Mapping and reconstruction of domoic acid-induced neurodegeneration in the mouse brain.

Domoic acid, a potent neurotoxin and glutamate analog produced by certain species of the marine diatom Pseudonitzschia, is responsible for several human and wildlife intoxication events. The toxin characteristically damages the hippocampus in exposed humans, rodents, and marine mammals. Histochemical studies have identified this, and other regions of neurodegeneration, though none have sought to map all brain regions affected by domoic acid. In this study, mice exposed (i.p.) to 4 mg/kg domoic acid for 72 h exhibited behavioral and pathological signs of neurotoxicity. Brains were fixed by intracardial perfusion and processed for histochemical analysis. Serial coronal sections (50 microm) were stained using the degeneration-sensitive cupric silver staining method of DeOlmos. Degenerated axons, terminals, and cell bodies, which stained black, were identified and the areas of degeneration were mapped onto Paxinos mouse atlas brain plates using Adobe Illustrator CS. The plates were then combined to reconstruct a 3-dimensional image of domoic acid-induced neurodegeneration using Amira 3.1 software. Affected regions included the olfactory bulb, septal area, and limbic system. These findings are consistent with behavioral and pathological studies demonstrating the effects of domoic acid on cognitive function and neurodegeneration in rodents.

Students' attitudes towards computer testing in a basic science course.

OBJECTIVES: The introduction of computerized testing offers several advantages for test administration, however, little research has examined students' attitudes toward computerized testing. This paper, reports the attitudes of 202 students in a first year cell biology and histology course toward computerized testing and its influence on their study habits over a three year period. DESIGN AND METHODS: Multiple choice and image-based extra credit examinations and summative image-based examinations have been successfully administered in the course. The results indicate that students readily accept computer exams and that their study habits were influenced in a positive manner by the computer administered extra-credit examinations. RESULTS: Our results provide further evidence that medical students like the use of computer administered examinations and that the examinations may actually accentuate the learning experience.

TAL1/SCL is expressed in endothelial progenitor cells/angioblasts and defines a dorsal-to-ventral gradient of vasculogenesis.

In this study we establish that TAL1/SCL, a member of the helix-loop-helix family of transcription factors, and an important regulator of the hematopoietic lineage in mice, is expressed in the endothelial lineage of avians. The earliest events of vascular development were examined using antibodies to TAL1/SCL, and the QH1 antibody, an established marker of quail endothelial cells. Analyses using double immunofluorescence confocal microscopy show that: (i) TAL1/SCL is expressed by both quail and chicken endothelial cells; (ii) TAL1/SCL expression precedes that of the QH1 epitope; and (iii) TAL1/SCL, but not QH1, expression defines a subpopulation of primordial cells within the splanchnic mesoderm. Collectively these data suggest that TAL1/SCL-positive/QH1-negative cells are angioblasts. Further, using TAL1/SCL expression as a marker of the endothelial lineage, we demonstrate that in addition to the previously described cranial-to-caudal gradient, there is a dorsal-to-ventral progression of vasculogenesis.

Activity correlates of cytochrome oxidase-defined compartments in granular and supragranular layers of primary visual cortex of the macaque monkey.

To determine if changes in metabolic capacity revealed by cytochrome oxidase (CO) histochemistry are related to sustained changes in energy-utilizing neuronal activity, we assayed CO levels and recorded multiunit firing rates along nearly tangential penetrations of V1 in seven adult macaque monkeys before and after single, monocular injections of TTX. Within as little as 14 h, TTX blockade began to reduce CO staining in zones of layer 4C that received dominant input from the injected eye. Since simple monocular occlusion has only minor effects on cortical CO levels (Trusk et al., 1990), the changes in activity that were specifically associated with CO depletion were isolated by comparing spike rates during monocular TTX blockade and during monocular occlusion. Five second samples of multiunit spike rate were obtained after 2-min adaptation to each of four adapting fields: black, gray, white, and textured. Results were similar for these four conditions. In layer 4C, ocular dominance zones with input from the TTX eye had ongoing spike rates that were 48% of the rates in zones with input from a normal but occluded eye. In six animals, it was possible to record activity at a single site before, during, and after the onset of TTX blockade. Background activity at these interpuff sites decreased as much as 3-fold in less than 1 h but stabilized within 3-4 h to an average of 53% of pre-TTX rates. These data support the interpretation that energy utilization linked to sustained spike rates partially regulates CO levels under normal conditions, at least in layer 4. Furthermore, changes in neuronal activity induced by retinal TTX preceded the detectable reduction in CO activity in V1 suggesting that the adjustment of CO levels was in response to the altered activity.

Effect of retinal impulse blockage on cytochrome oxidase-poor interpuffs in the macaque striate cortex: quantitative EM analysis of neurons.

One of the hallmarks of the primate striate cortex is the presence of cytochrome oxidase-rich puffs in its supragranular layers. Neurons in puffs have been classified as type A, B, and C in ascending order of cytochrome oxidase content, with type C cells being the most vulnerable to retinal impulse blockade. The present study aimed at analysing cytochrome oxidase-poor interpuffs with reference to their metabolic cell types and the effect of intraretinal tetrodotoxin treatment. The same three metabolic types were found in interpuffs, except that type B and C neurons were smaller and less cytochrome oxidase-reactive in interpuffs than in puffs. Type A neurons had small perikarya, low levels of cytochrome oxidase, and received exclusively symmetric axosomatic synapses. The largest neurons were pyramidal, type B cells with moderate cytochrome oxidase activity and were also contacted exclusively by symmetric axosomatic synapses. Type C cells medium-sized with a rich supply of large, darkly reactive mitochondria and possessed all the characteristics of GABAergic neurons. They were the only cell type that received both symmetric and asymmetric axosomatic synapses. Two weeks of monocular tetrodotoxin blockade in adult monkeys caused all three major cell types in deprived interpuffs to suffer a significant downward shift in the size and cytochrome oxidase reactivity of their mitochondria, but the effects were more severe in type B and C neurons. In nondeprived interpuffs, all three cell types gained both in size and absolute number of mitochondria, and type A cells also had an elevated level of cytochrome oxidase, indicating that they might be functioning at a competitive advantage over cells in deprived columns. However, type B and C neurons showed a net loss of darkly reactive mitochondria, indicating that these cells became less active. Thus, mature interpuff neurons remained vulnerable to retinal impulse blockade and the metabolic capacity of these cells remains tightly regulated by neuronal activity.

Effects of monocular enucleation, tetrodotoxin, and lid suture on cytochrome-oxidase reactivity in supragranular puffs of adult macaque striate cortex.

The laminar structure and cellular distribution of cytochrome-oxidase (CO) reactivity in supragranular puffs of striate cortex was examined in adult macaque monkeys surviving various periods of monocular enucleation, lid suture, and retinal impulse blockage with tetrodotoxin (TTX). Enucleation and TTX produced a rapid and severe loss in the size of the CO reactive region in puffs dominated by the removed or treated eye compared to slower and less marked reductions obtained in deprived puffs of lid-sutured monkeys. In all deprived animals, the cross-sectional areas of deprived puffs decreased most rapidly in the upper layers (2 and 3A). In long-term enucleated (60 wks) and TTX-treated (4 wks) monkeys, puff area was severely reduced in layer 3B, while reactivity in layer 3B appeared partially spared in lid-sutured monkeys. The density of the CO reaction product was significantly and evenly reduced throughout deprived puffs for all of the monkeys examined; however, this decrease was less severe in adult monkeys lid-sutured for 11 wks. Although no evidence for cell loss was obtained, all three forms of visual deprivation led to lower counts of neuronal perikarya with high levels of CO reaction product in both deprived puff and interpuff areas. This effect was less marked in the deprived puffs of monkeys lid-sutured for 2.5 and 3 yrs, suggesting recovery of CO activity in some neurons. Neurons in deprived puffs and interpuffs were generally similar in size to those in nondeprived regions, although CO-reactive cells were significantly smaller in the deprived puffs of monkeys enucleated for 28.5 or 60 wks. These results indicate that the metabolic response of neuronal elements in supragranular striate cortex depends upon the nature of the visual deficit. The partial sparing of CO reactivity in deprived puffs of lid-sutured monkeys may reflect the continued transmission of certain types of visual stimuli through a closed eyelid.

Effect of retinal impulse blockage on cytochrome oxidase-rich zones in the macaque striate cortex: I. Quantitative electron-microscopic (EM) analysis of neurons.

Our previous light-microscopic study indicates that unilateral retinal impulse blockage with tetrodotoxin (TTX) causes a reversible decrease of cytochrome oxidase (CO) in alternating rows of metabolically active zones (puffs) in the adult macaque striate cortex (Wong-Riley & Carroll, 1984b). The goal of the present study was to determine if TTX blockade adversely affects all neurons or only a subpopulation of neurons within the puffs. Three major neuronal types were identified based on mitochondrial CO activities and morphological characteristics. Type A neurons were the most prevalent, consisting of small pyramidal and nonpyramidal neurons that received only symmetrical axosomatic synapses. They had little cytoplasm and relatively low levels of CO activity, and showed the least change with TTX treatment. Type B cells were medium-to-large pyramidal neurons that received exclusively symmetrical axosomatic synapses and were moderately reactive for CO. Impulse blockage caused a decrease in mitochondrial size and packing density, but somal size remained within the control range. Type C cells were medium-sized nonpyramidal neurons contacted by both asymmetrical and symmetrical axosomatic synapses. They contained abundant darkly reactive mitochondria and presumably are metabolically the most active. This cell type suffered the greatest decrease in somal size and packing density of mitochondria, particularly the darkly reactive ones. A rare fourth cell type, type D, was a small, darkly reactive nonpyramidal variety that gave rise to somatodendritic synapses. Their low occurrence prevented statistical analysis under normal and TTX-treated conditions. These data indicate that retinal impulse blockade is most detrimental to the metabolically most active neurons in the adult primate cortical puffs. The alterations are not permanent, because the effects of TTX are fully reversible (Carroll & Wong-Riley, 1987).

Effect of retinal impulse blockage on cytochrome oxidase-rich zones in the macaque striate cortex: II. Quantitative electron-microscopic (EM) analysis of neuropil.

Unilateral retinal impulse blockage with tetrodotoxin (TTX) induces reversible shrinkage and decreased cytochrome oxidase (CO) activity in alternate rows of supragranular, CO-rich puffs in the adult macaque striate cortex (Wong-Riley & Carroll, 1984b: Carroll & Wong-Riley, 1987). The present study extended the findings to the electron-microscopic (EM) level to determine if various neuropil profiles in control puffs exhibit heterogeneous levels of CO activity, and whether specific processes were more susceptible to intravitreal TTX than others. Within the neuropil of control puffs, 60% of the total mitochondrial population resided in dendrites, and the majority of dendritic mitochondria were highly reactive for CO. Axon terminals forming symmetrical synapses also contained darkly reactive mitochondria, whereas those forming asymmetrical synapses possessed very few and mainly lightly reactive mitochondria. Unmyelinated axon trunks, myelinated axons, and glia all exhibited low levels of CO activity. Synaptic count revealed a 3:1 ratio of asymmetrical to symmetrical synapses. Intravitreal TTX for 2-4 weeks adversely affects dendrites and symmetrical terminals much more so than other neuropil processes. There was a general decrease in darkly and moderately reactive mitochondria and an increase in lightly reactive mitochondria throughout the puffs, especially in dendrites. This indicates that afferent blockade is more detrimental to processes of higher metabolic activity. Changes also differed between central and peripheral regions of puffs, and indications of axonal and synaptic reorganization were more evident in the latter. Thus, stabilization of neuronal structure and synapses appears to be activity-dependent even in the adult. A working model of these metabolic and morphological responses to chronic TTX is proposed.

Effect of heroin-conditioned auditory stimuli on cerebral functional activity in rats.

Cerebral functional activity was measured as changes in distribution of the free fatty acid [1-14C]octanoate in autoradiograms obtained from rats during brief presentation of a tone previously paired to infusions of heroin or saline. Rats were trained in groups of three consisting of one heroin self-administering animal and two animals receiving yoked infusions of heroin or saline. Behavioral experiments in separate groups of rats demonstrated that these training parameters imparts secondary reinforcing properties to the tone for animals self-administering heroin while the tone remains behaviorally neutral in yoked-infusion animals. The optical densities of thirty-seven brain regions were normalized to a relative index for comparisons between groups. Previous pairing of the tone to heroin infusions irrespective of behavior (yoked-heroin vs. yoked-saline groups) produced functional activity changes in fifteen brain areas. In addition, nineteen regional differences in octanoate labeling density were evident when comparison was made between animals previously trained to self-administer heroin to those receiving yoked-heroin infusions, while twelve differences were noted when comparisons were made between the yoked vehicle and self administration group. These functional activity changes are presumed related to the secondary reinforcing capacity of the tone acquired by association with heroin, and may identify neural substrates involved in auditory signalled conditioning of positive reinforcement to opiates.

Effects of heroin and cocaine on brain activity in rats using [1-14C]octanoate as a fast functional tracer.

Brain activity was measured autoradiographically using [1-14C]octanoate (OCTO) as a fast functional tracer in rats receiving either saline, heroin or cocaine. Regional optical densities were normalized to a relative optical density index for comparisons of OCTO labeling between treatment groups. Heroin significantly increased labeling in the dentate gyrus and cocaine increased density in the anterior cingulate cortex, globus pallidus, hippocampus CA3-4, lateral septum, hypothalamus and ventral tegmentum. Heroin and cocaine induced significant, but opposing effects in medial cortex and bed nucleus of the stria terminalis. Both drugs decreased labeling density in the nucleus accumbens and piriform cortex, and increased density in the substantia nigra, subthalamus, medial septum, claustrum, lateral hypothalamus and hippocampus CA2. These results demonstrate the ability of the OCTO method to discriminate the brief metabolic effects of different drug classes, and suggest that heroin and cocaine may activate a common functional system in the brain.

Effect of intravenous heroin and naloxone on regional cerebral blood flow in the conscious rat.

Regional cerebral blood flow (RCBF) was measured with the [14C]iodoantipyrine technique and quantitative autoradiography in awake, restrained rats shortly after intravenous injection of heroin, naloxone or naloxone before heroin. The RCBF observed in these animals was compared to those obtained in similarly treated, saline-injected rats. In an identically treated series of animals, no significant change in arterial blood gases, pH or bicarbonate was seen following any of the drug treatments at the equivalent time RCBF was determined. Blood flow increased an average 36% in 37 of the 40 areas measured 1 min after heroin injection. Significant increases were found in 21 areas including visual and piriform cortex, basal ganglia, diencephalon, limbic system, midbrain tegmentum, superior colliculus, periaqueductal gray, internal capsule and fornix. These elevations in blood flow were reversed in rats receiving heroin following naloxone pretreatment. RCBF decreased in 35 areas (mean = -12%) 4 min after naloxone injection; a 40% decrease in blood flow to entorhinal cortex was significant. These results suggest that opiate receptor stimulation by heroin increases functional activity within selected brain areas, and this effect is not limited to regions with dense populations of opiate receptors.