Brain research. Brain research protocols最新文献

The mouse main olfactory bulb (MOB) is commonly used as a mammalian model to study olfactory processing. The genetic techniques available with the mouse make its MOB a powerful model for analysis of neuronal circuitry. The mouse has been used as a mammalian model for all types of MOB neurons, but especially to study the activity of mitral cells. However, mouse mitral cell activity is most commonly studied in vitro. Therefore, we aimed to develop a protocol to record the activity of antidromically identified mitral cells in mouse in vivo. Currently, such a protocol does not exist. Using extracellular techniques, we report a protocol that is able to record neurons from all mouse MOB layers. Specifically, mitral cell single-units were identified by antidromic activation from the posterior piriform cortex, and their spontaneous activity was recorded for more than 30 min. This protocol is stable enough to record from single-units while buprenorphine was applied both topically to the surface of the MOB and injected systemically.

In our laboratory, we have developed methods in real-time detection and quantitative-polymerase chain reaction (Q-PCR) to analyse the relative levels of gene expression in post mortem brain tissues. We have then applied this method to examine differences in gene activity between normal white matter (NWM) and plaque tissue from multiple sclerosis (MS) patients. Genes were selected based on their association with pathology and through identification by previously conducted global gene expression analysis. Plaque tissue was obtained from secondary progressive (SP) patients displaying chronic active, as well as acute pathologies; while NWM from the same location was obtained from age- and sex-matched controls (normal patients). In this study, we used both SYBR Green I supplementation and commercially available mixes to assess both comparative and absolute levels of gene activity. The results of both methods compared favourably for four of the five genes examined (P < 0.05, Pearsons), while differences in gene expression between chronic active and acute pathologies were also identified. For example, a >50-fold increase in osteopontin (Spp1) and inositol 1-4-5 phosphate 3 kinase B (Itpκb) levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P < 0.05, unpaired t test). By contrast, there was no significant difference in the levels of the MS marker and calcium-dependent protease (Calpain, Capns1) in MS plaque tissue. In summary, Q-PCR analysis using SYBR Green I has allowed us to economically obtain what may be clinically significant information from small amounts of the CNS, providing an opportunity for further clinical investigations.

A protocol was developed for the stereological quantification of neurons expressing androgen receptor (AR) in the basolateral nuclear group (BNG) of the canine amygdaloid body. The Cavalieri method was used to estimate the BNG volume and the physical disector technique was applied for assessing the numerical densities and total numbers of both ordinary and AR-positive BNG neurons. The overall number of BNG neurons and the BNG volume were assessed on Nissl-stained sections, while AR was visualised using indirect immunohistochemistry. The morphological differentiation between neurons, astrocytes, and oligodendrocytes in these immunohistochemical sections was hampered by the cytoplasmic localisation of AR in these cells. Therefore, an additional criterion was developed based on the nuclear diameters of these cells. With the cutoff value of 7.4 μm, a sensitivity of 97.7% and specificity of 97.6% were obtained. A negative correlation was found between the BNG volume and the numerical density of its neurons, implicating that a large BNG will not necessarily have a higher number of neurons. Therefore, the numerical density or BNG volume should always be assessed in addition to the total number of neurons, justifying the use of the physical disector instead of the fractionator technique in the present study. However, higher coefficients of error were obtained for the total number of neurons with the physical disector method because of the indirect measurement of cell numbers. Therefore, the precision of the estimates must be high enough when using the disector method to compensate the precision loss caused by this indirect calculation of the total cell number.

White matter astrocytes have physiological functions which are distinct from those of astrocytes in gray matter. White matter becomes highly non-permissive to neurite growth after injury, but the role of white matter astrocytes in this process is incompletely understood. Current protocols for making primary astroglial cultures are inadequate for exploring the specific properties of white matter astrocytes in vitro. We describe a procedure for obtaining cultures of white matter astrocytes from the rodent corpus callosum. In this procedure, we take advantage of our previous finding that white, but not gray matter astrocytes express the calcium-binding protein S100A4. S100A4 expressing astrocytes are abundant in the corpus callosum, and we show that cultures, highly enriched in S100A4 expressing white matter astrocytes, can be reproducibly generated from this area. Key factors for successful cultures are (i) meticulous dissection of the corpus callosum from 4-day-old rats, and (ii) Percoll density gradient centrifugation to purify astrocytes. As a means of exploring the possible role of S100A4 in white matter astrocytes, we describe the use of the siRNA technique to eliminate the expression of S100A4 in our in vitro system.

The application of transcriptomics and proteomics approaches to accurately dissected anatomically-defined brain regions and sub-regions remains a central focus of current neurobiological investigations as well as a necessary step towards single-neuron neurogenomics and neuroproteomics. A protocol is described for the simple, rapid, and reproducible laser microdissection of brain regions and sub-regions for microarray-based gene expression analyses from individual rats or mice using two rounds of in vitro transcription (IVT). The results presented also demonstrate that the current Affymetrix GeneChip® arrays are well suited for this experimental design with high reproducibility and limited effects of the shortening of target RNA caused by the double IVT approach.

γ-hydroxybutyric acid (GHB) and its precursors, 1,4-butanediol (1,4-BD) and γ-butyrolactone (GBL), are recreational drugs widely abused in the US, Europe and Australasia. A severe withdrawal syndrome from GHB, 1,4-BD and GBL has been increasingly documented over the last years, necessitating the development of a reliable animal model for investigations of potential therapeutic approaches. The present study describes the induction and occurrence of audiogenic seizures as a sign of withdrawal from GHB and 1,4-BD in selectively bred Sardinian alcohol-preferring (sP) rats, treated with escalating doses of GHB (1.5–3.5 g/kg, twice daily; i.g.) or 1,4-BD (500–1000 mg/kg, twice daily; i.g.) for 9 consecutive days. Acute administration of the selective GABA_B receptor antagonist, SCH 50911, dramatically increased seizure occurrence. We propose that the inherent sensitivity of sP rats to different GHB-associated responses may have contributed to the unraveling of a phenomenon which was otherwise not recognizable in other rat strains.

Theta-burst stimulation (TBS: four pulses at 100 Hz repeated with 200 ms inter-burst-intervals) and another commonly used high-frequency stimulation protocol (HFS: 1 s burst of equally spaced pulses at 100 Hz) were compared for the magnitude of LTP produced in rat hippocampal slices. The total number of pulses applied during tetanus (TET) was either 40, 100, 200, or 300. In a conventional analysis of the last 10 min of the post-TET period, a two-way ANOVA revealed no difference either in LTP of the field excitatory post-synaptic potential (fEPSP) between TBS and HFS or differences across pulse number at 40, 100, or 200 pulses. At 300 pulses, there was a significant main effect by pulse number but not by protocol. A linear regression analysis showed that stimulation protocol accounted for only about 10% of the change in magnitude while pulse number contributed to 30% of the change. However, when an extended analysis of the same data was performed across the entire post-TET period with a repeated-measure ANOVA, a small but persistent increase in TBS over HFS at 200 pulses was significant. A difference between TBS and HFS at 300 pulses that occurred only during the early phase of LTP was also significant. These results suggest that, over a range of stimuli, the number of pulses in an induction protocol, rather than the pattern of stimulation, determines the magnitude of late phase LTP, while TBS produces greater potentiation than HFS in the early phase of LTP with higher TET number.

Aiming to map the distribution of spinally projecting, hypothalamic neurons containing neuronal nitric oxide synthase (nNOS), True Blue (TB) is injected into the rat spinal cord. After survival times of 7–14 days the animals are anaesthetized and perfused transcardially with a solution containing paraformaldehyde and sucrose. After dissection, the injection site is further fixed for 4–8 h, cut in a cryostat, and documented by computer-assisted digital photography. The brain region of interest is fixed for 4 h, rinsed in phosphate buffer for 48 h, sectioned, and photographically documented utilizing filter settings for visualization of TB. The brain sections are then immunohistochemically processed using a primary antibody against nNOS and a Texas Red (TR)-labelled secondary antibody and once again photographically documented, now using filter settings for visualization of TB and TR, respectively. Utilizing the Photoshop program, the TB containing cells can then be exactly aligned and the presence of TB and/or TR fluorescence in the same cell bodies are evaluated. This method for neurotransmitter-specific retrograde tracing derives its high sensitivity from the optimization of fixation/rinsing parameters, the use of appropriate fluorophores, and sequential digital microphotography.