Serotonin2C receptor localization in GABA neurons of the rat medial prefrontal cortex: implications for understanding the neurobiology of addiction.

Serotonin (5-HT) action via the 5-HT(2C) receptor (5-HT(2C)R) provides an important modulatory influence over neurons of the prefrontal cortex (PFC), which is critically involved in disorders of executive function including substance use disorders. In the present study, we investigated the distribution of the 5-HT(2C)R in the rat prelimbic prefrontal cortex (PrL), a subregion of the medial prefrontal cortex (mPFC), using a polyclonal antibody raised against the 5-HT(2C)R. The expression of 5-HT(2C)R immunoreactivity (IR) was highest in the deep layers (layers V/VI) of the mPFC. The 5-HT(2C)R-IR was typically most intense at the periphery of cell bodies and the initial segment of cell processes. Approximately 50% of the 5-HT(2C)R-IR detected was found in glutamate decarboxylase, isoform 67 (GAD 67)-positive neurons. Of the subtypes of GABA interneurons identified by expression of several calcium-binding proteins, a significantly higher percentage of neurons expressing IR for parvalbumin also expressed 5-HT(2C)R-IR than did the percentage of neurons expressing calbindin-IR or calretinin-IR that also expressed 5-HT(2C)R-IR. Since parvalbumin is located in basket and chandelier GABA interneurons which project to cell body and initial axon segments of pyramidal cells, respectively, these results raise the possibility that the 5-HT(2C)R in the mPFC acts via the parvalbumin-positive GABAergic interneurons to regulate the output of pyramidal cells in the rat mPFC.

A fuzzy logic approach to identifying brain structures in MRI using expert anatomic knowledge.

We report a novel computer method for automatic labeling of structures in 3D MRI data sets using expert anatomical knowledge that is coded in fuzzy sets and fuzzy rules. The method first identifies major structures and then uses spatial relationships to these landmarks to recognize other structures. This labeling process simulates the iterative process that we ourselves use to locate structures in images. We demonstrate its application in three data sets, labeling brain MRI by locating the longitudinal and lateral fissures and the central sulci and then determining boundaries for the frontal lobes. Our method is adaptable to the identification of other anatomical structures.

Chronic phencyclidine increases NMDA receptor NR1 subunit mRNA in rat forebrain.

The present study was designed to determine whether the sensitization of locomotor activity that results from chronic phencyclidine (PCP) administration is associated with altered NMDA receptor function or mRNA in rat brain. Female Sprague-Dawley rats were administered PCP (20 mg/kg, i.p.) once daily for 5 days. After withdrawal for 72 hr, challenge with 3.2 mg/kg PCP (i.p.) revealed a significant sensitization to the locomotor activating effect of PCP. In situ hybridization analysis with an oligonucleotide probe complementary to the mRNA encoding the NR1 subunit of the NMDA receptor demonstrated that chronic PCP treatment resulted in a marked increase in NR1 subunit mRNA in the forebrain. Quantitative image analysis revealed a significant increase in the labeling of NR1 mRNA in the olfactory tubercle, piriform cortex, frontal cortex, and anterior striatum. However, no significant difference between PCP and saline-treated rats was found in the hippocampus or cerebellum. In a parallel study, possible functional alterations in the NMDA receptor were assessed by measuring NMDA-stimulated release of [3H]DA from slices of the olfactory tubercle and piriform cortex. NMDA-stimulated release was not affected by chronic PCP treatment, but the inhibition of this release by PCP, 7-chlorokynurenic acid (7-CK), and DL-2-amino-5-phosphovaleric acid (AP-5) was significantly diminished by chronic PCP. This suggests that the behavioral plasticity associated with chronic PCP may be related to an altered subunit stoichiometry of NMDA receptors in selective forebrain regions.

Augmentation of locomotor activity by chronic phencyclidine is associated with an increase in striatal NMDA receptor function and an upregulation of the NR1 receptor subunit.

Phencyclidine (PCP) is a drug of abuse that produces schizophrenia-like symptoms in humans and increases locomotor activity and stereotypic behavior in rodents. PCP-induced alteration in rat locomotor activity is thought to be mediated by an inhibition of N-methyl-D-aspartate (NMDA) receptors in the striatum and other brain regions. In this study, rats treated chronically with PCP (20 mg/kg once per day for 5 days) showed a marked increase in locomotor activity following a PCP challenge (3.2 mg/kg) administered after either 3 or 8 days of withdrawal. In biochemical assays, the release of striatal [14C]GABA by NMDA was enhanced by about 77% by chronic PCP treatment, whereas [3H]ACh release was increased by about 31% in tissue from PCP-treated rats. Even though binding experiments with 1-[1-(2-thiethyl)cyclohexyl]piperidyl-3,4 3H(N) ([3H]TCP) showed no alteration in the Kd or Bmax in whole striatum, quantitative immunocytochemical experiments found an upregulation in the NR1 subunit in the cell bodies and neuropil of cortical and striatal regions of the forebrain following chronic PCP treatment. An increase in the size of NR1-immunoreactive cells in the forebrain was also observed following chronic PCP treatment. Together, these data may help in understanding the mechanisms underlying the adaptive response to chronic reduction in glutamatergic NMDA transmission that has been postulated to be involved in the etiology of schizophrenia.

Altered nuclear activation parameters of rat sperm treated in vitro with chromatin-damaging agents.

Standard semen measures do not assess the genetic integrity of sperm. A human sperm activation assay (HSAA) has proven very useful for assessing sperm quality and predicting pregnancy outcome. The HSAA involves incubating permeabilized sperm in cytoplasmic extracts of Xenopus laevis frog eggs. The extracts activate sperm nuclei, which undergo chromatin decondensation, DNA synthesis, and chromatin recondensation, mimicking events that occur after fertilization in vivo. However, no animal model sperm activation assay has been reported. We hypothesize that sperm activation assays will be useful for studying molecular mechanisms of sperm DNA repair by egg cytoplasm and for screening sperm for damaged DNA. Thus, the objectives of this study were to develop an in vitro rat sperm activation assay (RSAA) using cytoplasmic extracts of X. laevis frog eggs and to determine how chemically damaging the sperm chromatin would affect two sperm activation parameters, chromatin decondensation and DNA synthesis. We incubated demembranated rat sperm in a cytoplasmic extract of X. laevis frog eggs supplemented with tritiated thymidine triphosphate ([3H]TTP). The activated sperm nuclei underwent chromatin decondensation and DNA synthesis. Decondensation kinetics were examined using image analysis to measure the size of the sperm nuclei as they decondensed. DNA synthesis kinetics were examined using autoradiography of incorporated [3H]TTP. To investigate how chemical damage affects nuclear activation, we treated rat sperm in vitro with ethylene glycolbis(sulfosuccinimidyl-succinate; SEGS), a reversible crosslinking agent, or hydroxylamine (HA), a DNA base modifier. Treatment with SEGS blocked decondensation in a dose-dependent manner. In contrast, treatment with HA enhanced decondensation, induced gross chromatin abnormalities, and increased [3H]TTP incorporation into activated sperm nuclei, responses consistent with an attempt by the egg cytoplasm to repair DNA damage. These results suggest that the RSAA may be useful for detecting damaged sperm chromatin as a result of toxicant exposure.

NG-nitro-L-arginine methyl ester modifies the input function measured by dynamic susceptibility contrast magnetic resonance imaging.

In rat brain dynamic susceptibility contrast magnetic resonance (MR) images, vessels visible on the same scan plane as the brain tissue were used to measure the characteristics of the input function of the MR contrast agent gadopentetate dimeglumine. MR images were acquired 30 and 60 minutes after intravenous injections of 3 mg/kg and 15 mg/kg NG-Nitro-L-arginine methyl ester (L-NAME) (n = 9). The time of arrival (TOA) and the mean transit time corrected for TOA of the input function were increased by 3 mg/kg or 15 mg/kg L-NAME. The area of the input function was increased by 15 mg/kg L-NAME. In two animals, similar modifications of the input function induced by 20 mg/kg L-NAME were reversed by infusion of sodium nitroprusside. In two other animals, MABP was increased by phenylephrine to a similar extent as in L-NAME experiments, but did not induce the same modifications of the input function, showing that the action of L-NAME on the input function was not simply caused by an effect on MABP. These results show that the input function can be significantly altered by manipulations widely used in cerebrovascular studies. These input function changes have important implications for calculation of cerebral blood flow.

Perfusion deficit parallels exacerbation of cerebral ischemia/reperfusion injury in hyperglycemic rats.

Magnetic resonance imaging (MRI) techniques were used to determine the effect of preexisting hyperglycemia on the extent of cerebral ischemia/reperfusion injury and the level of cerebral perfusion. Middle cerebral artery occlusion (MCAO) was induced by a suture insertion technique. Forty one rats were divided into hyperglycemic and normoglycemic groups with either 4 hours of continuous MCAO or 2 hours of MCAO followed by 2 hours of reperfusion. Diffusion-weighted imaging (DWI) was performed at 4 hours after MCAO to quantify the degree of injury in 6 brain regions. Relative cerebral blood flow (CBF) and cerebral blood volume (CBV) were estimated using gradient echo (GE) bolus tracking and steady-state spin echo (SE) imaging techniques, respectively. Brain injury correlated with the perfusion level measured in both SE CBV and dynamic GE CBF images. In the temporary MCAO model, mean lesion size in DWI was 118% larger and hemispheric CBV was reduced by 37% in hyperglycemic compared with normoglycemic rats. Hyperglycemia did not significantly exacerbate brain injury or CBV deficit in permanent MCAO models. We conclude that preexisting hyperglycemia increases acute postischemic MRI-measurable brain cellular injury in proportion to an associated increased microvascular ischemia.

Magnetic resonance imaging quantitation of changes in muscle volume during 7 days of strict bed rest.

BACKGROUND: Prolonged bed rest results in a loss of leg lean body mass. Previous studies using bed rest as a model for microgravity have shown decreases in leg mass after 12 and 14 d, 5 and 17 wk. HYPOTHESIS: As magnetic resonance imaging (MRI) can provide a precise and non-invasive means of determining muscle volume, we sought to determine if changes in leg muscle volume could be detected in bed rest periods as short as 7 d. METHODS: Five young, healthy, male volunteers were subjected to 7 d of absolute bed rest. Each subject underwent MRI quantitation of segmental muscle volumes of the calves and thighs before and after bed rest. Eleven (calf) and nine (thigh) contiguous 1-cm thick transaxial images were generated over prescribed regions using a Technicare MRI imager with a 0.6T superconducting magnet and body coil. Image processing was performed using a generalized 8-bit medical image analysis package developed at University of Texas Medical Branch. Images were analyzed for muscle and non-muscle volumes (including fat, blood vessel, and bone marrow volumes). RESULTS: The MRI quantitation demonstrated bed rest-induced significant decreases in segmental thigh muscle (approximately 3.0%, p < 0.05) volume. CONCLUSIONS: We conclude that computerized image analysis of MRI images provides a sensitive tool capable of detecting leg volume changes of as little as 3.0% over a 7-d period of strict bed rest.

Classification of brain compartments and head injury lesions by neural networks applied to MRI.

An automatic, neural network-based approach was applied to segment normal brain compartments and lesions on MR images. Two supervised networks, backpropagation (BPN) and counterpropagation, and two unsupervised networks, Kohonen learning vector quantizer and analog adaptive resonance theory, were trained on registered T2-weighted and proton density images. The classes of interest were background, gray matter, white matter, cerebrospinal fluid, macrocystic encephalomalacia, gliosis, and "unknown." A comprehensive feature vector was chosen to discriminate these classes. The BPN combined with feature conditioning, multiple discriminant analysis followed by Hotelling transform, produced the most accurate and consistent classification results. Classification of normal brain compartments were generally in agreement with expert interpretation of the images. Macrocystic encephalomalacia and gliosis were recognized and, except around the periphery, classified in agreement with the clinician's report used to train the neural network.

Frontal lobe changes after severe diffuse closed head injury in children: a volumetric study of magnetic resonance imaging.

In view of the pathophysiology and biomechanics of severe closed head injury (CHI) in children, we postulated that the frontal lobes sustain diffuse injury, even in the absence of focal brain lesions detected by magnetic resonance imaging (MRI). This study quantitated the morphological effects of CHI on the frontal lobes in children who sustained head trauma of varying severity. The MRI findings of 14 children who had sustained severe CHIs (Glasgow Coma Scale score of < or = 8) were compared with the findings in a matched group of 14 children having sustained mild head injuries (Glasgow Coma Scale score of 13-15). The patients ranged in age from 5 to 15 years at the time of their MRIs, which were acquired at least 3 months postinjury. MRI findings revealed no focal areas of abnormal signal in the frontal lobes. Volumetric analysis disclosed that the total prefrontal cerebrospinal fluid increased and the gray matter volume decreased in the patients with severe CHI, relative to the mildly injured comparison group. Gray matter volume was also reduced in the orbitofrontal and dorsolateral regions of the brains of children with severe CHI, relative to the children who sustained mild head trauma. These volumetric findings indicate that prefrontal tissue loss occurs after severe CHI in children, even in the absence of focal brain lesions in this area. Nearly two-thirds of the children who sustained severe CHIs were moderately disabled after an average postinjury interval of 3 years or more, whereas 12 of the 14 patients with mild CHIs attained a good recovery (2 were moderately disabled) by the time of study.(ABSTRACT TRUNCATED AT 250 WORDS)

Location and geometric description of carpal bones in CT images.

The carpal regions of ten cadaver extremities were imaged by CT. The images were combined into a 3-dimensional model of the carpus using a technique based on a dynamic programming algorithm to find an optimal estimate of the location of the bone boundaries in the CT images. The resulting set of surface points on each bone was used to compute volumes and principal and antipodal axes for the bones. A spatial coordinate system was established based on the positions of the centroids of three bones in the distal carpal row. The angular orientations of all carpal bones were determined with respect to this system. The principal axes for the same bone among ten wrist specimens proved to be more widely dispersed than the antipodal axes for the same bones. The antipodal axes also correspond more closely to an intuitive notion of the "longest axis" of the bones. We conclude that the antipodal axis is a more reliable and useful measure of bone orientation than the principal axis.

Surface area measurement of pressure sores using wound molds and computerized imaging.

OBJECTIVE: To develop a method to measure wound surface area. DESIGN: Validity study. SETTING: Inpatient service. PARTICIPANTS: Four patients with decubitus ulcers. MEASUREMENTS: A new imaging process estimating wound surface area and volume by NMR spectroscopy of a mold of the pressure ulcer and a comparison measurement of volume of the mold by water displacement. Measurements made serially totalled 17. RESULTS: Measurement of the volume of the mold by the computer system correlated strongly with measurement of the volume by water displacement. Surface area of the pressure sore mold correlated strongly with volume of the mold raised to the two-thirds power. CONCLUSIONS: It is possible for the first time to measure surface area of decubitus ulcers. This may provide a way of determining accurately the dose of newly proposed topical treatments.

Measurement of carpal bone geometry by computer analysis of three-dimensional CT images.

The aim of this project was noninvasively to analyze and quantitate the geometry, load transfer characteristics, and spatial relationships of the carpal bones by using a new three-dimensional CT scan reconstruction technique. The determination of mechanical parameters such as distances between centroids and between bone surfaces, carpal alignment, volumes, surface areas, and contact areas can provide the basis for comparison between normal wrists and wrists with a variety of progressive instability patterns, types of fracture, pathologic and posttraumatic states, and different simulated surgical procedures. This new technology has demonstrated a volumetric accuracy of 94% and a linear accuracy of 97%. Simultaneous analysis of all articulating surfaces of multiple joints can be performed in cadavers and in patients because of the noninvasive nature of the imaging reconstruction technique. This new research offers much more information than has previously been available. It also promises direct application to the clinical setting and eliminates several limitations and questions that were inescapable with previous technology.

The evolution of acute stroke recorded by multimodal magnetic resonance imaging.

Events associated with an evolving cerebral infarction were studied using multiple magnetic resonance imaging (MRI) techniques at 4.7 T in a rat model of middle cerebral artery occlusion. High resolution perfusion images revealed a core of absent perfusion surrounded by a zone of slow, but measurable perfusion. Only the core of severest perfusion deficit demonstrated restricted water diffusion as early as 1 hr, consistent with "cytotoxic" cellular edema in the most vulnerable region. Within 24 hours, the area of restricted diffusion encompassed the entire region destined to become infarcted. In spin-echo images, hypointensity, likely reflecting deoxygenated hemoglobin, was visible in the ischemic hemisphere. Edema accumulated over 72 hr primarily in the surrounding slowly perfused rim, consistent with the concept of "vasogenic" edema. These studies demonstrate that multimodal MRI can visualize events which define the ischemic penumbra--deoxygenation, maintenance of transmembrane ionic gradients, reduced flow, and delayed cell death. These experiments noninvasively visualized differential hemodynamic and biochemical processes within the core and perifocal penumbra and will allow quantitation over time of the relationship between blood flow, cytotoxicity and edema in stroke.

Measurement of brain compartment volumes in MR using voxel composition calculations.

A computer method was developed for brain compartment volume measurement in MR images. The method is a statistical averaging technique, in which each voxel is viewed as a mixture of adjacent tissues in a measurable proportion. This method is based on sampling representative tissue intensities and then interpolating intermediate intensities. It can automatically correct for volume averaging artifacts occurring in voxels that contain heterogeneous tissues.

Simulated scaphoid proximal pole fracture.

Five fresh cadaver upper extremities were studied with use of a static positioning frame, pressure-sensitive film, a microcomputer-based videodigitizing system, and a Sun station image analysis system to assess the load bearing characteristics of the scaphoid in the proximal carpal joint. Specimens were studied in their normal condition, after a proximal pole osteotomy of the scaphoid, and after resection of the proximal pole of the scaphoid. The amount of contact area born through the scaphoid fossa was essentially the same whether the scaphoid was intact, or after a simulated scaphoid fracture of its proximal pole, or after resection of the proximal pole. The scaphoid contact area and pressure, although overall relatively constant, was redistributed after osteotomy, resulting in increased contact area under the distal fragment and no change or a slight decrease in the contact area under the proximal fragment of the scaphoid. After resection of the proximal fragment, all scaphoid contact area and pressure was born by the distal scaphoid fragment. The contact area and pressure characteristics of the lunate remained unchanged in all conditions compared with the normal condition. There were no significant changes in the locations of the centroids of the scaphoid segments and the lunate in any of the conditions tested.

Magnetic resonance imaging and neuropsychological findings in human immunodeficiency virus infection.

Neurobehavioral functioning and magnetic resonance imaging (MRI) were investigated in 25 patients with various Centers for Disease Control (CDC) stages of human immunodeficiency virus (HIV) infection and in a control group of seven normal subjects. Unequivocal slowing of information processing speed and cerebral atrophy were related to the stage of HIV infection, with patients in CDC group IV exhibiting the most abnormal findings. Slowing of response speed was directly related to the severity of cerebral atrophy.

Differential effects of p-chlorophenylalanine on indoleamines in brainstem nuclei and spinal cord of rats. II. Identification of immunohistochemically stained structures using computer-assisted image enhancement techniques.

Following intraperitoneal injection of p-chlorophenylalanine (PCPA, 400 and 600 mg/kg) on 3 consecutive days, the brainstem and lumbar cord of rats were removed, frozen-sectioned and immunohistochemically stained (PAP method) for serotonin (5-HT). Using computer-assisted image analysis, the density of 5-HT staining in control, 400 and 600 mg/kg PCPA groups was determined. The mean number of pixels (representing 5-HT staining) was determined in 6 areas in the brainstem containing 5-HT cell bodies (nuclei raphe pallidus, raphe obscurus, rostral and caudal raphe magnus, raphe dorsalis and paragigantocellularis lateralis) and in the dorsal and ventral spinal cord. The results suggest a differential depletion of 5-HT within brainstem nuclei following PCPA treatment in that the most marked dose-related reductions were observed in nucleus raphe obscurus and caudal nucleus raphe magnus. Furthermore, a computer program designed to isolate terminal structures in the spinal cord identified a differential depletion of 5-HT terminals in the dorsal horn versus the ventral horn. The present study describes 3 analytical approaches combining immunohistochemistry with the computer-assisted image analysis technique and allows comparison between groups of animals which received the same or different drug treatments.

Two-dimensional and three-dimensional movement of human polymorphonuclear leukocytes: two fundamentally different mechanisms of locomotion [corrected].

Patients with an inherited deficiency of the adherence glycoproteins LFA-1, Mac-1, and p150,95 are unable to mobilize polymorphonuclear leukocytes (PMNLs) to peripheral sites of inflammation. LFA-1/Mac-1/p150,95-deficient PMNL exhibited profoundly impaired movement stimulated by chemotactic factors when the cells were required to move over two-dimensional surfaces. Less impairment of movement was demonstrated in three-dimensional movement through cellulose filters. A possible explanation for this difference in cell translational mobility is that movement in cellulose filters is less adherence dependent than movement over a two-dimensional plastic surface. Movement of PMNL in collagen gels is known to be relatively independent of adherence. No deficiency of translational mobility of PMNL from LFA-1/Mac-1/p150,95-deficient patients was observed in collagen gels. Antibodies against the common beta subunit effectively blocked two-dimensional movement but had little effect on three-dimensional movement through cellulose filters or collagen gel matrices. HL-60 cells were employed as a model to investigate the effects of adherence on cell movement. Treatment of HL-60 cells with phorbol myristate acetate resulted in the appearance of Mac-1 and p150,95 on the cell surface. Concurrently, the cells exhibited increased adherence to glass and plastic. In spite of increased adherence, HL-60 cells showed no translational movement, indicating factors other than the ability to adhere were important in cell motility. These experiments implied that PMNLs undergo two fundamentally different kinds of motion, one adherence dependent (two-dimensional movement) and the other largely adherence independent (three-dimensional movement). These findings are consistent with the view that egress of PMNLs from the vascular space is adherence dependent. Movement through extravascular tissues may be adherence independent.

Drug effects on the 5-HT response of Schistosoma mansoni.

Several vertebrate 5-HT antagonists at concentrations around 0.1 mM reduced 5-HT-induced increases in the motor activity of the parasitic blood fluke Schistosoma mansoni. The order of potency for 5-HT response antagonism was haloperidol greater than cyproheptadine greater than mianserin greater than trazodone greater than spiperone greater than methysergide. Nisoxetine, a 5-HT uptake inhibitor in vertebrate preparations, was also a potent antagonist of the 5-HT response in schistosomes. The potent antischistosomal praziquantel reduced the 5-HT response similarly to the other antiserotonergic drugs, but at much lower concentrations, beginning around 0.1 microM. The 5-HT agonist quipazine stimulated worm activity at 1-0.1 mM when applied alone, but reduced the 1 mM 5-HT response when quipazine and 5-HT were administered concurrently. Dopamine (DA) alone had no effect on the overall activity of S. mansoni. Although no drug was found to have absolute species specificity, quantitative differences were observed between the relative activity of drugs in schistosomes and vertebrates.