Journal of Neuroscience Methods

We describe a method for assessing the periodic elements of variations in a time series and illustrate it with examples drawn from infant cardiac beat-to-beat intervals. Compared with averaging techniques, the procedure has the advantage of providing quantification of periodical elements in a non-stationary time series. Moreover, the procedure is robust to artifacts such as those which frequently contaminate beat-to-beat interval data. The method examines successive increments of the time-series plot, and when they become negative or positive, peaks and troughs are noted in the curve. Two successive troughs confine a wave which may be described by its amplitude and period. The set of all waves, terminated by the sequence of troughs, is defined as the "high-frequency component" of the series. Waves of the next low-frequency component are delineated when only the high-frequency peaks (or troughs) are considered. Thus, low-frequency peaks are defined as peaks of the curve formed by the high-frequency peaks, and lower-frequency troughs are the troughs of the curve formed by the high-frequency troughs. The process iterates to assess variations at lower and lower frequencies and any specific frequency component is being characterized by the median and interquartile range of its wave amplitudes and wave periods.

Both 0.2 T and 9.4 T MRI systems were used to image a mouse model of glioma. RF coils were designed for both fields. A spin-echo, multi-echo pulse sequence was used to determine T(2) relaxation times of both brain and tumor tissues. Contrast-to-noise ratio was calculated based on the selected echo time. The results showed that 0.2 T is suitable for mouse model imaging, however total scan time must be long to achieve high enough SNR. T(2) relaxation times of the tumor and brain tissues can be measured at 0.2 T and are 2.1 and 1.8 times respectively longer at 0.2 T than at 9.4 T. Contrast to noise ratio for tumor and brain was better at high field than at the low field. We concluded that 0.2 T may be used to study mouse model of glioma using spin echo pulse sequence, yet the total scan time is long (about 40 min), resolution is lower (∼250 μm × 250 μm) and slice thickness (3mm) must be large enough to obtain sufficient SNR.

Recently we presented a method to label the neuropeptide substance P with a 1.4-nm gold particle covalently bound at the N-terminus that can be used for demonstrating its binding sites in histological sections. In this study we examined whether the peptides neuropeptide Y, somatostatin, calcitonin gene-related peptide and bradykinin can be labelled in the same way. Polyacrylamide gel electrophoresis revealed a reduction in mobility for peptide-gold conjugates over gold particles alone consistent with peptide binding. In cryostat sections of the rat lumbar spinal cord, the peptides showed a distinct binding pattern in the grey matter corresponding to data of studies using autoradiographic methods. Therefore, we conclude that this simple and fast method can be used for labelling peptides in general to demonstrate their binding sites in histological sections, provided the peptide binds by its C-terminus.

Alpha 7 subunit-containing nicotinic acetylcholine receptors (alpha7* nAChR) are involved in a variety of functions in the mammalian brain, including modulating neurotransmitter release and synaptic plasticity. Identifying the precise cellular distribution of alpha7* nAChRs with respect to the local neurochemical environment is crucial to understanding these biological roles. Current strategies for localising alpha7* nAChRs at the subcellular level have limitations. Anti-alpha7 subunit antibodies detect both assembled and unassembled subunits whereas biotinylated alphabungarotoxin (alphaBgt) only binds to assembled alpha7* nAChRs, but interpretation of labelling is marred by co-detection of endogenous tissue biotin. To overcome these problems, we have characterised a novel 1.4 nm gold alphaBgt conjugate used to directly localise alpha7* nAChR. Gold conjugation does not significantly decrease binding affinity, and gold alphaBgt specifically labels alpha7* nAChR in both unfixed and aldehyde-fixed tissue at the light and electron microscope levels, labelling being abolished in the presence of excess competing toxin. At the ultrastructural level, gold alphaBgt is associated with neuronal membranes and located at axon-dendritic synapses in the rat hippocampus CA1 stratum radiatum. These results reveal gold alphaBgt to be a valuable new tool in elucidating the functional neuroanatomy of alpha7* nAChR in the central nervous system.

The purpose of the present animal experiment was to determine whether source images from dynamic susceptibility contrast-enhanced perfusion weighted imaging (DSC-PWI) at a 1.5T MR scanner, performed early after photochemically induced thrombosis (PIT) of cerebral middle artery (MCA), is feasible to predict final cerebral infarct size in a rat stroke model. Fifteen rats were subjected to PIT of proximal MCA. T2 weighted imaging (T2WI), diffusion-weighted imaging (DWI), and contrast-enhanced PWI were obtained at 1 h and 24 h after MCA occlusion. The relative lesion size (RLS) was defined as lesion volume/brain volume x 100% and measured for MR images, and compared with the final RLS on the gold standard triphenyl tetrazolium chloride (TTC) staining at 24 h. One hour after MCA occlusion, the RLS with DSC-PWI was 24.9 +/- 6.3%, which was significantly larger than 17.6 +/- 4.8% with DWI (P < 0.01). At 24 h, the final RLS on TTC was 24.3 +/- 4.8%, which was comparable to 25.1 +/- 3.5%, 24.6 +/- 3.6% and 27.9 +/- 6.8% with T2WI, DWI and DSC-PWI respectively (P > 0.05). The fact that at 1 h after MCA occlusion only the displayed perfusion deficit was similar to the final infarct size on TTC (P > 0.05) suggests that early source images from DSC-PWI at 1.5T MR scanner is feasible to noninvasively predict the final infarct size in rat models of stroke.

Magnetic resonance imaging (MRI) offers a noninvasive technique for studying neurodegenerative events in the rat brain, however, most of the studies are performed on small bore purpose dedicated MR scanners of limited availability and at high cost. The present study explored the feasibility of using a clinical whole body MR-scanner to perform imaging in rat brain and specifically in models of Parkinson's (PD) and Huntington's disease (HD). For that purpose rats were placed into a specially designed PVC device equipped with a flexible surface coil-and T2-weighted spin echo sequences were acquired on a Siemens Magnetom Vision at 1.5 T. In the experimental protocols of PD and HD, animals underwent 6-hydroxydopamine (6-OHDA) and quinolinic acid (QA) injections, respectively and were subsequently grafted with fetal tissue. T2-weighted images showed a small hyperintense area at the 6-OHDA lesion site and a diffuse hyperintensity in the striata with QA lesions. Transplants were seen as a hypointense area surrounded by a hyperintense rim on T1-weighted images. Moreover, disturbances of the blood-brain-barrier and its time of restoration could be monitored. In conclusion, high-resolution in vivo imaging of small animals is feasible with clinical MR-scanners and hence allows the study of various experimental protocols.

Confounding low-frequency fluctuation (LFF) physiological noise is a concern for functional connectivity analyses in blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI). Using estimates of LFF physiological noise derived from measured cardiac and respiration signals, noise can be filtered from the time series thus improving the results of functional connectivity analysis. The ability of spatial independent component analysis (ICA) to separate LFF physiological noise from the default mode network (DMN), which overlap each other spatially and occur at similar frequencies, has remained an open question. We aimed to define the net effect of physiological correction for spatial ICA DMN detection at 1.5 T by statistically testing obtained ICASSO centrotype DMN maps before and after physiological correction. Comparisons with 21 subjects were performed for ICA model orders 20, 30 and 40 and no statistically significant spatial difference was found after physiological correction, although slight DMN reduction in precuneus or sagittal sinus was detected in all dimensionalities. A confounding factor in the analysis is the susceptibility of the ICA decomposition for data changes yielding different DMN splitting between and after physiological correction conditions without comparable true change in the data. This issue is mitigated at higher ICA model orders. The results suggest that subject-level DMN can for some subjects be optimized by physiological correction, but on the group-level this contribution is minor.

The field of spinal cord injury research is an active one. The pathophysiology of SCI is not yet entirely revealed. As such, animal models are required for the exploration of new therapies and treatments. We present a novel technique using available hospital MRI machines to examine SCI in a mouse SCI model. The model is a 60 kdyne direct contusion injury in a mouse thoracic spine. No new electronic equipment is required. A 1.5T MRI machine with a human wrist coil is employed. A standard multisection 2D fast spin-echo (FSE) T2-weighted sequence is used for imaging the mouse. The contrast-to-noise ratio (CNR) between the injured and normal area of the spinal cord showed a three-fold increase in the contrast between these two regions. The MRI findings could be correlated with kinematic outcome scores of ambulation, such as BBB or BMS. The ability to follow a SCI in the same animal over time should improve the quality of data while reducing the quantity of animals required in SCI research. It is the aim of the authors to share this non-invasive technique and to make it available to the scientific research community.

Magnetic resonance (MR) imaging in animal models is usually performed in expensive dedicated small bore animal scanners of limited availability. In the present study a standard clinical 1.5 T MR scanner was used for morphometric and dynamic contrast-enhanced susceptibility-weighted MR imaging (DSC-MRI) of a glioma model of the rat brain. Ten male Wistar rats were examined with coronal T2-weighted, and T1-weighted images (matrix 128 x 128, FOV 64 mm) after implantation of an intracerebral tumor xenografts (C6) using a conventional surface coil. For DSC-MRI a T2*-weighted sequence (TR/TE=30/14 ms, matrix 64 x 64, FOV 90 mm; slice thickness of 1.5mm) was performed. Regions of interest were defined within the tumor and the non-affected contralateral hemisphere and the mean transit time (MTT) was determined. Tumor dimensions in MR predicted well its real size as proven by histology. The MTT of contrast agent passing through the brain was significantly decelerated in the tumor compared to the unaffected hemisphere (p<0.001, paired t-test), which is most likely due to the leakage of contrast agent through the disrupted blood brain barrier. This setup offers advanced MR imaging of small animals without the need for dedicated animal scanners or dedicated custom-made coils.

The reliable identification of astrocytes for physiological measurements was always time-consuming and difficult. Recently, the fluorescent dye sulforhodamine 101 (SR101) was reported to label cortical glial cells in vivo [Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 2004;1:31-7]. We adapted this technique for use in acute rat hippocampal slices at early postnatal stages (P3, 7, 15) and in young adults (P24-27) and describe a procedure for double-labeling of SR101 and ion-selective dyes. Using whole-cell patch-clamp, imaging, and immunohistochemistry, we characterized the properties of SR101-positive versus SR101-negative cells in the stratum radiatum. Our data show that SR101, in contrast to Fura-2 or SBFI, only stains a subset of glial cells. Throughout development, SR101-positive and SR101-negative cells differ in their basic membrane properties. Furthermore, SR101-positive cells undergo a developmental switch from variably rectifying to passive between P3 and P15 and lack voltage-gated Na+ currents. At P15, the majority of SR101-positive cells is positive for GFAP. Thus, our data demonstrate that SR101 selectively labels a subpopulation of glial cells in early juvenile hippocampi that shows the typical developmental changes and characteristics of classical astrocytes. Owing to its reliability and uncomplicated handling, we expect that this technique will be helpful in future investigations studying astrocytes in the developing brain.

We have constructed a new 1020-site optical system for simultaneous recording of transmembrane electrical activity, using a 34 x 34-element photodiode array. This new apparatus permits analyses of the spatio-temporal pattern of neural activity, such as action potentials and postsynaptic potentials, in the central nervous system, at higher spatial and temporal resolutions.

A histochemical method for staining CNS zinc by the stoichiometric formation of zinc: quinoline fluorescent chelates is described. Four congeners of quinoline have been tested, and two found to be useful for histochemistry. The procedure is a one-step method, suitable for fresh-frozen and fixed tissue sections alike. The quinoline fluorescence selectively labels the CNS regions (such as hippocampus, amygdala) shown by prior histochemical procedures to be rich in histochemically reactive zinc in axon boutons and therefore appears to be a specific marker for the bouton zinc. Microfluorometric data indicate that the fluorochrome can be used for quantitative estimates of CNS zinc pools as well as qualitative studies of localization.

Animal models of stroke, notably transient middle cerebral artery occlusion (MCAo), are used to assess the efficacy of pharmacological and transplant treatments. Long-term studies (>1 month) of the functional effects of treatments in animal models are required to predict treatments likely to improve dysfunctions associated with stroke damage. These pre-clinical studies require (1) optimum post-operative care to ensure long-term survival, (2) methods for assignment of rats to groups with equivalent impairments to reduce variability and enhance detection of treatment effects, and (3) behavioural tests that detect long-term stable deficits. For long-term functional assessment, a battery of behavioural tests sensitive to a range of deficits observed after MCAo was developed. The bilateral asymmetry test evaluated the time course of sensory neglect. Deficits of motor integration were examined in the footfault test, and motor bias was assessed by pharmacological stimulation of rotation. The water maze was used to detect long-term deficits in spatial information processing. Long-term differences between control and MCAo animals in this battery of tests indicate that the protocol provides an efficient assessment suitable for evaluating treatment outcomes in pre-clinical studies of stroke, and that the post-operative care procedure and method of assignment to groups were effective.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Adult animals continue to produce new neurons in the dentate gyrus of hippocampus. Until now, the principal method of studying neurogenesis has been to inject either tritiated thymidine or 5'-Bromo-2-deoxyuridine (BrdU) intraperitoneally followed by autoradiographic or immunohistochemical detection methods respectively. However, such exogenous markers may produce toxic effects. Our objective was to determine whether Ki-67, a nuclear protein expressed in all phases of the cell cycle except the resting phase, can be used as an alternative, endogenous marker. Using immunohistochemistry, we examined Ki-67 and BrdU expression pattern in rats. Ki-67 was expressed within the proliferative zone of the dentate gyrus and its expression pattern mimicked that of BrdU when examined soon after exogenous BrdU administration. Quantitative comparison of BrdU and Ki-67-positive cells showed 50% higher numbers of the latter when examined 24 h after the BrdU injection. This was expected, since BrdU can be incorporated into DNA only during the S-phase of the mitotic process, whereas Ki-67 is expressed for its whole duration. Experimental increases (by ischemia) or reductions (by radiation) in the number of mitotic cells produced parallel changes in BrdU and Ki-67 signals. Thus, Ki-67 is an effective mitotic marker and has most of the benefits of BrdU and none of the costs. This study provides evidence for Ki-67 to be used as a marker of proliferation in the initial phase of adult neurogenesis.

Microdialysis measurements of extracellular substances under hyperbaric conditions were manifold used in several investigations. However, to our knowledge there is no analysis, which verified the applicability of microdialysis pumps under hyperbaric conditions. Thus, a goal of this study was to investigate the reliability of the microdialysis pump (MDP) CMA 107 in a hyperbaric environment up to 2.4bar absolute pressure. The CMA 107 with a perfusion rate of 2microL/min was stored in a decompression chamber. The ambient pressure was increased from 1 to 2.4bar absolute within 15min, maintained for 90min and then decreased to 1bar within 15min. The vials were changed every 15min, weighed before as well as after collecting the sample volume and the absolute recovery of glutamate, pyruvate, lactate, glucose and glycerol was determined. The same setup was performed under normobaric conditions. The pumping capacity was 1.7% greater than expected under normobaric conditions, 36.5% less than expected in the compression phase, 10.5% less than expected in the isopression phase and 26.3% greater than expected in the decompression phase under hyperbaric conditions. The absolute recoveries under hyperbaric conditions were affected during the isopression phase with a deviation from -6 to +20% compared to normobaric environments. The study demonstrated that an absolute ambient pressure up to 2.4bar did influence the pumping capacity and the reliability of the absolute recovery. These results need to be taken into consideration when interpreting microdialysis studies performed under hyperbaric conditions.

Most of functional magnetic resonance imaging (fMRI) experiments have been performed on horizontal bore magnets. Here, we present practical aspects of fMRI based on single-shot, spin-echo echo-planar imaging (EPI) using a widely available, cost effective 89 mm bore vertical 11.7 T microimager. It was demonstrated that reproducible, high-quality fMRI data can be obtained from alpha-chloralose anesthetized adult rat brain. Both coronal and the more extended horizontal EPI images were acquired to measure blood oxygenation level dependent (BOLD) responses to electrical stimulation of fore- and hindpaws. The BOLD patterns observed match the known representations of fore- and hindpaws in the somatosensory cortex in rats. Preliminary results on BOLD signal enhancement using aminophylline are also presented.

Knockout mouse models allow preparation of primary neuronal cultures from distinct brain regions in order to investigate the underlying neuronal pathomechanisms of human metabolic diseases associated with severe, regionally distinct brain pathologies (e.g. Zellweger syndrome, the most severe form of a peroxisomal biogenesis disorder). However, homozygous mouse pups with Zellweger syndrome usually die shortly after birth. Therefore, in this study, we established optimized protocols for the simultaneous preparation and cultivation of serum-free primary neuronal cultures from distinct brain regions (medial neocortex, hippocampus and cerebellum) from individual newborn (P0.5) C57Bl/6J mice. For each of the three types of neuronal cultures, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, mode of trituration, seeding density and composition of the culture medium. As indicated by indirect immunofluorescence using antibodies against NeuN, GFAP and CNPase, the purity of the distinct neuronal cultures was high. The percentage of oligodendrocytes was less than 1% in all neuronal cultures. Only 5% astrocytes were present in cortical, 7% in hippocampal and 10% in cerebellar cultures. Cytosine arabinofuranoside (AraC) treatment reduced the percentage of astrocytes only significantly in hippocampal cultures, however, increased the percentage of apoptotic neurons in hippocampal and cortical cultures.

Quantifying mechanisms underlying extracellular signaling by the neurotransmitter dopamine (DA) is a difficult task, particularly in the complex extracellular microenvironment of the intact brain. In this study, two methods for evaluating release and uptake from DA dynamics monitored by real-time voltammetry are described. Both are based on a neurochemical model characterizing electrically evoked levels of DA as a balance between these opposing mechanisms. The theoretical basis of what is called here nonlinear regression and single curve analyses is given. Fitting simulated data tests the reliability of the methods. The two analyses are also compared with an experimental data set describing the effects of pharmacologically inhibiting the DA transporter in the caudate-putamen (CP) and nucleus accumbens (NAc). The results indicate that nonlinear regression and single curve analyses are suitable for quantifying release and uptake mechanisms underlying DA neurotransmission. Additionally, the most important experimental finding of this technical study was the independent confirmation of high affinity (approximately 0.2 microM) DA uptake in the intact striatum.

Optical imaging over extended periods of time in non-human primates presents serious challenges because the dura mater must be removed to expose the cortical surface. We present a novel nylon imaging chamber with a transparent artificial dura implant, which allows repeated, long-term optical recordings from the cortex. The cylinder of the chamber is inserted into a cranial trephination and held in place with a minimum of screws and acrylic cement. A round patch of artificial dura with a perpendicular wall protects the cortical surface and slows re-growth of dural tissue within the chamber. A cap, manufactured from the same material as the cylinder, is screwed into the chamber and seals it completely. Over a period of 1-4 months, the chamber required a minimum of maintenance and stayed infection-free without local antibiotic application. We repeatedly performed optical imaging in the same animal with the advantages of shortened preparation time. To permit precise alignment and comparison of maps obtained from different imaging sessions, we developed a program that calculated a 2-dimensional spatial transformation between maps of different magnifications, translations, and distortions. We suggest that these methods provide a practical solution to long-term optical imaging in the anesthetized or alert monkey. The exclusive use of non-metallic materials offers the benefit of a lighter and more compact implant, and the possibility to perform MRI scans after chamber implantation.

The usefulness of three retrograde fluorescent dyes for tracing injured peripheral axons was investigated. The rat sciatic was transected bilaterally and the proximal end briefly exposed to either Fast Blue (FB), Fluoro-Gold (FG) or to Diamidino Yellow (DY) on the right side, and to saline on the left side, respectively. The nerves were then resutured and allowed to regenerate. Electrophysiological tests 3 months later showed similar latencies and amplitudes of evoked muscle and nerve action potentials between tracer groups. The nerves were then cut distal to the original injury and exposed to a second (different) dye. Five days later, retrogradely labelled neurones were counted in the dorsal root ganglia (DRGs) and spinal cord ventral horn. The number of neurones labelled by the first tracer was similar for all three dyes in the DRG and ventral horn except for FG, which labelled fewer motoneurones. When used as second tracer, DY labelled fewer neurones than FG and FB in some experimental situations. The total number of neurones labelled by the first and/or second tracer was reduced by about 30% compared with controls. The contributions of cell death as well as different optional tracer combinations for studies of nerve regeneration are discussed.

There is increasing interest in examining neuron-glial interactions in the developing and the mature central nervous system. Here, we describe a system that allows examination of interactions mediated by diffusible molecules even when such interactions involve more than 2 cell populations. Our procedure involves culturing highly enriched populations of neurons and glial cells on separate coverslips. Such coverslips can be readily moved from one petri dish to another, thus enabling the investigator to subject the neurons and the glial cells to different experimental manipulations. The coverslips can then be placed in any desired combination within a petri dish. This gives one great flexibility in examining neuron-glial interactions. Using this approach, we unequivocally demonstrate that astrocytes protect neurons from glutamate excitotoxicity.

An interface has been constructed for the Nicolet 1170 signal averager to allow computer control of most of the instrument's functions, because the standard computer interface available from the manufacturer does not permit control over a number of front panel switches necessary for automatic data transfer. Computer control can be obtained with the addition of one printed circuit card, containing 3 integrated circuits, that plugs into an empty motherboard socket in the Nicolet mainframe, and a few simple backplane wiring changes. The digital I/O interface supplied by the manufacturer is still required, but this interface does not allow easy access to the desired signals on the control connector. Therefore, construction of a new interface in a separate enclosure is more desirable. The computer requires two digital I/O ports for data transfer and control. A number of Pascal routines have been written for computer control of the Nicolet.

We have developed an automated, high-throughput behavioral screening method for detecting hearing defects in zebrafish. Our assay monitors a rapid escape reflex in response to a loud sound. With this approach, 36 adult zebrafish, restrained in visually isolated compartments, can be simultaneously assessed for responsiveness to near-field 400 Hz sinusoidal tone bursts. Automated, objective determinations of responses are achieved with a computer program that obtains images at precise times relative to the acoustic stimulus. Images taken with a CCD video camera before and after stimulus presentation are subtracted to reveal a response to the sound. Up to 108 fish can be screened per hour. Over 6500 fish were tested to validate the reliability of the assay. We found that 1% of these animals displayed hearing deficits. The phenotypes of non-responders were further assessed with radiological analysis for defects in the gross morphology of the auditory system. Nearly all of those showed abnormalities in conductive elements of the auditory system: the swim bladder or Weberian ossicles.

This paper describes improvements and further characterization of a ceramic-based multisite microelectrode for in vivo measurements of L-glutamate. Improvements include increased recording area, insulation deposition using photolithography for more uniform recording sites and forming the microelectrodes using a diamond saw providing smoother microelectrode edges. The new microelectrodes are triangular in shape, 1 cm in length and taper from 1 mm to a 2-5 microm tip. Details on performing in vivo measurements are given, including microelectrode preparation, pitfalls of the recording method and approaches to enhance reproducibility of the technique. The detection limit for L-glutamate was lowered to approximately 0.5 microM and a self-referencing recording technique was utilized to remove interferents as well as decrease noise. Applications of the microelectrodes to study L-glutamate uptake and release in rat prefrontal cortex, cortex, cerebellum and striatum are included.

Intracerebral microdialysis is used extensively as a research tool in the investigation of the neurochemical and metabolic changes that occur following acute brain injury. Microdialysis has enabled elucidation of intra-cerebral levels of substances such as lactate, pyruvate and glycerol but, as yet, has not been used effectively to recover macromolecules from the human brain. Traumatic brain injury is known to result in the generation of cytokines and neurotrophins into extracellular fluid compartment of the brain, with effects on neuronal damage and repair. We have developed a technique of in vivo sampling of the interstitial fluid of the brain of patients with severe head injuries which has allowed the measurement of IL-1beta, IL-6 and nerve growth factor. This report confirms the safety and effectiveness of this modified microdialysis method in the clinical setting of a neurological intensive care unit. The technique provides a timely addition to the armamentarium of the clinical scientist and will potentially lead to a greater understanding of neuroinflammation following acute traumatic brain injury.

In vitro autoradiography of a 11C-labelled ligand, Ro 15-1788, was used in saturation experiments in order to quantify benzodiazepine receptor binding in whole human brain hemisphere cryo-sections. A special incubation chamber was developed with the aim of performing standardized quantitative studies with short-lived positron emitting isotopes. 11C-labelled ligand binding was studied in temporal cortex, cerebellum, white manner and pons incubated in vitro. White manner, lacking benzodiazepine receptor binding, was used as an estimated of nonspecific binding, for calculation of Saturability of binding was demonstrated in the neocortical and cerebellar regions. Radioactivity counting of incubated adjacent tissue sections was done as a control. Computerized densitometry of the autoradiograms gave similar results as the tissue counting. A comparison with in vivo saturation experiments using the same 11C-labelled ligand and positron emission tomography in healthy human subjects also gave binding characteristics of the same magnitude. The 11C-autoradiograms showed a good spatial resolution (about 180 microns). 11C-autoradiography with suitable radioligands should be a valuable technique of screening the distribution and characteristics of neuroreceptors in the human brain. This quantitative method will provide the PET investigator with preliminary binding data, and may thus supply valuable information concerning positioning in PET and concerning significant regions of interest.

The present work tests the feasibility of using the most recently developed positron emission tomograph detector technology to image positron-emitting radioligands in small experimental animals. A prototype imaging device, using two opposing multicrystal, high-resolution (approximately 4 mm) block detectors of bismuth germanate to produce a 2-dimensional image in the centre of the field of view, is described. To evaluate the probe's potential as a non-invasive experimental tool, the dynamic regional distribution of the established opiate receptor ligand, [11C]diprenorphine was determined in rat brain following intravenous injection. The distribution of counts in the images was consistent with the localisation of diprenorphine binding sites and the specificity of the signal obtained was confirmed by administration of non-radioactive diprenorphine and naloxone. Although the signal-to-noise ratio was reduced compared with data obtained by post mortem dissection, the dynamic data acquisition capabilities of the system demonstrate the feasibility of monitoring the kinetics of ligand binding in individual animals and encourages further design of a small-diameter detector system with tomographic capabilities.

The quantitation of regional cerebral in vivo opioid receptor rate constants using [11C]diprenorphine and positron emission tomography (PET) using 3 types of protocol (presaturation, pulse-chase naloxone displacement and tracer-only protocols) together with measurements of regional cerebral blood flow is described in normal volunteers. Arterial blood was sampled continuously for radioactivity and was corrected for metabolites and plasma/blood partition of radioactivity to provide a continuous plasma input function. A compartmental model involving 3 tissue compartments was used to describe the regional cerebral pharmacokinetics of the tracer. The compartments comprised: (1) free plus rapidly exchanging non-specifically bound ligand, (2) specifically bound, naloxone displaceable ligand, and (3) a kinetically distinguishable non-specifically bound pool. Regional estimates of fractional rate constants relating to specific binding were obtained using naloxone in a pulse-chase design of tracer displacement. Less precise estimates of these rate constraints were obtained from single-tracer-only studies, but when binding was expressed as the tissue total volume of distribution relative to plasma there was good correlation with regional values obtained from pulse-chase studies performed in the same individuals. The application of these protocols to the measurement of indices of regional-specific opioid receptor binding in the human brain is discussed.

The results are described of the cerebral uptake and heterogeneous retention of [11C]diprenorphine after intravenous injection in 4 normal volunteers. This potent opioid antagonist (Kd = 0.2 nM) was chosen because of its safety, lack of side-effects at trace doses in human pilot studies, rapid cerebral uptake and high percentage (80-90%) specific binding in animal in vivo studies. High uptake of [11C]diprenorphine was demonstrated in regions such as the thalamus, caudate nucleus, temporal, frontal and parietal cortices, which are known from postmortem studies to have high concentrations of opioid receptors. A stable level of activity or a very slow decline in activity was observed between 20 and 50 min after injection in areas such as the caudate nucleus and thalamus. Conversely, rapid washout of activity was observed in the occipital cortex, which is known to have low opioid receptor concentrations. Some 80-90% of maximum binding was naloxone reversible. These results with a ligand that is safe and without side-effects, suggest that this technique is suitable for studies of opioid physiology in man.

Middle cerebral artery occlusion (MCAO) is the most commonly used method to study the neurological and histological outcomes and the pathological mechanisms of ischaemic stroke. The current work compares sensorimotor and cognitive deficits and the infarct volume in rats following a transient 90- or 120-min MCAO, which allows the appropriate behavioural tests to be chosen based on the goal and design of the experiment. In the beam-walking test, we found significant differences between the 90- and 120-min MCAO groups in the number of foot faults made with the impaired hindlimb on post-stroke days 3, 7 and 14. In the cylinder test, a difference between the 90- and 120-min groups was observed on post-operation day 14. The responses to tactile and proprioceptive stimulation were impaired to a similar extent after 90- and 120-min MCAO in the vibrissae-evoked forelimb-placing and limb-placing tests. Moreover, we found significant memory impairment in the 120-min MCAO group 6 days after the acquisition trial. The brain tissue damage was significantly higher after 120-min occlusion of the MCA compared with 90-min occlusion; the infarct volumes were 13% and 25% of the contralateral hemispheres, respectively. In conclusion, both the 90- and 120-min occlusion models result in a significant impairment of sensorimotor, tactile and proprioceptive function, but memory impairment is only observed in the 120-min MCAO group. The beam-walking and cylinder tests detected neurological dysfunction after the 120-min MCAO, whereas the limb-placing and vibrissae-evoked forelimb-placing tests were able to evaluate the neurological dysfunction in rats after 90- and 120-min MCAO.

Binding of the cocaine analog 3 beta-(4-[125I]iodophenyl)tropane-2 beta-carboxylic acid isopropyl ester ([125I]RTI-121) to filters was studied in order to assess its contribution to labeling dopamine transporters on rat striatal synaptosomal membranes in filtration assays. Filter binding (FB) decreased with increasing Na+. Cocaine (30 and 100 microM) substantially reduced the FB at low Na+ with much less of an effect at higher Na+. Similar results were observed with K+. At 10 mM Na+, RTI-121 (1 microM) displaced the FB to the same degree as cocaine (100 microM); mazindol (10 microM), BTCP (1 microM), and dopamine (1 mM) did so to a lesser degree; and GBR12935 (1 microM) did not. If the specific binding was calculated without deducting the FB displaced with cocaine (DFB), the DFB accounted for 15-19% of the 'specific binding' at 10 mM Na+ in the assay. This additional binding population resulted in an upward curvilinear Scatchard plot and incorrect estimation of equilibrium binding parameters and ion potencies. At 10 mM Na+, without deduction of DFB, the high-affinity component had a Kd of 3.4 nM and Bmax of 2.4 pmol/mg protein, and the respective values for the low-affinity component were 84 nM and 16 pmol/mg protein; when DFB was deducted, one component was observed with a Kd of 4.4 nM and Bmax of 3.3 pmol/mg protein. The presence of higher Na+ in the assay diminished these artifacts. Thus, at 150 mM Na+, without deduction of DFB, there was one binding component with a Kd of 3.9 nM and Bmax of 4.6 pmol/mg protein; these values became 3.3 nM and 3.8 pmol/mg protein when DFB was deducted.

Cerebral glucose metabolism has been used as a marker of cerebral maturation and neuroplasticity. In studies addressing these issues in young non-human primates, investigators have used positron emission tomography (PET) and [18F]2-fluoro-2-deoxy-D-glucose (FDG) to calculate local cerebral metabolic rates of glucose (1CMRG1c). Unfortunately, these values were influenced by anesthesia. In order to avoid this confounding factor, we have established a method that permits reliable measurements in young conscious vervet monkeys using FDG-PET. Immature animals remained in a conscious, resting state during the initial 42 min of FDG uptake as they were allowed to cling to their anesthetized mothers. After FDG uptake, animals were anesthetized and placed in the PET scanner with data acquisition beginning at 60 min post-FDG injection. FDG image sets consisted of 30 planes separated by 1.69 mm, parameters sufficient to image the entire monkey brain. Our method of region-of-interest (ROI) analysis was assessed within and between raters and demonstrated high reliability (P < 0.001). To illustrate that our method was sensitive to developmental changes in cerebral glucose metabolism, quantitative studies of young conscious monkeys revealed that infant monkeys 6-8 months of age exhibited significantly higher 1CMRG1c values (P < 0.05) in all regions examined, except sensorimotor cortex and thalamus, compared to monkeys younger than 4 months of age. This method provided high resolution images and 1CMRG1c values that were reliable within age group. These results support the application of FDG-PET to investigate questions related to cerebral glucose metabolism in young conscious non-human primates.

The mitochondrial potential is an essential regulator in cellular physiology and detection of this parameter in living cells is still under discussion. Here we present a protocol which allows the use of rhodamine 123 as a probe for quantifying the mitochondrial potential. To avoid dequenching artefacts the detection area is limited to the area above the nucleus. In co-cultured rat hippocampal astrocytes and neurons, we analysed the mitochondrial accumulation of the cationic fluorescent dye rhodamine 123 (Rh123). Application of the uncoupler carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone (FCCP, 4 micromol/L) together with the ATP-synthase inhibitor oligomycin (Oli, 10 micromol/L) induced an immediate fluorescence increase of Rh123-loaded mitochondria. This effect is due to the well-known fluorescence dequenching caused by the reduction in concentration of Rh123 in the mitochondria after depolarization. However, above the nucleus an increase in fluorescence was registered. Due to the absence of mitochondria in the area above the nucleus this fluorescence increase is most likely caused by the Rh123 release from mitochondria. Pre-treatment of cells with antimycin A abolished the response to FCCP/Oli. Furthermore, a 10-min exposure to 50 mmol/L K+, which causes a plasma membrane depolarization in neurons, did not significantly change the FCCP/Oli-mediated Rh123 release measured above the nucleus of neurons. However, application of 100 micromol/L glutamate enhanced the effect of FCCP/Oli both in astrocytes and neurons. This enhancement is interpreted as an increase in mitochondrial potential above the control potential. Thus, this Rh123 method described here allows a cell type-specific determination of changes of mitochondrial polarization.

Considerable progress has been made in small animal single photon emission computed tomography (SPECT) imaging in the field of Parkinson's disease. In preclinical research, there is an increasing demand for in vivo imaging techniques to apply to animal models. Here, we report the first protocol for dopamine transporter (DAT) SPECT in common marmosets using the radioligand (123)I-N-ω-fluoropropyl-2β-carbomethoxy-3β-{4-iodophenyl}nortropane ((123)I-FP-CIT). Serial SPECT images were obtained on an upgraded clinical scanner to determine the distribution kinetics of (123)I-FP-CIT in the marmoset brain. After intravenous injection of approximately 60MBq of the radiotracer (123)I-FP-CIT, stable and specific striatal uptake was observed for at least 4h. Analysis of plasma samples showed rapid disappearance of the radiotracer from blood plasma within a few minutes after application, with activity declining to 4.1% of the administered activity. Structural magnetic resonance imaging (MRI) at 400μm resolution provided the details of the underlying anatomy. In a marmoset model of Parkinson's disease, which was generated by unilateral injections of 6-hydroxydopamine (6-OHDA) into the nigro-striatal projection pathway, complete loss of striatal DAT binding in combination with behavioral deficits was observed. The presented study demonstrates that (123)I-FP-CIT SPECT is a suitable tool to investigate DAT integrity in preclinical studies on common marmosets.

This protocol presents an improved method for SPECT imaging based on multi-pinhole techniques, applied to the visualisation of neurotracers in small animal models. Three types of collimators with 6-pinhole apertures adapted to special requirements for the imaging of the brain of mice and rats and to full body imaging in mice are employed in the experiments. A conventional triple-headed TRIAD/Trionix SPECT system was upgraded with pyramidal supports and shieldings onto the multi-pinhole collimators were installed. The system was employed for the assessment of the uptake of [123I]FP-CIT and [123I]IBZM, well known tracers of dopamine transport and dopamine D2/D3 receptors, respectively. Requirements regarding the applied radioactivity are reported, as well as further conditions determining the effectiveness of the detection of the uptake of [123I]FP-CIT and [123I]IBZM. The measurements in mice required only 20-25% of the activity described in previous studies. Dynamic measurements are presented, with a time resolution as high as 10 min in the brain of rats. Due to the lower signal intensity obtained for mice, the time resolution was 42min for [123I]FP-CIT, with a ratio ROI/background of 5.4, and 17 min for [123I]IBZM, with the ratio ROI/background of 4.5 (1.6-7.4).

A new apparatus has been developed that integrates an animal restrainer arrangement for small animals with an actively tunable/detunable dual radio-frequency (RF) coil system for in vivo anatomical and functional magnetic resonance imaging of small animals at 4.7 T. The radio-frequency coil features an eight-element microstrip line configuration that, in conjunction with a segmented outer copper shield, forms a transversal electromagnetic (TEM) resonator structure. Matching and active tuning/detuning is achieved through fixed/variable capacitors and a PIN diode for each resonator element. These components along with radio-frequency chokes (RFCs) and blocking capacitors are placed on two printed circuit boards (PCBs) whose copper coated ground planes form the front and back of the volume coil and are therefore an integral part of the resonator structure. The magnetic resonance signal response is received with a dome-shaped single-loop surface coil that can be height-adjustable with respect to the animal's head. The conscious animal is immobilized through a mechanical arrangement that consists of a Plexiglas body tube and a head restrainer. This restrainer has a cylindrical holder with a mouthpiece and position screws to receive and restrain the head of the animal. The apparatus is intended to perform anatomical and functional magnetic resonance imaging in conscious animals such as mice, rats, hamsters, and marmosets. Cranial images acquired from fully conscious rats in a 4.7 T Bruker 40 cm bore animal scanner underscore the feasibility of this approach and bode well to extend this system to the imaging of other animals.

Viral vectors have attracted great interest as vehicles for gene therapy. Due to concerns regarding continued viral gene expression in several systems, new approaches have been sought for gene transfer in the nervous system. This article reviews the general concepts and basic biology of defective viral vectors. These are vectors which can package into a viral coat but contain no viral genes, thereby allowing efficient gene transfer in the absence of viral gene expression in target cells. The defective herpes simplex virus (HSV) vector has been applied to numerous interesting questions in neurobiology. The inability to completely eliminate helper viruses has raised concern regarding the application of this vector to human disease. The adeno-associated virus (AAV) vector has recently been introduced into the nervous system. This vector harbors no viral genes, however helper viruses can also be completely eliminated from the system. Although the smaller size may limit the range of applications for this vector, it has received great interest as a potential agent for gene therapy in the nervous system. Potential future directions are discussed as well.

Radio-iodinated fasciculin 2 (Fas2), a polypeptide anticholinesterase toxin from Mamba venom, was used as a new probe for localizing and quantifying acetylcholinesterase (AChE) at mouse neuromuscular junctions (NMJs) by quantitative electron microscope autoradiography. We demonstrate that 125I-Fas2 binds very specifically to the NMJs of mouse sternomastoid muscles, with very little binding to other regions in the muscles. Junctional AChE-site densities obtained from the autoradiograms were similar to those previously obtained for the same muscles using 3H-DFP. The use of 125I-Fas2 with EM-autoradiography is simpler and provides higher resolution and sensitivity, as well as considerably lower non-specific binding than previously attainable with 3H-DFP. The advantages and limitations of this procedure are discussed.

We have developed a double labeling immunocytochemical method utilizing peroxidase conjugated Fab fragments and 125I-labeled protein A to localize two neuronal markers on the same light or electron microscopic section with primary antibodies raised in the same animal species. The technique is applicable to the study of chemical connectivity in the brain, as illustrated by data obtained in the hypothalamus using rabbit polyclonal antisera against tyrosine hydroxylase (TH), phenylethanolamine-N-methyltransferase (PNMT), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP). Moreover, due to a high level of sensitivity and resolution, the technique offers considerable advantages over many previously developed dual labeling immunocytochemical methods for the demonstration of transmitter axonal co-localizations. Utilizing the peroxidase Fab/[125I]protein A method, we present here the first direct evidence that PNMT is present in many endings also containing NPY in the thalamic and hypothalamic paraventricular nuclei and in the arcuate nucleus. The method also may be combined as required with other labeling methods for localizing more than two neurochemical markers on one and the same electron microscopic section.

This article describes a video digitizing system designed for measuring film optical density. The system, which is based on a 6-bit (64 gray level) digitizer, solid state video camera, and Apple II microcomputer, digitizes a rectangular area selected by the operator and converts the gray level values into preselected standard units. In order to develop autoradiographic standard curves for quantitative autoradiography with 125I-insulin, liver slices labeled with 125I-insulin and plastic sections containing known amounts of tritium were apposed to the same sheet of LKB Ultrofilm for exposures of 1-7 days. The results indicate that 3H plastic standards can be used to calibrate QAR of 125I-labeled ligands with LKB Ultrofilm. The Apple system was also used to measure binding of 125I-insulin to the external plexiform layer (EPL) in slices of the rat olfactory bulb. Results suggest that the EPL has two binding sites for insulin, a high affinity site with Kd = 1.0 X 10(-8) M and a low affinity site having a Kd = 1.4 X 10(-5) M.

[125I]standards prepared from grey and white matter from rat brain have been compared to commercially available polymer-based standards using two types of radiation-sensitive film for use in quantitative receptor autoradiography. There was a linear relationship between the natural log of optical density and radioactivity (dpm/mm2) for both types of tissue standard exposed to Hyperfilm [3H] or betamax from 1-14 days. No difference between either the slopes or intercepts of these lines for each period of exposure was identified. There was also no significant difference between the slopes and intercepts of tissue-based standards and the lowest 5 activity levels of polymer standards. Over the complete range of ten radioactivity levels of polymer standards, the linear relationship was lost and a quadratic function was the best model for describing the relationship between 1n optical density and radioactivity. Moreover, a linear relationship between section thickness over the range frequently used in receptor studies (5-20 microns) was observed. These results suggest that the lower activity levels of polymer standards are comparable to tissue standards and can be used for quantitative receptor autoradiography.

We have developed and characterized a method for the rapid autoradiographic determination of receptor sites for the non-competitive NMDA receptor antagonist, MK-801, using an iodinated form of the compound, (+)-3-[125I]-iodo-MK-801. The binding site was shown to exhibit those criteria necessary for its definition as a receptor site, i.e., the binding was saturable, of high affinity, easily reversible, and stereospecific. Saturation analysis of binding to thin brain sections revealed a Bmax of 108.1 +/- 10.5 fmol/mg protein and a Kd of 383 +/- 67 pM. The pharmacology of the interaction of the ligand with the binding site yielded good correlation between the potency of various substances to complete for the binding site and their ability to act as antagonists of NMDA. Autoradiographs of thin coronal brain sections using (+)-3-[125I]-iodo-MK-801 yielded high quality images in 24-48 h with a distribution of binding sites paralleling that reported for the tritiated form of the ligand, i.e., with high densities in the hippocampus, cerebral cortex and lateral septum. Other areas with significant binding included parts of the thalamus, the amygdala and the olfactory tubercules. Furthermore, due to its high specific activity, this ligand lends itself to the study of regions not rich in MK-801 binding sites, such as the diencephalon.

To avoid repeated production of 125I tissue mash standards and to decrease inter-experiment variability, a set of commercially available, high activity plastic 3H standards was calibrated for use with 125I autoradiographs. Standards with a range of 2.0-7390 microCi 3H/g plastic were used since they produced autoradiographs of suitable optical density (darkness) at exposure times of 4 to more than 72 h. Tissue mash standards at 12 concentrations of 125I were produced by homogenizing rat brains and adding 125I. These were frozen and sectioned at thicknesses ranging from 6 to 120 microns and mounted on microscope slides. Each set of tissue standards, along with slides bearing the plastic 3H standards, was opposed to film for various times. The calibration of the 3H standards was done separately for each time point and section thickness. Autoradiographs were analyzed using a computer assisted image analysis system. The standard curve generated for each set of 125I-labelled tissue standards was correlated with the 3H standards. It was found that the relationship between the 3H plastic standards and the 125I tissue mash standards was not simple and depended on exposure time, thickness of the section, as well as the concentration of radioactivity in a given standard. However, this accurate calibration of the 3H standards yields long-lived standards for 125I autoradiography useful over a wide range of radionuclide concentrations, tissue thicknesses and exposure times.

We describe the characterization of high affinity [125I-Tyr0]-human CRF binding to purified recombinant human CRF-binding protein (CRF-BP) using a scintillation proximity assay (SPA). For this stable nonseparation technique developed in 96 well microtiter plates, biotinylated CRF-BP is captured by streptavidin-coated SPA beads for the detection of bound [125I-Tyr0]-CRF. Unbound [125I-Tyr0]-CRF represented little or no signal in the assay. Total binding observed was greater than 5000 cpm with a nonspecific signal of < 100 cpm determined in the presence of excess unlabeled human CRF. A comparison of the SPA method with a charcoal precipitation method confirmed that the biotinylation procedure did not adversely affect affinity of the CRF-BP for [125I-Tyr0]-CRF. Saturation binding analysis yielded an apparent equilibrium dissociation constant (Kd) of 208 +/- 5.0 pM (+/- S.D., n = 3). An inhibition constant (Ki) for unlabeled CRF was calculated to be 0.22 +/- 0.03 nM (+/- S.D., n = 8) and a pharmacological profile for eight CRF-related neuropeptides gave a rank potency similar to previously reported results. Finally, the assay variability was assessed with intra- and inter-plate coefficients of variation which were less than 5% each.

The radiosynthesis of (1R)-(+)-1-phenyl-3-methyl-7-[125I]iodo-8-hydroxy- 2,3,4,5-tetrahydro-1H-3-benzazepine (commonly referred to as SCH23982) and its use as a high affinity D1 dopamine antagonist ligand have been reported previously. We now provide a simple and inexpensive protocol for the rapid and efficient synthesis of this radioligand based on the Cloramine-T-catalyzed reaction between the commercially available precursor (R)-(+)-1-phenyl-3-methyl- 8-hydroxy-2,3,4,5-tetrahydro-1H-3-benzazepine and carrier-free sodium [125I]iodide. [125I]SCH23982 is separated rapidly (within 20 min) from the precursor and reaction byproducts (e.g., chlorinated precursor, SCH23390) by reverse-phase HPLC on a C-8 column. The major iodinated product has been identified as SCH23982 based on co-chromatography with authentic SCH23982, UV spectral characteristics, and biological activity. The chromatographic effluent containing the active product is adsorbed on a C-18 Sep-Pak cartridge to remove mobile-phase constituents and permit it to be eluted and diluted to the desired concentration; this technique is used also for periodic repurification. Our synthesis protocol results in final purified product that incorporates ca. 50% of the initial 125I (tested using starting quantities of 1-10 mCi Na125I); the final product has a specific activity of ca. 2500 +/- 350 Ci/mmol. Data from in vitro receptor autoradiographic and homogenate studies with this radioligand are consistent with previously reported values in terms of expected receptor distribution, affinity, and density (KD of 1.0 nM, Bmax of 1400 fmol/mg protein in rat striatal membranes).

We have applied a newly developed SYBR green-based real-time RT-PCR assay for quantification of full-length and truncated neurokinin-1 receptor (NK1R) mRNA expression in nine regions of human brain tissues obtained from 23 subjects who died with no evidence of neurological or neurodegenerative disease. The following brain regions were examined: cingulate cortex, cerebellum, nucleus accumbens, caudate nucleus, putamen, pons, hippocampus, locus coeruleus, and basal ganglia. The SYBR green-based real-time PCR was more sensitive than TaqMan probe-based real-time PCR in amplifying both full-length and truncated NK1R mRNA. The real-time RT-PCR assay had excellent specificity and sensitivity, with a dynamic range of detection between 100 and 1,000,000 copies of the NK1R cDNA per reaction. The truncated NK1R mRNA levels were more abundant than those of the full-length NK1R in most of the regions examined and there was no significant difference in the truncated NK1R mRNA levels among the nine regions studied. There was, however, a significant difference in the expression of full-length NK1R mRNA levels among the nine regions (P=0.0024), and the putamen region expressed the highest full-length NK1R mRNA. Further studies are needed in order to examine the differences between full-length and truncated NK1R in signal transduction and functional consequences in order to delineate the significance of the co-presence of the two forms of NK1R in the human brain.