Brain research. Brain research protocols最新文献

Subcellular localisation of proteins and peptides yields fundamental information about cell functioning. Immunoelectron microscopy is a powerful tool to achieve this goal, but combining good tissue preservation with strong immunoreactivity is a great challenge in electron microscopy. We have applied a novel approach, using high-pressure freezing (HPF) followed by cryosubstitution, to prepare the pituitary gland of the amphibian Xenopus laevis for immunogold-electron microscopy. In this way, we investigated the subcellular distribution of brain-derived neurotrophic factor and the amphibian neurohormone mesotocin in the pituitary neural lobe, and the peptide hormone α-melanophore-stimulating hormone and its protein precursor proopiomelanocortin in melanotrope cells of the pituitary intermediate lobe. In contrast to conventional chemical fixation (followed by cryosubstitution), HPF not only revealed strong immunoreactivity of the secretory products, but also provided excellent ultrastructural preservation of cell organelles, including secretory granule subtypes. We conclude that HPF followed by cryosubstitution provides a preparation technique of choice when both optimal tissue ultrastructure and strong immunoreactivity are required.

In vitro studies of glucose and lactate utilization have been performed in acute hippocampal slices or dissociated neurons and glia. While some studies concluded that lactate and glucose are equivalent substrates to support evoked synaptic activity, others showed decreased synaptic activity in the presence of lactate as compared to glucose. We found diminished neural activity in the presence of lactate in hippocampal slice cultures. We developed a method to examine the oxidation rates of ¹⁴C-labeled substrates by hippocampal slice cultures. The rate of ¹⁴CO₂ production from either ¹⁴C-glucose or ¹⁴C-lactate remained unchanged for 6 h suggesting that slice cultures are metabolically stable. While the glucose oxidation rate saturated between 2.8 and 10 mM, lactate oxidation rate had not saturated at 10 mM. These data suggest that organotypic slice cultures provide a method to examine elements of cerebral metabolism in vitro.

A unique interventional approach for modulating sensory signaling involves targeting neurons in the sensory ganglia through use of pharmacological or gene therapies. This has previously been accomplished in the trigeminal system via stereotactic ganglionic microinjections. However, this procedure can be time consuming and requires expensive stereotactic equipment. The current paper describes a percutaneous approach for injecting the trigeminal ganglion with an electrical-stimulation needle inserted through the infraorbital foramen, infraorbital canal and foramen rotundum. The needle tip was positioned in the ganglion by eliciting a twitch in the ipsilateral masseter following electrical stimulation. The technique was imaged using computerized tomography and methylene blue dye to verify the site of the injection. We validated this technique by reproducing the results from our prior study that involved injection of resiniferatoxin (RTX) into the trigeminal ganglion using the stereotactic approach. Both techniques for intraganglionic injection of RTX produced a specific deletion of C-fiber neurons, as demonstrated by inhibition of neurogenic-induced plasma extravasation and behavioral assays. Thus, we propose a novel and simple technique for studying mechanisms of peripheral sensory modulation of orofacial pain via direct application of drugs, tracers or viral vectors around trigeminal sensory neuronal cell bodies. This technique minimizes trauma to brain structures that may have an impact on pain perception.

The rostral migratory stream is one of the few regions of the adult mammalian central nervous system in which cellular migration and proliferation have been described. Most rostral migratory stream cells divide rapidly and hence different proliferation markers have been employed to identify them. Nitrogen base substitutes, such as tritiated thymidine or 5-bromo-2′-deoxyuridine (BrdU), together with endogenous molecules, such as Proliferating Cell Nuclear Antigen (PCNA), are the cell cycle markers most widely employed. Protocols for BrdU and PCNA localization are both plentiful and diverse, but to date no optimized protocol for obtaining trustworthy double staining of both markers has been described. In this work, we propose optimized protocols for achieving both single staining and the joint detection of BrdU and PCNA in the rodent brain using double-immunofluorescence procedures. The double labeling described allows the discrimination of different cell cycle stages in migratory cells from the mouse brain.

Serum starvation of astrocytes for a period of time followed by refeeding has been proposed as a method to produce synchronized astrocytes. Here, it is proposed that the method neither synchronizes cells nor satisfies rigorous criteria for cell synchronization. The proposed non-selective, whole-culture synchronization protocol cannot, in theory, synchronize cells. The cells produced by the proposed serum starvation/refeeding protocol do not reflect the properties of any particular cell during the cell cycle. Cells produced by the published protocol will have a wide distribution of cell sizes, and therefore, the cells produced by starvation/refeeding will not model cells of any specific age during the division cycle. Thus, the proposed protocol will not produce a synchronized culture.

A methodological study on subject-specific regression analysis (SSRA) exploring the correlation between the neural response and the subjective evaluation of emotional experience in eleven healthy females is presented. The target emotions, i.e., amusement and sadness, were induced using validated film clips, regional cerebral blood flow (rCBF) was measured using positron emission tomography (PET), and the subjective intensity of the emotional experience during the PET scanning was measured using a category ratio (CR-10) scale. Reliability analysis of the rating data indicated that the subjects rated the intensity of their emotional experience fairly consistently on the CR-10 scale (Cronbach alphas 0.70–0.97). A two-phase random-effects analysis was performed to ensure the generalizability and inter-study comparability of the SSRA results. Random-effects SSRAs using Statistical non-Parametric Mapping 99 (SnPM99) showed that rCBF correlated with the self-rated intensity of the emotional experience mainly in the brain regions that were identified in the random-effects subtraction analyses using the same imaging data. Our results give preliminary evidence of a linear association between the neural responses related to amusement and sadness and the self-evaluated intensity of the emotional experience in several regions involved in the emotional response. SSRA utilizing subjective evaluation of emotional experience turned out a feasible and promising method of analysis. It allows versatile exploration of the neurobiology of emotions and the neural correlates of actual and individual emotional experience. Thus, SSRA might be able to catch the idiosyncratic aspects of the emotional response better than traditional subtraction analysis.