase in the amount of mitotically active and proliferating cells with the topical application of the alpha7 nAChR agonist. However, these previous studies were performed using fluorescent immunolabeling and subsequent confocal microscopy, thus limiting the number of antibodies that can be multiplexed. As a result, we have developed a flow cytometry method that allows for the multiplexing and analysis of multiple external and internal markers in dissociated retinal cells. In this paper, a step-by-step protocol is described for the labeling of multiple retinal cell types such as retinal ganglion cells, rod photoreceptors, and Müller glia, concurrently with Müller glia–derived progenitor cells that arise after treatment with PNU-282987.

Palmitoylation is a unique and reversible posttranslational lipid modification (PTM) that plays a critical role in many cellular events, including protein stability, activity, membrane association, and protein–protein interactions. The dynamic nature of palmitoylation dictates the efficient sorting of various retinal proteins to specific subcellular compartments. However, the underlying mechanism through which palmitoylation supports efficient protein trafficking in the retina remains unclear. Recent studies show that palmitoylation can also function as a signaling PTM, underlying epigenetic regulation and homeostasis in the retina. Efficient isolation of retinal palmitoyl proteome will pave the way to a better understanding of the role(s) for palmitoylation in visual function. The standard methods for detecting palmitoylated proteins employ ³H- or ¹⁴C-radiolabeled palmitic acid and have many limitations, including poor sensitivity. Relatively recent studies use thiopropyl Sepharose 6B resin, which offers efficient detection of palmitoylated proteome but is now discontinued from the market. Here, we describe a modified acyl resin–assisted capture (Acyl-RAC) method using agarose S3 high-capacity resin to purify palmitoylated proteins from the retina and other tissues, which is greatly compatible with downstream processing by LC-MS/MS. Unlike other palmitoylation assays, the present protocol is easy to perform and cost-effective.

Graphical overview

The retina is a thin neuronal multilayer responsible for the detection of visual information. The first step in visual transduction occurs in the photoreceptor outer segment. The studies on photoreception and visual biochemistry have often utilized rod outer segments (OS) or OS disks purified from mammalian eyes. Literature reports several OS and disk purification procedures that rarely specify the procedure utilized to collect the retina from the eye. Some reports suggest the use of scissors, while others do not mention the issue as they declare to utilize frozen retinas. Because the OS are deeply embedded in the retinal pigmented epithelium (RPE), the detachment of the retina by a harsh pull-out can cause the fracture of the photoreceptor cilium. Here, we present a protocol maximizing OS yield. Eye semi-cups, obtained by hemisecting the eyeball and discarding the anterior chamber structures and the vitreous, are filled with Mammalian Ringer. After 10–15 min of incubation, the retinas spontaneously detach with their wealth of OS almost intact. The impressive ability of the present protocol to minimize the number of OS stuck inside the RPE, and therefore lost, compared with the classic procedure, is shown by confocal laser scanning microscopy analysis of samples stained ex vivo with a dye (MitoTracker deep red) that stains both retinal mitochondria and OS. Total protein assay of OS disks purified by either procedure also shows a 300% total protein yield improvement. The advantage of the protocol presented is its higher yield of photoreceptor OS for subsequent purification procedures, while maintaining the physiological features of the retina.

The vestibular sensory apparatus contained in the inner ear is a marvelous evolutionary adaptation for sensing movement in 3 dimensions and is essential for an animal’s sense of orientation in space, head movement, and balance. Damage to these systems through injury or disease can lead to vertigo, Meniere’s disease, and other disorders that are profoundly debilitating. One challenge in studying vestibular organs is their location within the boney inner ear and their small size, especially in mice, which have become an advantageous mammalian model. This protocol describes the dissection procedure of the five vestibular organs from the inner ear of adult mice, followed by immunohistochemical labeling of a whole mount preparation using antibodies to label endogenous proteins such as calretinin to label Type I hair cells or to amplify genetically expressed fluorescent proteins for confocal microscopic imaging. Using typical lab equipment and reagents, a patient technician, student, or postdoc can learn to dissect and immunolabel mouse vestibular organs to investigate their structure in health and disease.

Thermotaxis behaviors in C. elegans exhibit experience-dependent plasticity of thermal preference memory. This behavior can be assayed either at population level, on linear temperature gradients, or at the individual animal level, by radial isothermal or microfluidic tracking of orientation. These behaviors are low-throughput as well as variable, due to the inherent sensitivity to environmental perturbations. To facilitate reproducible studies, we describe an updated apparatus design that enables simultaneous runs of three thermal preference assays, instead of single-run assays described previously. By enabling parallel runs of control and experimental conditions, this set-up enables more throughput and rigorous assessment of behavioral variability.

The search for safe and efficient chronic pain treatments is dampened by the lack of reliable models that faithfully reproduce current pharmacological treatments for chronic spontaneous pain in humans. Preclinical models often assess the antinociceptive efficacy of non-contingent pharmacological treatments evaluated in the short-term. Here, we provide a protocol of contingent operant self-medication in mice, which allows the estimation of spontaneous pain relief and drug abuse liability in models of persistent pain. This paradigm requires preliminary habituation and animal handling, followed by training of mice in operant conditioning boxes, to allow subsequent analgesic drug self-administration. After the initial acquisition of food-maintained operant behavior, a chronic pain sensitization is induced. Posterior intravenous jugular catheterization and coupling of operant conditioning boxes to perfusion pumps allow quantification of operant responding for intravenous drug self-administration. All mice show an initial operant drug self-administration behavior associated with the previous food-maintained operant training. This initial operant responding is extinguished after administration of ineffective treatments, but continues when the compounds have analgesic efficacy or intrinsic reinforcing properties. The identification of a significant drug self-administration selectively expressed in mice exposed to the chronic pain condition is indicative of analgesic drug effects, whereas persistent self-administration in control mice is indicative of abuse liability. The present protocol provides the behavioral and surgical procedures needed to assess spontaneous pain relief and potential for abuse of pharmacological treatments, through contingent analgesic self-medication in mice.

Graphic abstract:

Experimental design. Animals are subjected to a 5-day food self-administration protocol with a fixed ratio of reinforcement of 1 (FR1, 1 interaction with the active nose-poke causes the release of 1 reinforcer/infusion), to acquire the operant behavior. After this training, mice are subjected to the chronic pain or sham procedure, and four days later an intravenous (i.v.) catheterization is performed, to allow self-administration with the selected compound or its vehicle. Three days after the catheterization, animals start the drug/vehicle self-administration protocol at FR1. The patency of the catheter is evaluated with the thiopental test after the last self-administration session. Adapted from Bura et al. (2018).

Spiral ganglion neurons (SGN) are the primary neuronal pathway for transmitting sensory information from the inner ear to the brainstem. Recent studies have revealed significant biophysical and molecular diversity indicating that auditory neurons are comprised of sub-groups whose intrinsic properties contribute to their diverse functions. Previous approaches for studying the intrinsic biophysical properties of spiral ganglion neurons relied on patch-clamp and molecular analysis of cultured somata that were disconnected from their pre-synaptic hair cell partners. In the absence of the information provided by cell-to-cell connectivity, such studies could not associate biophysical diversity with functional sub-groups. Here we describe a protocol for preparing, recording, and labeling spiral ganglion neurons in a semi-intact ex-vivo preparation. In these preparations, the cell bodies of spiral ganglion neurons remain connected to their hair cell partners. The recordings are completed within 4 hours of euthanasia, alleviating concerns about whether long culture times and culture conditions change the intrinsic properties of neurons.

Assessment of corticospinal excitability (CSE) is an essential component of experiments designed to induce or study neuronal plasticity in the motor system. Common examples are paired associative stimulation (PAS), theta-burst stimulation (TBS), intensive motor training, or any methods aimed at potentiating the corticomotor system in the hope of promoting better recovery after neurological insult. To date, rodent models of CSE assessment have mostly been completed under anaesthesia, which greatly affects the level of CSE, as well as the mechanisms of plasticity. Experiments in awake animals are difficult because the ongoing state of behavior affects the excitability of the motor system and complicates the assessment of CSE. To address this issue, we have designed a novel approach for CSE assessment in awake behaving rodents, enabling a reliable measure of evoked motor responses obtained from cortical microstimulation in repeatable conditions of ongoing motor activity. The system relies on chronically implanted intracortical and intramuscular electrodes and a custom-made software control system, enabling the user to require that precise parameters of EMG activity be met before cortical stimulation probes are delivered. This approach could be used for further studies of PAS, TBS or other interventions requiring the assessment of CSE under repeatable conditions. We provide fabrication schematics and a list of materials for the implant, as well as instructions for running a custom-made MATLAB codebase, customizing the PAS protocol, and performing the complete analysis of experimental data. We hope these tools can further facilitate animal research in the field of neuroplasticity and neurorehabilitation.