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Background Stereoelectroencephalographic (SEEG) recordings can be performed before final resective surgery in some drug-resistant patients with focal epilepsies. For good SEEG signal interpretation, it is important to correctly identify the brain tissue in which each contact is inserted. Tissue classification is usually done with the coregistration of CT scan (with implanted SEEG electrodes) with preoperative MRI. New method Brain tissue classification is done here directly from SEEG signals obtained at rest by a linear discriminant analysis (LDA) classifier using measured SEEG signals. The classification operates on features extracted from Bode plots obtained via non-parametric frequency domain transfer functions of adjacent contacts pairs. Classification results have been compared with classification from T1 MRI following the labelling procedure described in Deman et al. (2018), together with minor corrections by visual inspection by specialists. Results With the data processed from 19 epileptic patients representing 1284 contact pairs, an accuracy of 72 ± 3% was obtained for homogeneous tissue separation. To our knowledge only one previous study conducted brain tissue classification using the power spectra of SEEG signals, and the distance between contacts on a shaft. The features proposed in our article performed better with the LDA classifier. However, the Bayesian classifier proposed in Greene et al. (2020) is more robust and could be used in a future study to enhance the classification performance. Conclusions and significance Our findings suggest that careful analysis of the transfer function between adjacent contacts measuring resting activity via frequency domain identification, could allow improved interpretation of SEEG data and or their co-registration with subject’s anatomy.

Neuromodulation plays a critical role in regulating brain function and its dysregulation is implicated in the pathogenesis of numerous neurological and psychiatric disorders. However, only in the last few years have optical tools become available to probe the spatial and temporal profiles of neuromodulator signaling, including dopamine, with the requisite resolution to uncover mechanisms of neuromodulation. In this review, we summarize the current state of synthetic nanomaterial-based optical nanosensors for monitoring neurotransmission with high spatial and temporal resolution. Specifically, we highlight how synthetic nanosensors can elucidate the spatial distribution of neuromodulator release sites and report the temporal dynamics and spatial diffusion of neuromodulator release. Next, we outline advantages and limitations of currently available nanosensors and their recent application to imaging endogenous dopamine release in brain tissue. Finally, we discuss strategies to improve nanosensors for in vivo use, with implications for translational applications.

Highlights • Development of inhalation-based anaesthesia for early postnatal (P0−2) mice. • 3D-printed mould allows anaesthetic maintenance for neonatal surgery. • Improved mouse welfare through reliable neonatal inhalation anaesthesia. • Rapid procedure for brain transduction of mouse litter in under 2 h., Background Investigating brain function requires tools and techniques to visualise, modify and manipulate neuronal tissue. One powerful and popular method is intracerebral injection of customised viruses, allowing expression of exogenous transgenes. This technique is a standard procedure for adult mice, and is used by laboratories worldwide. Use of neonatal animals in scientific research allows investigation of developing tissues and enables long-term study of cell populations. However, procedures on neonatal mice are more challenging, due to the lack of reliable methods and apparatus for anaesthesia of these animals. New method Here, we report an inhalation-based protocol for anaesthesia of neonatal (P0−2) mice and present a custom 3D-printed apparatus for maintenance of anaesthesia during surgical procedures. Our optimised method of anaesthesia enables a rapid method of stereotactic injection in neonatal mice for transduction of brain tissue. Results and comparison with existing methods This approach significantly enhances animal welfare and facilitates wider and simpler use of neonatal rodents in scientific research. We demonstrate this procedure for targeted labelling of specific brain regions, and in vivo modification of tissue prior to organotypic culture. Conclusions Our protocol for reliable delivery of inhalational anaesthetics can be readily adopted by any laboratory and will enable safer use of neonatal rodents across a diverse spectrum of scientific disciplines. Application to stereotactic injections allows a rapid and efficient method for modification of brain tissue.

Background High resolution neuronal information is extraordinarily useful in understanding the brain's functionality. The development of the Golgi-Cox stain allowed observation of the neuron in its entirety with unrivalled detail. Tissue clearing techniques, e.g., CLARITY and CUBIC, provide the potential to observe entire neuronal circuits intact within tissue and without previous restrictions with regard to section thickness. New method Here we describe an improved Golgi-Cox stain method, optimised for use with CLARITY and CUBIC that can be used in both fresh and fixed tissue. Results Using this method, we were able to observe neurons in their entirety within a fraction of the time traditionally taken to clear tissue (48 h). We were also able to show for the first-time that Golgi stained tissue is fluorescent when visualized using a multi-photon microscope, allowing us to image synaptic spines with a detail previously unachievable. Comparison with existing methods These novel methods provide cheap and easy to use techniques to investigate the morphology of cellular processes in the brain at a new-found depth, speed, utility and detail, without previous restrictions of time, tissue type and section thickness. Conclusions This is the first application of a Golgi-Cox stain to cleared brain tissue, it is investigated and discussed in detail, describing different methodologies that may be used, a comparison between the different clearing techniques and lastly the novel interaction of these techniques with this ultra-rapid stain.

Background Implanted osmotic minipumps are commonly used for long-term, brain-targeted delivery of a wide range of experimental agents by being connected to a catheter and a cannula. During the stereotactical surgery procedure, the cannula has to be placed correctly in the x-y directions and also with respect to the injection point in the z-direction (deepness). However, the flat fixation base of available cannula holders doesn’t allow an easy, secure fixation onto the curve-shaped skull. New method We have developed a modified method for a better fixation of the cannula holder by using an easy-to-produce, skull-shaped silicone spacer as fixation adapter. Results We describe the application and its fast and reliable production in the lab. Comparison with existing method(s) Superglue or cement is currently being used as the method of choice. However, the curve-shaped skull surface does not fit well with the flat and rigid cannula adapter which leads to fixation problems over time causing wide infusion channels and often also to leakage problems from intracerebrally applied agents towards the surface meninges. As another consequence of the inappropriate fixation, the cannula may loosen from the skull before the end of the experiment or it causes damage to the brain tissue, harming the animals with leading to a failure of the whole experiment. Conclusions The easy-to-produce spacer facilitates the crucial step of long-term, stereotactic brain infusion experiments with intracerebral catheters in a highly secure and reproducible way.

Background The histopathological study of brain tissue is a conventional method in neuroscience. However, procedures specifically developed to recover intact hypothalamic-pituitary brain specimens, are not available. New method We describe a detailed protocol for obtaining intact rat brain with pituitary-hypothalamus continuity through an intact infundibulum. The brain is collected via a ventral approach through removing the skull base. Membranous structures surrounding the hypothalamus-pituitary system can be preserved, including vasculature. Results We report a retaining sphenoid and dura technique to obtain intact hypothalamic-pituitary brain preparations, and we confirm the practicability of this method. By combination of this technique with histological analysis or 3D brain tissue clearing and imaging methods, the functional morphology structure of the hypothalamus-pituitary can be further explored. Comparison with existing method The current procedure is limited in showing the connection between the hypothalamus and the pituitary. Our procedure effectively protects the integrity of the fragile infundibulum and thus prevents the pituitary from separating from the hypothalamus. Conclusions We present a convenient and practical approach to obtain intact hypothalamus-pituitary brain specimens for subsequent histopathological evaluation.

Background: Fixation of brain tissue is a common practice which allows preservation of tissue and aids in preventing structural and chemical abnormalities. However, fixation procedures may disrupt the levels of biometals such as zinc when compared to tissue that is fresh-frozen. Thus, we sought to determine if any differences in free-zinc levels exist between perfused and fresh-frozen tissue. Zinc is an essential biometal critical for cellular communication and memory and exists in both bound and free forms; the latter playing critical roles in synaptic communication. New method: C57BL/6 J mice were divided into two water types: those given lab water and those given water supplemented with 10 ppm zinc carbonate. Perfusion was carried out with 4% paraformaldehyde on half of the animals in each water group to assess the impact on levels of free Zn as measured through Zinpyr-1 fluorescence. Results: There were significant differences in Zn fluorescence values between Zn-supplemented and lab water groups as well as between perfused and fresh-frozen tissues in the dentate gyrus and CA3 regions of the hippocampus, regions critical in learning & memory. Comparison with existing methods: These results show that when determining a method for euthanasia, any future histological techniques involving assessment of metal content should first be considered. Conclusions: Researchers must be cautious with the way in which tissue is collected and treated since this can lead to misleading conclusions when linking changes in behavior and relative levels of trace metals.

Background There is a need for effective computational methods for quantifying the three-dimensional (3-D) spatial distribution, cellular arbor morphologies, and the morphological diversity of brain astrocytes to support quantitative studies of astrocytes in health, injury, and disease. New method Confocal fluorescence microscopy of multiplex-labeled (GFAP, DAPI) brain tissue is used to perform imaging of astrocytes in their tissue context. The proposed computational method identifies the astrocyte cell nuclei, and reconstructs their arbors using a local priority based parallel (LPP) tracing algorithm. Quantitative arbor measurements are extracted using Scorcioni's L-measure, and profiled by unsupervised harmonic co-clustering to reveal the morphological diversity. Results The proposed method identifies astrocyte nuclei, generates 3-D reconstructions of their arbors, and extracts quantitative arbor measurements, enabling a morphological grouping of the cell population. Comparison with existing methods Our method enables comprehensive spatial and morphological profiling of astrocyte populations in brain tissue for the first time, and overcomes limitations of prior methods. Visual proofreading of the results indicate a >95% accuracy in identifying astrocyte nuclei. The arbor reconstructions exhibited 3.2% fewer erroneous jumps in tracing, and 17.7% fewer false segments compared to the widely used fast-marching method that resulted in 9% jumps and 20.8% false segments. Conclusions The proposed method can be used for large-scale quantitative studies of brain astrocyte distribution and morphology.

Background Mechanical properties of the brain tissue are crucial to understand the mechanisms of traumatic brain injury (TBI). Injured brain tissue could induce changes of mechanical properties and anatomical structures. However, limited data is available for the injured tissue. New Method We developed a custom-built device to introduce controlled cortical impact (CCI) to brain with controlled impact velocity and direction. A study protocol for measuring the viscoelastic properties of injured brain tissue was also developed. Micro-scale morphological changes of the vasculature were quantified by analyzing confocal images of the brain tissue using CLARITY method. Results Results showed significant differences of the instantaneous shear modulus of the impact region from different impact angles. However, no significant differences were found for long-term shear modulus by varying the impact angles and velocities. Analysis of the vasculature showed an increased radius of the vessels in the injured tissue compared with that in the control group. Comparison with existing methods A combination of three different impact velocities and three different impact angles were adopted for producing injury to the brain. In addition, viscoelastic properties were compared between the injured and non-injured regions. The corresponding morphological changes of the vasculature system were also investigated. Conclusions The instantaneous shear modulus at the impact region was significantly different for the three impact angles. Compared to that of the control group, increased radius of the vasculature was also observed in the injured brain tissue. Results indicated that the biomechanical and structural changes of the injured tissue were closely related to the impact angles and velocities. Viscoelastic measurements could also help validation of computational models.

Single cell intracellular recordings in-vivo at deep brain structures are seldomly accompanied by nearby optogenetics or drug application. The use of such tools is limited as both light and drugs cannot penetrate deep inside brain tissue. Hence, the optical fiber or drug delivery pipette needs to be placed within the brain close to the recording pipette. So far, however, this has required highly accurate hardware to achieve. These complications have now been solved by new approaches enabling intracellular recordings both for optogenetics and pharmacological application by the use of a single manipulator. In this manuscript we review these technologies - their pros, cons and implications.

Background Cerebrospinal fluid (CSF) collection is currently the only feasible means to obtain biological fluid for the quantitative determination of biomarkers that may reflect disease activity within the brain. Studies in mouse models of human neurological disease benefit from ascertainment and subsequent analysis of brain tissue that is not afforded in human patients. The CSF provides a translational forum, however, due to the practical constraints presented by the mouse’s small size, CSF is often ignored in experimental mouse models. New method Here we report a method for the controlled, precise and predictable collection of 10 μL of CSF from the lateral ventricles of adult mice using stereotaxic equipment and a micro-syringe pump. Results Collected CSF was clear and manifested the consistency of water and moreover, quantification of a disease-specific biomarker for the neurodegenerative disorder, mucopolysaccharidosis type IIIA (MPS IIIA) was possible in this small volume of CSF. In the naturally occurring mouse model of MPS IIIA, that faithfully recapitulates the human form of the disease, this biomarker was present at concentrations of >100 pmol/mL and undetectable in wild-type mice. Comparison with existing method Advantages of this method over the most commonly used cisterna magna collection technique include increased CSF sample volume (10 μL) and reduced blood contamination. Conclusion The ability to collect CSF from mouse models of neurological disease enables a forum for translating research outcomes in experimental models to the human equivalent in which CSF collection is also possible.

A major challenge in the histological sectioning of brain tissue is achieving accurate alignment in the standard coronal, horizontal, or sagittal planes. Correct alignment is desirable for ease of subsequent analysis and is a prerequisite for computational registration and algorithm-based quantification of experimental data. We have developed a simple and low-cost technique for whole-brain cryosectioning of rodent brains that reliably results in a precise alignment of stereotactic coordinates. The system utilises a 3-D printed model of a mouse brain to create a tailored cavity that is used to align and support the brain during freezing. The alignment of the frozen block is achieved in relation to the fixed edge of the mold. The system also allows for two brains to be frozen and sectioned simultaneously. System components, procedural steps, and examples of the end results are presented.

Continuous monitoring of brain tissue partial pressure of oxygen (ptiO(2)), thermal-diffusion regional cerebral blood flow (TD-rCBF) and cerebral perfusion pressure (CPP) allows the calculation of flow- and oxygen-related autoregulation indices ORx and FRx. The influence of temporal phase shifts on ORx and FRx due to a delay in the response time of ptiO(2) and TD-rCBF has received little attention. We investigated the impact of phase shifts between changes in CPP and the corresponding ptiO(2) and TD-rCBF responses on the degree of correlation of ORx and FRx. In five patients with aneurysmal subarachnoid hemorrhage continuous multimodal neuromonitoring was performed for 7-10 days. In each patient the phase shift of the ptiO(2)- and TD-rCBF-response after spontaneous positive or negative CPP fluctuations during two 1-h time periods of disturbed cerebral autoregulation was determined. For these periods, ORx and FRx were calculated as Pearson correlation coefficients before (ORx and FRx) and after (ORx(sync) and FRx(sync)) temporal synchronization of ptiO(2), TD-rCBF and CPP. The mean temporal phase shift after CPP fluctuations was 65±11 s for ptiO(2) and 12±4 s for TD-rCBF. Before synchronization, ORx and FRx were determined at 0.52±0.3 and 0.59±0.3, respectively. After synchronization, ORx(sync) and FRx(sync) correlated significantly stronger than unsynchronized indices (ORx(sync): 0.66±0.3; FRx(sync): 0.62±0.3; p0.01 vs. ORx and FRx, respectively). These findings suggest that ORx and FRx are subject to temporal latency shifts of ptiO(2) and TD-rCBF in regard to spontaneous CPP fluctuations. Temporal synchronization for the calculation of both ORx and FRx may permit continuous monitoring of these indices with higher sensitivity.

One of the major challenges of connectomics is obtaining a physical map of the neurons that comprise a circuit and the sites within the whole mouse brain. However, there is no report that addresses the preparation of whole mouse brain tissue for microsectioning. In this paper, such tissue is prepared by a modified Golgi-Cox method in which the staining time is prolonged to half a year, the darkening solution is changed to 1% LiOH, and the brain is embedded in resin. Projections of several coronal sections are reconstructed by serial 1-μm sectioning and simultaneous imaging of the specimen. This approach ensures that the stained neurons are present throughout the whole mouse brain from superficial to deep layers and that the neuronal soma and traces of the processes can be distinguished in local magnification.

Udgivelsesdato: 2009-Sep-15 Improving histomorphometric analysis of the human neocortex by combining stereological cell counting with immunohistochemical visualisation of specific neuronal and glial cell populations is a methodological challenge. To enable standardized immunohistochemical staining, the amount of brain tissue to be stained and analysed by cell counting was efficiently reduced using a fractionator protocol involving several steps of sub-sampling. Since no mathematical or statistical tools exist to predict the variance originating from repeated sampling in complex structures like the human neocortex, the variance at each level of sampling was determined empirically. The methodology was tested in three brains analysing the contribution of the multi-step sampling procedure to the precision on the estimated total numbers of immunohistochemically defined NeuN expressing (NeuN(+)) neurons and CD45(+) microglia. The results showed that it was possible, but not straight forward, to combine immunohistochemistry and the optical fractionator for estimation of specific subpopulations of brain cells in human neocortex.

This study reports an online temperature correction method for determining tissue oxygen partial pressure P(tO2) in the striatum and a novel simultaneous measurement of brain P(tO2) and temperature (T(brain)) in conjunction with global oxygen consumption V(O2) in non-sedated and non-anesthetized freely moving Arctic ground squirrels (AGS, Spermophilus parryii). This method fills an important research gap-the lack of a suitable method for physiologic studies of tissue P(O2) in hibernating or other cool-blooded species. P(tO2) in AGS brain during euthermy (21.22+/-2.06 mmHg) is significantly higher (P=0.016) than during hibernation (13.21+/-0.46 mmHg) suggests brain oxygenation in the striatum is normoxic during euthermy and hypoxic during hibernation. These results in P(tO2) are different from blood oxygen partial pressure P(aO2) in AGS, which are significantly lower during euthermy than during hibernation and are actually hypoxic during euthermy and normoxic during hibernation in our previous study. This intriguing difference between the P(O2) of brain tissue and blood during these two physiological states suggests that regional mechanisms in the brain play a role in maintaining tissue oxygenation and protect against hypoxia during hibernation.

Background Laser capture microdissection (LCM) is an established technique for the procurement of enriched cell populations that can undergo further downstream analysis, although it does have limitations. Expression microdissection (xMD) is a new technique that begins to address these pitfalls, such as operator dependence and contamination. New method xMD utilises immunohistochemistry in conjunction with a chromogen to isolate specific cell types by extending the fundamental principles of LCM to create an operator-independent method for the procurement of specific CNS cell types. Results We report how xMD enables the isolation of specific cell populations, namely neurones and astrocytes, from rat formalin fixed-paraffin embedded (FFPE) tissue. Subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirms the enrichment of these specific populations. RIN values after xMD indicate samples are sufficient to carry out further analysis. Comparison with existing method xMD offers a rapid method of isolating specific CNS cell types without the need for identification by an operator, reducing the amount of unintentional contamination caused by operator error, whilst also significantly reducing the time required by the current basic LCM technique. Conclusions xMD is a superior method for the procurement of enriched cell populations from post-mortem tissue, which can be utilised to create transcriptome profiles, aiding our understanding of the contribution of these cells to a range of neurological diseases. xMD also addresses the issues associated with LCM, such as reliance on an operator to identify target cells, which can cause contamination, as well as addressing the time consuming nature of LCM.

BACKGROUND: Studies with continuous wave near infrared spectroscopy (CW-NIRS) have shown little difference in brain oxygenation of dead compared to live subjects. We determined brain oxyhemoglobin (OHb) and deoxyhemoglobin (HHb) concentrations in healthy volunteers and cadavers using frequency domain near infrared spectroscopy (FD-NIRS). METHODS: Regional OHb and HHb, brain oxygen saturation (SO2), and total hemoglobin (tHb) were determined. Nine patients who died in the hospital were evaluated by FD-NIRS in the morgue 7-96 h after death was confirmed. Ten volunteers served as a control group. RESULTS: Absolute concentrations of brain tissue OHb and HHb were 24.9 +/- 9.1 uM and 13.8 +/- 32 uM, respectively, in live subjects. In dead subjects, OHb was 1.3 +/- 2.1 uM and HHb was 30.8 +/- 14.4 uM (both P < 0.05 compared to live). OHb showed a 90% decrease within 7h of death. There was a significant trend for a continued decrease in OHb from 7 to 96 h. CONCLUSION: OHb decreased and HHb increased in dead patients compared to live volunteers. Depletion of OHb primarily occurred within 7 h of death but continued gradually over 96 h. FD-NIRS was a novel technique for determining OHb and HHb changes following death.

An important concern about microdialysis methodology is the histological validation of the dialysis probe implantation site in brain tissue of rodents (rat, mouse). Several methods have been described on standard histological staining (i.e., cresyl violet, formalin fixation, fast green perfusion, etc.). However, this methodology is time consuming. These requirements are not compatible with a histological validation prior to analysis of microdialysis samples. Here, we developed a new method to locate the track of the dialysis probe in the rodent brain. This method is based on a digital photomicrograph of a coronal section of the rodent frozen brain. The fitting of an appropriate coronal diagram of the rats' and mice' brain atlas with this photomicrograph, allowed us to locate precisely and quickly the track of the dialysis probe.

Golgi impregnation is unique in its ability to display the dendritic trees of large numbers of individual neurons. However, its reputation for inconsistency leaves many investigators reluctant to embrace this methodology, particularly for the study of formalin-fixed human brain tissue. After reviewing the literature, testing a variety of technical variations, and discussing the procedure with experienced practitioners, we have concluded that much of the unpredictability can be removed by matching the Golgi technique to the conditions that were used for fixation of the tissue. Briefly fixed tissues worked best with the rapid Golgi technique, which includes osmium during the initial chromation step, and with the Golgi-Cox method, which includes mercuric chloride during chromation. For tissues that have been fixed for several years or even for several decades, superior results are obtained with the Golgi-Kopsch technique, using multiple changes of a chromation solution that contains paraformaldehyde. In the Golgi-Kopsch technique, pH should be used to monitor the reduction of Cr6+ to Cr3+, which is a crucial determinant of successful chromation. With any Golgi technique, agitation throughout the impregnation helps to avoid precipitates and to improve the quality of impregnation. When the appropriate method is chosen, Golgi impregnation is a useful technique for the neuropathologist.

Background Synaptoneurosomes have become an important tool for studying synaptic proteins. The filtered synaptoneurosomes preparation originally developed by Hollingsworth et al. (1985) is widely used and is an easy method to prepare synaptoneurosomes. The hand processing steps in that preparation, however, are labor intensive and have become a bottleneck for current proteomic studies using synaptoneurosomes. For this reason, we developed new steps for tissue homogenization and filtration that transform the preparation of synaptoneurosomes to a high-throughput, semi-automated process. New method We implemented a standardized protocol with easy to follow steps for homogenizing multiple samples simultaneously using a FastPrep tissue homogenizer (MP Biomedicals, LLC) and then filtering all of the samples in centrifugal filter units (EMD Millipore, Corp). Results and comparison with existing methods The new steps dramatically reduce the time to prepare synaptoneurosomes from hours to minutes, increase sample recovery, and nearly double enrichment for synaptic proteins. These steps are also compatible with biosafety requirements for working with pathogen infected brain tissue. Conclusions The new high-throughput semi-automated steps to prepare synaptoneurosomes are timely technical advances for studies of low abundance synaptic proteins in valuable tissue samples.

Because of concern that the stunning of cattle with captive bolt guns (CBGs) could, if used on an animal with bovine spongiform encephalopathy (BSE), cause embolism of infective brain tissue and carcass contamination, the Ministry of Agriculture, Food and Fisheries commissioned research to assess the risk of haematogenous dissemination of CNS material after stunning. We have devised two methods to investigate this risk. The first involves the concentration of embolic tissue in buffy coat Cytoblocks that can be embedded for sectioning, microscopy and immunocytochemistry. The second method is an ELISA for the presynaptic protein, syntaxin 1B. The methods were validated by analysis of several bovine tissues, including blood samples deliberately contaminated with brain. We then studied jugular venous blood obtained before and after the stunning of 60 cattle with CBGs. Samples obtained, after stunning, from five of the cattle contained CNS tissue within the Cytoblocks and yielded positive syntaxin assays. Syntaxin was also detected in samples from one other animal that had been stunned with a pneumatically operated CBG. The described methods should allow an assessment of the risk of neuroembolism associated with different types of CBG and may also be useful in other contexts.

A method is described which enables the spatial pattern of discrete objects in histological sections of brain tissue to be determined. The method can be applied to cell bodies, sections of blood vessels or the characteristic lesions which develop in the brain of patients with neurodegenerative disorders. The density of the histological feature under study is measured in a series of contiguous sample fields arranged in a grid or transect. Data from adjacent sample fields are added together to provide density data for larger field sizes. A plot of the variance/mean ratio (V/M) of the data versus field size reveals whether the objects are distributed randomly, uniformly or in clusters. If the objects are clustered, the analysis determines whether the clusters are randomly or regularly distributed and the mean size of the clusters. In addition, if two different histological features are clustered, the analysis can determine whether their clusters are in phase, out of phase or unrelated to each other. To illustrate the method, the spatial patterns of senile plaques and neurofibrillary tangles were studied in histological sections of brain tissue from patients with Alzheimer's disease.

In vitro receptor autoradiography is a widely used technique for determining the distribution of radioligand binding sites. By using this technique it is possible to investigate alterations in receptor number and affinity caused by trauma or a disease state. To date, however, the largest sections prepared for in vitro autoradiography have been from single human hemispheres, with the adjacent hemisphere being used for neuropathological investigations. Therefore, a method for cutting large cryosections of human whole brain tissue is described. Whole brains obtained less than 2 days postmortem were frozen at -80 degrees C. 1.5-2 cm coronal slices were cut from the brain and embedded and frozen in a carboxymethylcellulose solution. Sections 40 microm in size were sliced from the frozen block at -16 degrees C in a whole body cryostat. The sections were lifted by means of a nylon membrane backing material and subsequently incubated with tritiated ligand to produce autoradiograms of each whole brain coronal section. [(3)H]paroxetine was used in the present study as an example.

Neurodegeneration is the underlying cause of a vast majority of neurological disorders and often a result of brain trauma, stroke, or neurotoxic insult. Here we describe a simple method for labeling degenerating neurons in unfixed brain tissue samples. This method could provide a new avenue for identifying and harvesting degenerative neurons from unfixed brain tissues for subsequent molecular analyses.

The peptidome encompasses all the peptides present in a particular cell, tissue or organism at a particular point in time. Neuropeptidomics studies the peptidome of the nervous system and will become increasingly important in neuroscience research. Novel peptides can be discovered and, when applied to disease models, key players in pathophysiological mechanisms will be identified. That way, they can serve as drug targets or biomarkers. Presently, different extraction protocols are in use, but no consensus has been reached on what fixation and extraction protocol is best suited for brain tissue. Therefore, in this article we compare different methods for quenching of proteolytic activity (snap-freezing of whole mouse in liquid nitrogen immediately after cervical dislocation, freezing of the dissected brain in 2-methyl-butane and heat denaturation of the tissue by microwave treatment) in combination with different extraction methods. The protocol that combines submersion in liquid nitrogen with extraction in 0.25% acetic acid results in the highest number of unique identifications, a high conservation of posttranslational modifications, the best reproducibility between duplicate samples and the best comparison with former studies on mouse brain peptides. For these reasons, we recommend the use of this protocol in future neuropeptidomics studies.

The thickness of a brain tissue slice preparation governs the amount of time required for substances to diffuse from the bathing solution to preparation. Slice thickness may increase during the experiment, e.g., in cases of hypoxia where osmotic pressure within the tissue changes, enabling water to enter the preparation. With increasing slice thickness diffusion paths from the bath to central layers of the preparation increase possibly resulting in an insufficient O z supply to central layers. Therefore, the actual slice thickness should be monitored during the experiment especially in cases where osmolarity is changed or during hypoxia. This paper describes a simple method to monitor the actual slice thickness using ion profiles measured by ion selective micro-electrodes driven at a constant rate of approximately 10 Am/s (sample rate ca. 10/s). The method is based on steep changes in the concentration gradients at the upper and lower surfaces of the preparation induced by simple diffusion in the presence of concentration gradients between the non-tortuous bath and the tortuous tissue. The thickness of the preparation is derived from the location of the steep gradient changes as reflected by the registered profile.

Neuronal staining techniques have played a crucial role in the analysis of neuronal function. Several different staining techniques have been developed to allow morphological analyses of neurons. DiOlistic labeling, in which beads are coated with a lipophilic dye and then ballistically ejected onto brain tissue, has recently been introduced as a useful and simple means to label neurons and glia in their entirety. Although diOlistic labeling provides detailed information on the morphology of neurons, combining this approach with other staining methods is a significant advance. We have developed protocols that result in high quality diOlistically- and retrogradely-labeled or diOlistically-immunohistochemically labeled neurons. These dual-label methods require modification of fixation parameters and the restricted use of detergents for tissue permeabilization, and are readily applicable to a wide range of tracers and antibodies.

Internally quenched fluorogenic substrates are commonly used for measuring enzyme activity in biological samples and allow high sensitivity and continuous real-time measurement that is well suited for high throughput analysis. We describe the development and optimisation of an immunocapture-based assay that uses the fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)) and allows the specific measurement of insulin-degrading enzyme (IDE) activity in brain tissue homogenates. This fluorogenic substrate can be cleaved by a number of enzymes including neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1) and angiotensin-converting enzyme (ACE), as well as IDE, and we have previously shown that discrimination between these individual enzymes is not readily achieved in tissue homogenates, even in the presence of selective inhibitors and pH conditions. We tested a panel of IDE antibodies to isolate and capture IDE from brain tissue homogenates and found that immunocapture with antibody to the inactive domain of IDE prior to the addition of fluorogenic substrate allows sensitive (linear at 156-2500ng/ml) and specific measurement of IDE activity and negligible cross-reactivity with NEP, ACE or ECE-1. This assay should allow the measurement of IDE enzyme levels in a variety of biological tissues and may be useful in study of diseases such as Alzheimer's disease and insulin-dependent diabetes.

Deficiency of docosahexaenoic acid (DHA) and other omega-3 (omega3) fatty acids may constitute an alterable risk factor for Alzheimer's disease (AD). Mechanisms of potential involvement of DHA in the disease process have been postulated primarily from studies in vitro and in mouse models of AD. Information on the fatty acid profile of the brain in AD itself is limited and in some respects contradictory. Interpretation of the findings is complicated by the diversity of methods used in previous studies and a lack of information as to the effect of post-mortem delay on the results. Here we report the development of a simple and highly reproducible method that enables relatively high-throughput measurement of the fatty acid composition in samples of brain tissue and using this method we have demonstrated that there is no significant change in fatty acid composition under conditions designed to model post-mortem delay of up to 3 days at 4 degrees C (or even at room temperature). The development of this method and the observation that delay of up to 3 days has no effect on fatty acid content will facilitate further studies of fatty acid composition on large cohorts of post-mortem brains.

In sections of paraformaldehyde fixed brain tissue, stained using immunohistochemical methods, the distribution of staining within the sections is not uniform. Whilst stained cells are seen at the top and bottom surfaces, the central thicknesses of the sections contain little or no immunoreactivity. This presents a major problem for quantification, as each section contains a population of cells that is not visualized by the staining method. Following extensive investigation of this phenomenon, we report that the failure of full thickness, immunohistochemical staining is not a failure of the immunohistochemical methodology per se, nor is it related directly to the thickness of the sections used. Rather, the problem lies in the chemistry of the tissue itself, and originates during fixation of the tissues using paraformaldehyde-based perfusion methods, which render the cell membranes impermeable to one or more components of the staining protocol. We show that this impermeability is affected by addition of membrane-disrupting agents to the fixative, and by a reduction of exposure to paraformaldehyde during fixation. The present investigation contributes to the development of new fixation protocols, optimised for use in both immunohistochemical methods and morphometric analyses.

Chronically implanted recording electrode arrays linked to prosthetics have the potential to make positive impacts on patients suffering from full or partial paralysis. Such arrays are implanted into the patient's cortical tissue and record extracellular potentials from nearby neurons, allowing the information encoded by the neuronal discharges to control external devices. While such systems perform well during acute recordings, they often fail to function reliably in clinically relevant chronic settings. Available evidence suggests that a major failure mode of electrode arrays is the brain tissue reaction against these implants, making the biocompatibility of implanted electrodes a primary concern in device design. This review presents the biological components and time course of the acute and chronic tissue reaction in brain tissue, analyses the brain tissue response of current electrode systems, and comments on the various material science and bioactive strategies undertaken by electrode designers to enhance electrode performance.

Previous studies have demonstrated the utility of the gold chloride method for en bloc staining of a bisected brain in mice and rats. The present study explores several variations in the method, assesses its reliability, and extends the limits of its application. We conclude that the method is very efficient, highly robust, sufficiently accurate for most purposes, and adaptable to many morphometric measures. We obtained acceptable staining of commissures in every brain, despite a wide variety of fixation methods. One-half could be stained 24 h after the brain was extracted and the other half could be stained months later. When staining failed because of an exhausted solution, the brain could be stained successfully in fresh solution. Relatively small changes were found in the sizes of commissures several weeks after initial fixation or staining. A half brain stained to reveal the mid-sagittal section could then be sectioned coronally and stained again in either gold chloride for myelin or cresyl violet for Nissl substance. Uncertainty, arising from pixelation of digitized images was far less than errors arising from human judgments about the histological limits of major commissures. Useful data for morphometric analysis were obtained by scanning the surface of a gold chloride stained block of brain with an inexpensive flatbed scanner.

We describe a minimally invasive method for administering diffusible substances to computer-targeted regions of the primate brain. Treatments enter the brain by diffusion or under pressure from the portion of a curvilinear implant that lies within the region of interest. During the implantation surgery, a guide-tube cannula is passed through a cranial burr-hole and concentric, telescoping needles are extended from the cannula in sequence to trace a pre-planned course through brain tissue. After the leading end of the longest needle emerges through another burr-hole, the surgeon fastens a hollow dialysis fiber to its tip and draws the fiber into the brain by retracting the telescoping device in an orderly sequence. The surgery affects brain tissue only along the course of the fiber, causing about the same acute damage as the stereotactic introduction of an ordinary straight needle. The 'extended local access fiber' is relatively soft, flexible, and biocompatible. It remains permanently in the brain, with its semipermeable portion lodged in the target region. Leading and trailing portions are accessible from outside the brain. Experiments indicate a broad range of possible trajectories and confirm that substances delivered by access fiber can have physiological effects even 10 weeks after implantation.

Autofluorescence of aldehyde-fixed neural tissue often obscures perikaria and fine processes labeled with fluorescent anterograde or retrograde tracers. In particular, this autofluorescence hinders the detectability of fine axonal projections labeled with the convenient anterograde tracer, tetramethylrhodamine dextranamine. Background fluorescence was notably reduced by immersion of free-floating brain tissue sections in a solution of sodium borohydride (NaBH4, 0.1%), a chemical which is known to neutralize Schiffs bases through reduction of amine-aldehyde compounds into non-fluorescent salts. The reversible and renewable immersion technique was most effective in paraformaldehyde-fixed tissue where the preservation quality was improved such that labeled axons remained detectable for more than 1 year after initial preparation.

This paper describes a technique for microwave-accelerated fixation, washing and pre-embedding immunohistochemistry of insect brains. The temperature of the specimens is controlled with a water-cooling device. This device also serves as a permanent water load for microwave energy absorption and as a stirrer to avoid uneven energy distribution. The set-up described here allows continuous irradiation of specimens at full power, for unlimited time, without overheating. A considerable reduction in processing time could thus be achieved, along with improved immunolabelling.

Immunoassays for quantitative determinations of the S-100 protein, the glial fibrillary acidic protein, the neuron specific enolase and the neurofilament proteins with molecular weight of 68 and 200 kDa in hot SDS sonicated rat brain extracts have been developed and characterized. The assays utilize a dot immunobinding technique, poly- or monoclonal antibodies and 125 I-protein A. The SDS-sonication procedure was not found to affect the radioactivity recovery in the assay of the soluble S-100 protein or the neuron specific enolase. All 5 antigens can be measured with a within-assay variance below 10%. Even at a coefficient of variation less than or equal to 5%, the working ranges are approximately 30–100-fold with regard to the different antigens. It was found that gelatin-coated nitrocellulose membranes considerably increase the recovered radioactivity in the assay of the purified bovine S-100 protein, possibly by protein-protein interaction. This effect was not observed when SDS-sonicated rat brain extracts were assayed. The assay appears to be reproducible, convenient and rapid, and provides a high degree of precision in the determination of large number of samples.

This report describes in detail the assembly and use of miniature cannulas for intracerebral injections in freely moving rats. Guide and injection cannulas were prepared from polyimid-coated fused silica capillaries as routinely in use for gas chromatography, in this way avoiding the permanent contact of stainless-steel with brain tissue. The outer diameter of the guide capillary measured 300 microns, that of the injection capillary 170 microns. In spite of their fragile appearance, the capillaries were robust and easy to handle. In the same animal, up to 20 consecutive injections were possible. In the course of 2 years 204 cannulas directed to the amygdala or the lateral ventricle were implanted, and only in 8 rats (4%) the inner capillary broke and plugged the guide cannula, making injections impossible. Histological examination of brains implanted for up to 2 months demonstrated only blunt scar formation along the cannula's track. No changes in transmitter levels (biogenic amines, amino acids) could be detected in chronically implanted rat brains except for a slight increase in serotonergic activity in the contralateral piriform cortex. Intra-amygdaloid injection of 0.32 microliters 0.9% saline resulted in a mild and transient reduction of GABA levels in both piriform cortices, without any detectable persistent histopathological consequences.

In the present report we describe the construction of a systemic vascular perfusion device designed to perfuse small mammals with fixatives under a constant, controlled hydrostatic pressure. The device is powered by compressed-air. The construction of the device allows the interchange of vessels containing various perfusates during the perfusion, without having to interrupt the flow or alter the hydrostatic pressure. Large volumes of fixatives can be perfused in a relatively short time, leading to good preservation of brain tissue, both for light and electron microscopical purposes.

A method is described which allows intracranial injection of drug solutions through glass micropipettes (tip diameter 15 micron) in the freely moving rat by use of air pressure. Compared with conventional injection methods through steel cannulae this method has the advantages of (1) minimal destruction of brain tissue, (2) precise injection of small volumes of solutions (1 nl to several microliters), (3) simultaneous recording of local EEG activity, and (4) usage of multipipette assemblies.

A method is described which allows the Golgi-impregnation of neurons within a ‘single’ section or ‘slice’ of brain tissue 60–200 μm thick. The method may be combined with a variety of histochemical procedures allowing the identification of specific neural elements in the light-microscope and the subsequent examination of their synaptic relationships in the electron-microscope. The technique makes it possible to control visually the extent of the Golgi-impregnation and allows the re-impregnation of single sections. The procedure is illustrated with examples from the cerebellum, cortex, hippocampus, and thalamus of the rat.

A method is described for reversibly inactivating small, precisely localized regions of brain tissue with injections of nanoliter quantities of the local anesthetic lidocaine hydrochloride. The injections are made through a combined recording-injection probe consisting of a glass micropipette onto whose outer surface is plated a metallic cylinder for recording extracellular action potentials. The recording cylinder for recording extracellular action potentials. The recording cylinder, located a known distance from the pipette tip, picks up a continuous, large-amplitude, multiunit response which can be used to accurately position the tip according to physiological criteria. It also provides a means of determining the duration of the anesthetic block and estimating its spreat. This devise has been used to selectively block 200--400 micrometers regions of individual laminae of the lateral geniculate nucleus centered within 50 micrometers of a retinotopically defined target site. The blocks last from 3 to 10 min and can be repeated many times at the same location.