Preventing neuronal damage and inflammation in vivo during cortical microelectrode implantation through the use of poloxamer P-188.

OBJECTIVE: The aim of this study was to test the efficacy of Poloxamer P188 to reduce cell death and immune response associated with mechanical trauma to cells during implantation of a chronic recording electrode. APPROACH: Ceramic multi-site recording electrodes were implanted bilaterally into 15 adult male Long-Evans rats. One of each pair was randomly assigned to receive a coating of Poloxamer while the other was treated with saline. The extent of neuron loss, and glial cell recruitment were characterized at 2, 4 and 6 weeks post-implantation by stereologic analysis. MAIN RESULTS: At 2 and 4 weeks post-implantation, Poloxamer-coated implants showed significantly fewer glial cells and more neurons in the peri-electrode space than controls; however, this significance was lost by 6 weeks. SIGNIFICANCE: These findings are the first to suggest that Poloxamer has neuroprotective effects in vivo; however, at a fixed loading dose, these effects are limited to approximately 1 month post-implantation.

Thyroid hormone distribution in the mouse brain: the role of transthyretin.

Transthyretin is the major thyroxine-binding protein in the plasma of rodents, and the main thyroxine-binding protein in the cerebrospinal fluid of both rodents and humans. The choroid plexus synthesizes transthyretin and secretes it to the cerebrospinal fluid. Although it was suggested that transthyretin might play an important role in mediating thyroxine transfer from the blood into the brain across the choroid plexus-cerebrospinal fluid barrier, newer findings question this hypothesis. Because thyroid hormone passage across brain barriers is a precondition for its action in the CNS, and because brain is an important target of thyroid hormone action, we investigated the role of transthyretin in mediating thyroid hormone access to and distribution within the brain in a transthyretin-null mouse model system. In this report we describe the results derived from use of film autoradiography, a technique that yields definitive morphological results. Film autoradiograms were prepared at 3 and 19 h after intravenous injection of either high specific activity [(125)I]thyroxine or [(125)I]triiodothyronine. Image analyses were designed to demonstrate regional changes in hormone distribution, and to highlight alterations in iodothyronine delivery from ventricles to brain parenchyma. We find no qualitative or quantitative differences in these parameters between the transthyretin-null and the wild-type mouse brain after either [(125)I]thyroxine or [(125)I]triiodothyronine administration. The data presented here now provide definitive evidence that, under standard laboratory conditions, transthyretin is not required for thyroid hormone access to or distribution within the mouse brain. This study also provides the first map of iodothyronine distribution in the brain of the mouse.

Functional MR imaging of the human cervical spinal cord.

BACKGROUND AND PURPOSE: Although research with functional MR imaging of the brain has proliferated over the past 5 years, technical limitations, such as motion, chemical shift, and susceptibility artifacts, have impeded such research in the human spinal cord. The purpose of this investigation was to determine whether a reliable functional MR imaging signal can be elicited from the cervical spinal cord during simple motor activity. METHODS: Subjects performed three different motor tasks that activate different segments of the spinal cord. Gradient-echo-planar imaging on a 1.5-T clinical unit was used to image cervical spinal cords of human subjects. Another group of subjects was imaged while performing isometric exercise to study the relationship between the blood oxygenation level-dependent (BOLD) signal and applied force. RESULTS: Task-dependent BOLD activity was detected in all subjects. Signal amplitude varied between 0.5% and 7%. Moreover, a linear relationship was found between the applied force and the signal amplitude during isometric exercise. While regions of activation were distributed throughout the spinal cord, concentrated activity was found in the anatomic locations of expected motor innervation. CONCLUSION: The functional MR imaging signal can be reliably detected with motor activity in the human cervical spinal cord on a 1.5-T clinical unit. The location of neural activation has an anatomic correspondence to the myotome in use. The strength of the BOLD signal is directly proportional to the level of muscular activity.

Novel method to quantify neuropil threads in brains from elders with or without cognitive impairment.

Pathological alterations in dendrites and axons (i.e., neuritic pathologies) occur in the normal aging brain as well as in brains from elders with mild cognitive impairment and neurodegenerative dementia. These alterations may correlate with clinical measures of cognitive abilities, but the contribution of neuropil threads (NTs), which constitute 85-90% of cortical tau pathology, has not been clear because of the lack of quantitative methodologies. We combined quantitative fractionation and image analysis to devise a strategy for measuring the burden of tau-rich NTs in the entorhinal and perirhinal cortex of brains from elders with and without cognitive impairment, including dementia due to Alzheimer's disease (AD). On the basis of data presented here using this novel strategy, we conclude that this quantitative imaging technique will facilitate efforts to determine the behavioral correlations of neuritic lesions in AD and other brain disorders.

Combined c-fos and 14C-2-deoxyglucose method to differentiate site-specific excitation from disinhibition: analysis of maternal behavior in the rat.

On the basis of evidence that 14C-2-deoxyglucose (2-DG) autoradiography indicates activity at axonal terminals, whereas c-fos immunocytochemistry indicates activity of neuronal cell bodies, we combined these techniques in adjacent histological brain sections to assess excitatory and disinhibitory synaptic relations in selected sites in female rats in which maternal behavior was elicited by natural parturition, sensitization (7- to 10-day cohabitation with foster pups), or hysterectomy. All individuals in these three groups expressed maternal behavior immediately before 2-DG injection. Controls were non-maternal virgins. Parturient and Hysterectomized groups: elevation (compared with controls) in both 2-DG and c-fos activity in medial preoptic area (MPOA) indicated an increase in its input and output activity, i.e., an excitatory interaction; the MPOA was previously shown to be critical for maternal behavior. Sensitized group: a decrease in 2-DG activity of vomeronasal nuclei (bed nucleus of the accessory olfactory tract, BAOT, and medial amygdala, ME, replicating our previous study) and an elevation in c-fos activity, jointly indicate disinhibition of these nuclei, that were previously shown to modulate pup-chemostimulation-induced sensitization. All other sites showed evidence of excitatory input-output relationships (i.e., joint increase in both 2-DG and c-fos activity), e.g., bed nucleus of the stria terminalis (BNST), lateral habenula (LHAB), central gray (CG), thalamus (THAL), septum (SEPT), and ventral tegmental area (VTA). The present study demonstrates the feasibility of measuring 2-DG and c-fos activity jointly in adjacent sections of the same brain, thereby providing evidence to distinguish between localized excitation and disinhibition.

Automatic matching of homologous histological sections.

The role of neuroanatomical atlases is undergoing a significant redefinition as digital atlases become available. These have the potential to serve as more than passive guides and to hold the role of directing segmentation and multimodal fusion of experimental data. Key elements needed to support these new tasks are registration algorithms. For images derived from histological procedures, the need is for techniques to map the two-dimensional (2-D) images of the sectional material into the reference atlas which may be a full three-dimensional (3-D) data set or one consisting of a series of 2-D images. A variety of 2-D-2-D registration methods are available to align experimental images with the atlas once the corresponding plane of section through the atlas has been identified. Methods to automate the identification of the homologous plane, however, have not been previously reported. In this paper we use the external section contour to drive the identification and registration procedure. For this purpose, we model the contours by B-splines because of their attractive properties the most important of which are: 1) smoothness and continuity; 2) local controllability which implies that local changes in shape are confined to the B-spline parameters local to that change; 3) shape invariance under affine transformation, which means that the affine transformed curve is still a B-spline whose control points are related to the object control points through the transformation. In this paper we present a fast algorithm for estimating the control points of the B-spline which is robust to nonuniform sampling, noise, and local deformations. Curve matching is achieved by using a similarity measure that depends directly on the parameters of the B-spline. Performance tests are reported using histological material from rat brains.

Region-specific tritium enrichment, and not differential beta-absorption, is the major cause of 'quenching' in film autoradiography.

Tritium quenching refers to the situation in which estimates of tritium content generated by film autoradiography depend on the chemical composition of the tissue as well as on the concentration of the radioisotope. When analysing thin brain sections, for example, regions rich in lipid content generate reduced optical densities on x-ray film compared with lipid-poor regions even when the total tissue concentration of tritium in those regions is identical. We hypothesize that the dried thickness of regions within sections depends upon the relative concentrations and types of lipid within the regions. Areas low in white matter dry thinner than areas high in white matter, leading to a relative enrichment of tritium in the thinner regions. To test this model, a series of brain pastes were made with different concentrations of grey and white matter and impregnated with equal amounts of tritium. The thickness of dried sections was compared with percentage of white matter and apparent radioactive content as determined by autoradiogram analysis. The results demonstrated that thickness increased, and apparent radioactivity decreased, with higher percentages of white matter. In the second experiment, thickness measurements from dried sections were successfully used to correct the apparent radioisotope content of autoradiograms created from tritium containing white- and grey-matter tissue slices. We conclude that within-section thickness variation is the major physical cause for 'tritium quenching'.

Dual activity maps in primate visual cortex produced by different temporal patterns of zif268 mRNA and protein expression.

The inducible nature of the immediate-early genes (IEGs) c-fos and zif268 allows their products to be used as activity markers in the brain. The utility of such markers in general is restricted because they can resolve only neurons activated by a single stimulus. To overcome this limitation, we have developed a double-label technique that exploits the dissimilar time course of zif268 mRNA and protein induction, allowing them to be separately induced by two different stimuli and independently stained to provide a visual display of neurons that are responsive to each stimulus. Two powerful features of this new imaging technique-the possibility of staining separate populations of activated neurons and the ability to visualize them at the cellular level-should extend IEG applications in biological activity mapping.

Automatic vessel segmentation and quantification of the rat aortic ring assay of angiogenesis.

BACKGROUND: Pharmacologic control of angiogenesis is a promising new approach to the treatment of a variety of pathologic conditions including cancer. The recently developed in vitro rat aortic ring model provides a simple, reproducible assay for discovering angiogenic agonists and antagonists. However, quantification of results in this assay is time consuming, tedious, and subjective, because it involves visual inspection of images and manually counting the newly formed microvessels extending from the cultured aortic ring. This report describes an automated image analysis-based procedure for quantification of this assay that overcomes these difficulties. EXPERIMENTAL DESIGN: The designed image processing algorithm segments the vessels from gray scale images. A high-pass filter is used, and the results are separated into nonvascular and vessel compartments based on object size and shape. Quantification relies on identification of vessels intersecting a closed transect set a fixed distance from the aortic ring. The number and the total area of these vessels are determined. The entire operation has been automated and packaged in an application called Vessels. RESULTS: The correlation between computer-determined vessel area/vessel number and visual microvessel count is high (r2 = 0.91 and r2 = 0.86, respectively). CONCLUSION: Vessels offers high-speed, fully automatic batch processing including production of a hard copy for documentation. The application runs on the Apple family of computers. On a Quadra 800, the application can process approximately 30 images/hour, which is approximately 2.5 times faster than manual quantification of this assay.

Bioengineering approach to non-invasive measurement of body composition.

Measurement of body fat percentage is essential for medical care and research. The "gold standard" method for humans is underwater weighing, which is clearly inappropriate for infants, sick people and non-human animals. The corresponding criterion method for animals is comminution of the carcass followed by extraction of the fat with a volatile solvent such as ether. Our goal has been to develop a method for body composition (fat percentage) for use in animals and humans which is non-invasive and minimally intrusive, independent of variation in body conformation and fat distribution, and reasonable in cost. In one variant, our approach to this problem has been to move Archimedes' principle "on to dry land." The subject's volume is determined by measuring the differential buoyancy in comfortably breathable light (low density) and heavy atmospheres. In another, we use "structured light," in which a pattern of illumination is cast on the patient. The image is acquired using a video camera and the geometrical spatial coordinates of a large number of points on the surface of the subject are acquired. This permits the computation of the surface area and volume of the subject; which, combined with the weight, determines the fat percentage.

Advances in image processing for autoradiography.

The next generation of techniques for image processing in autoradiography will extend the range for what is already a broad band method for functional mapping. Among the new tools under development are techniques to correct for tritium quenching. With these, high resolution, accurate 2-deoxyglucose mapping will be feasible. Also being developed are automatic segmentation algorithms which will permit rapid surveys of a large number of structures in a large number of brains. Together with recent refinements in other aspects of image processing of autoradiograms, these tools will provide both better quantification accuracy and faster analysis speed.

Role of the Mauthner cell in sensorimotor integration by the brain stem escape network.

The Mauthner neurons have become synonymous with the C start evasive response of fishes. C starts are a two-part movement pattern. First, the fish bends its body so that it has a C-like profile (stage 1) when viewed from above. Second, the fish rapidly accelerates away from its starting position (stage 2). Until recently, it has been possible to determine the contribution of Mauthner cell activity to the expression of this behavior. In this paper we focus on three of our recent papers that address this issue. Our work combines high-speed digital image analysis of the C start with chronic Mauthner cell and electromyographic recordings, lesions of the Mauthner cells, and stimulation of single Mauthner axons in swimming fishes. This work shows that the firing of the Mauthner cell results in a short-latency body contraction that orients the initial stage of the C start away from the direction of the threatening stimulus. The direction of the escape trajectory, however, is more finely tuned to stimulus angle than can be explained by the firing of just the Mauthner cell and its post-synaptic followers. Precise control of trajectory must, therefore, require participation of other neurons. These neurons together with the Mauthner cell form a system that we term the brain stem escape network. We have identified candidate neurons of this network which can now be studied at the single-cell level. Because of both its accessibility for neurophysiological study and its neuroanatomical simplicity, we assert that the brain stem escape network is a useful preparation for understanding fundamental processes of sensorimotor integration in the brain stem.

The motor output of the Mauthner cell, a reticulospinal command neuron.

We electrically stimulated individual Mauthner (M-) cells to determine their motor contribution to C-starts of swimming goldfish. In comparison with sensory-evoked C-starts, M-reflexes triggered by electrical stimulation of single M-cells were significantly weaker and less variable. Stage 1 turns were both longer in duration and smaller in angle for the M-reflex when compared with the sensory-evoked C-start. This translates to an average reduction of 22% in angular velocity during stage 1. Likewise, during stage 2, the distance moved by the fish was reduced by 15% and the absolute value of stage 2 turning angle was reduced by 47%. In addition, the normal mechanical or neural coupling between stages 1 and 2 appeared to be altered for the M-reflex. From this and our other recent studies, we conclude that there must be two primary groups of reticulospinal neurons in the escape triggering network. The first group includes the M-cell and determines the initial left-right direction of the response and the extent of stage 1 angle. From previous EMG recordings we know that the second group of neurons can fire within 5-15 ms (average, 9 ms) after the stage 1 cells. These determine the onset time and direction of stage 2. Together the coupling of the two primary groups results in the full propulsive force and turning flexibility of the C-start.

Lateralization and adaptation of a continuously variable behavior following lesions of a reticulospinal command neuron.

This study utilizes digitized cinematic data and lesions of individual Mauthner (M-) cells, large medial reticulospinal command neurons, to examine their role in goldfish C-starts elicited by displacement stimuli. Our results show a major difference in response lateralization in animals with only one M-cell compared to those with both cells intact, or both cells absent. Animals with one M-cell responded by turning to the side opposite the remaining M-cell in 94% of the trials, whereas those with both M-cells intact or both cells absent responded with equal probability to both sides. When the M-cells were absent, the responses were on the average 4 ms longer in latency. This difference may confer a behaviorally significant advantage to the M-cell in blocking other networks that can trigger C-starts. Nevertheless, with the exception of latency, the central program producing the escape behavior adapts automatically to the absence of both M-cells: animals with bilateral M-cell lesions continued to produce the full spectrum of kinematic performance levels seen in intact animals.

Flexible body dynamics of the goldfish C-start: implications for reticulospinal command mechanisms.

As a model for learning how reticulospinal networks coordinate movement, we have analyzed the function of the Mauthner (M-) neurons in the escape response of the goldfish. We used water displacements of 3-6 micron to elicit C-start escape responses. These responses consist of 2 fundamental movements that grade into each other: Stage 1 lasts 15-40 msec and rotates the body 30 degrees-100 degrees about the center of mass; stage 2 is an axial acceleration that moves the center of mass 2-6 cm. Combined, the 2 stages result in trajectory turns ranging from 15 degrees to 135 degrees. Thus, these data show that M-initiated C-starts are not fixed movement patterns. The durations of stage 1 body muscle EMGs were correlated with turn angles achieved during stage 1. Since variable stage 1 EMGs are not seen when the M-cell is triggered by itself, other circuits, independent of the M-cell, must control the extent of the initial turn, and consequently escape trajectory. Furthermore, turning angles of stages 1 and 2 were correlated, allowing escape trajectory to be predicted, on average, 26 msec after movement started. This suggests that the commands for escape trajectory should be organized by the end of stage 1. In concert with this, the time of onset of the stage 2 EMG preceded the stage 2 onset by a range with a mean of 28.4 msec, typically putting the stage 2 command at the beginning of stage 1 movement. Thus, stage 2 initiation does not require motion-dependent feedback. Our findings indicate that the Mauthner cell initiates the first of a series of motor commands that establish the initial left-right decision of the escape sequence from the side of the stimulus, whereas parallel circuits simultaneously organize the command controlling the escape angle.