Modification of AgNOR staining to reveal the nucleolus in thick sections specified for stereological and pathological assessments of brain tissue.

BACKGROUND: Various stains have been devised to reveal degenerative or reactive cell phenotypes, or the disintegrative and/or neuropathic lesions associated with Alzheimer's, Parkinson's, and Pick's diseases, Down's syndrome, or chemical toxicity. Utilization of silver staining has allowed researchers to elucidate neural pathways promoting a greater understanding of the functional connections between brain regions. All of these methods employing silver can be characterized as 'directed staining technologies'. NEW METHODS: The argyrophilic proteins (AgNOR) staining protocol was modified to stain nucleoli in thick sections prepared for stereological evaluation of brain tissue. Nucleoli appeared as black dots against a pale amber background. Tissue sections were counterstained with Toluidine Blue, or reduced-strength Tyrosine Hydroxylase immunohistochemistry to facilitate visualization of basic cellular morphology and regional nucleus identification. Here, we present a modified method for nucleolar staining in free-floating thick sections of brain embedded in a gelatin matrix. The modifications in our procedure include incubation in HCl to denature ('unravel') the DNA, a bleaching step to reduce non-specific background silver staining, and counterstaining with Toluidine Blue or reduced-strength tyrosine hydroxylase immunohistochemistry. COMPARISON WITH OLD METHODS: Prior to the development of immunohistochemistry, silver staining was used primarily to identify pathological profiles and trace axon pathways; however, in many cases, a combination of silver staining and immunohistochemistry are required to fully visualize pathomorphology. The mechanism of these stains requires the binding of silver ions to cellular components and the subsequent reduction of the ions to metallic silver. Dilutions of TH primary antibody were evaluated to maximize identification of neurons and the nucleolus amongst the soma and processes present in the thick section. The use of stereology as a tool to estimate cell number has become increasingly prevalent in neuroscience experiments. As requirements for the preparation of experimental tissue have been refined, researchers have begun to use thicker sections, between 40 to 80 microns, to increase the number of optical planes available for analysis. These thick sections require modified staining protocols to assure complete penetration of stains throughout the tissue section. CONCLUSIONS: This method is particularly useful in nucleolar identification for Stereology, and automated counting methods. Use of the nucleolus avoids some of the problems associated with use of the nucleus. The nucleolus is smaller than the nucleus and is less susceptible to transection during sectioning. It has a higher density than the nucleus and is easier to visualize. It is generally darker staining than the immunohistochemical reaction product that provides the identification marker for the cells to be counted. Examples of the method in several brain sections of the rat are shown, though the method has been also proven in other mammalian models.

Cerebral beta-amyloid angiopathy in aged squirrel monkeys.

Cerebral beta-amyloid angiopathy (CAA) is an age-related disorder of the brain vasculature that is involved in up to 20% of non-traumatic cerebral hemorrhage in humans. CAA is a risk factor for cognitive decline, and may exacerbate the dementia of Alzheimer's disease. Progress in discovering the cause and potential therapies for this disorder has been hindered by the paucity of animal models, particularly models of idiopathic CAA. The squirrel monkey (Saimiri spp) develops significant CAA in the natural course of aging. To evaluate the suitability of Saimiri as a model of human CAA, we studied the distribution and composition of Abeta subtypes in CAA and parenchymal (senile plaque) deposits in the brains of aged squirrel monkeys, as well as the relationship between vascular beta-amyloid deposition and comorbid vasculopathies that occur in aged humans. Our findings show that: 1) CAA consists ultrastructurally of classical amyloid fibrils and is the principal type of cerebral beta-amyloidosis in squirrel monkeys; 2) The two primary isoforms of Abeta (Abeta40 and Abeta42) coexist in most microvascular and parenchymal lesions of Saimiri, although Abeta40 tends to predominate in larger arterioles; 3) CAA and parenchymal plaques overlap to a considerable degree in most affected brain areas, and are distributed symmetrically in the two hemispheres; 4) Both CAA and plaques are particularly abundant in rostral regions and comparatively sparse in the occipital lobe; 5) Capillaries are especially vulnerable to CAA in squirrel monkeys; and 6) When CAA is severe, it is associated with a small, but significant, increase in other vasculopathies, including microhemorrhage, fibrinoid extravasation and focal gliosis. These findings, in the context of genetic, vascular and immunologic similarities between squirrel monkeys and humans, support the squirrel monkey as a biologically advantageous model for studying the basic biology of idiopathic, age-related CAA, and for testing emerging therapies for human beta-amyloidoses such as Alzheimer's disease.

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.

Short-term, D2 receptor blockade induces synaptic degeneration, reduces levels of tyrosine hydroxylase and brain-derived neurotrophic factor, and enhances D2-mediated firing in the ventral pallidum.

Repeated treatments with neuroleptics are associated with biochemical and morphological alterations in forebrain neurons as well as an upregulation of D2-mediated changes in neuronal function. The present study evaluated the histological and physiological effects of three once-daily treatments with two chemically divergent neuroleptics, haloperidol (1 mg/kg i.p./day) and eticlopride (3 mg/kg i.p./day), measured in rats 24 h after the last injection. It was determined that this short-term antagonism of D2-like receptors induced fiber and terminal degeneration and significantly decreased tyrosine hydroxylase (TH) and brain-derived neurotrophic factor (BDNF) immunoreactivity in the ventral pallidum (VP), as determined by optical density measurements. While other forebrain regions demonstrated changes in TH and BDNF, the neurodegeneration profile was unique to the VP. This was accompanied by an enhancement in the efficacy of the D2 agonist quinpirole to increase spiking rate of VP neurons recorded in chloral hydrate-anesthetized rats. These data indicate that short-term treatments with D2 antagonists are sufficient to induce changes in the biochemical and morphological profiles uniquely within the VP. Moreover, the functional ramifications of these changes appear to include profound alterations in the way dopamine regulates neuronal activity in this region.

Neurodegeneration in Niemann-Pick type C disease mice.

Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disorder associated with intracellular cholesterol and glycolipid trafficking defects. Two separate genes, NPC1 and NPC2, have been linked to NP-C. NPC1 encodes a polytopic membrane-bound protein with a putative sterol-sensing domain. NPC2 has been recently identified as epididymal secretory glycoprotein 1. The NPC1 protein functions in the vesicular redistribution of endocytosed lysosomal cargo, but how its inactivation leads to neurodegeneration is not known. The neurological symptoms of NP-C typically appear after a period of normal early development and reflect progressive degeneration of widespread brain regions. Here we have delineated the pattern of neurodegeneration in NP-C mice, whose genetic defect has been shown to be an inactivating mutation of the mouse NPC1 gene. The results reveal a spatially and temporally specific pattern of degeneration of nerve fibers followed by degeneration of neuronal cell bodies beginning as early as day 9 and continuing throughout life. We have recently showed that in the primate brain, the NPC1 protein is localized predominantly within perisynaptic astrocytic processes. The present observations suggest that a functional disturbance in NPC1 could disrupt vesicular transport of cholesterol, glycolipids and possibly other endocytic cargo in glia, which is critical for maintaining the integrity of neurons.

Binge ethanol consumption causes differential brain damage in young adolescent rats compared with adult rats.

BACKGROUND: Adolescents respond differently to alcohol than adults. Furthermore, binge drinking in young adolescents is becoming increasingly common. METHODS: To determine if the effects of binge drinking on brain damage are different in juveniles compared with adults, the effects of a 4 day binge ethanol treatment (e.g., 4 days of 4 times per day 15% ethanol intragastrically, approximately 9-10 g/kg/day ethanol) were investigated in adolescent-juvenile rats (JVN) 35 days old and compared with adult (ADT) rats 80 to 90 days old. Brain damage was measured by using the amino cupric silver stain of de Olmos et al. (1994). RESULTS: Significant brain damage was found in both groups. The olfactory bulbs were equally damaged in both groups; however, the associated frontal cortical olfactory regions were damaged only in JVN. The anterior portions of the piriform and perirhinal cortices also were damaged only in JVN rats. Quantitation of silver-stained frontal areas in binge ethanol-treated JVN rats ranged from 400% to 1,260% of control values. For example, in anterior perirhinal cortex, silver stain increased from 48 +/- 14 to 444 +/- 114 (mm2 x 10(3) argyrophilic area; p < 0.01) in JVN control and binge ethanol-treated animals, respectively. In contrast, posterior perirhinal cortex showed greater damage in adults, being 236 +/- 76 vs. 875 +/- 135 (mm2 x 10(3) argyrophilic area; p < 0.005) in JVN and ADT, respectively. CONCLUSIONS: The young-adolescent brain shows differential sensitivity to alcohol-induced brain damage compared with adults.

Selective neurotoxic effects of nicotine on axons in fasciculus retroflexus further support evidence that this a weak link in brain across multiple drugs of abuse.

When administered continuously for several days at relatively low plasma levels, a variety of drugs of abuse with strong dopaminergic actions induce degeneration in axons traveling from the lateral habenula through the sheath of fasciculus retroflexus to midbrain monoaminergic nuclei. With some of these drugs, such as cocaine, this is virtually the only degeneration induced in brain. Nicotine given continuously also selectively induces degeneration in fasciculus retroflexus, but in the other half of the tract: the cholinergic axons running from medial habenula in the core of the tract to the interpeduncular nucleus. Fasciculus retroflexus appears to be a weak link in brain for diverse drugs of abuse when administered incessantly for several days. Alterations in this tract would be predicted to be especially important for the genesis of the symptomatology which develops during drug binges, residual effects of such binges, and the processes underlying relapse.

Application of silver degeneration stains for neurotoxicity testing.

Silver staining procedures have been used in numerous ways to render a variety of physical and biological features visible. In biological tissue, histologic protocols use silver to visualize diverse structures or features, such as reticulin, melanin, fungi, chromosome bands, nucleolar organizing regions, and different features in the nervous system. A comparison of the specific steps in these protocols indicates that the silver is "directed" to stain any given feature by the type of fixation, the pretreatment ("mordanting"), the composition of the silver-containing solution(s), and the form of development (reduction). Since the mechanisms of staining have not been understood historically (nor are they now), each method was developed by trial and error. Keystone methods such as those of Bodian and Bielschowsky exploit the nervous system's affinity for silver (argyrophilia). The beginning of a new era in brain research came with the recognition that distinct silver-impregnated morphologic changes occurring in damaged axons could be used for tracing axon pathways in experimental animals with specifically placed lesions. Improvements in staining methods used to selectively impregnate the disintegrating axons but to leave normal axons unstained were achieved by Nauta and Gygax (early workers with these procedures) and spawned a host of method variations known as the "Nauta" methods. Of these, the Fink-Heimer and de Olmos cupric-silver methods were able to unambiguously demonstrate disintegrating synaptic terminals, thereby allowing complete tracing of axon pathways. The late 1970s and 1980s witnessed innovative applications of these techniques. The silver methods once used to trace axon pathways became indicators of the extreme endpoint of neurotoxicity: disintegrative degeneration of neurons induced by neurotoxic chemicals that were administered systemically. The hallmark of neurotoxic substances is the selectivity with which each destroys specific populations or subpopulations of neurons. The high contrast and sensitivity of the silver degeneration stains greatly facilitate the screening process to detect these affected populations, especially when there is no basis for knowing where in the brain to look for damage. More recently, in addition to expanded use in screening for neurotoxic effects, the silver degeneration stains are being used to chart the neuron populations undergoing programmed cell death in the developing brain. Other newly developed silver methods have been refined to show nondisintegrative degeneration, such as the plaques,and tangles of Alzheimer's disease.

MK-801 neurotoxicity in cupric silver-stained sections: lesion reconstruction by 3-dimensional computer image analysis.

Routine histopathologic evaluation of the brain (paraffin embedding, hematoxylin and eosin staining) makes it difficult for an investigator to identify the overall location and relative extent of lesions as they relate to neural substructures. Moreover, it is very difficult to convey this information to others who are less familiar with neuroanatomy. This study combined a 3-dimensional imaging program with a cupric silver stain for neuronal degeneration in order to determine the location and extent of a focal lesion produced by MK-801 (dizocilpine maleate), a glutamate receptor antagonist that induces necrosis in a small population of neurons in the cortex of rats. A male Sprague-Dawley rat was treated with a subcutaneous dose of MK-801 (10 mg/kg) and was perfused with fixative through the left ventricle 3 days after treatment, a time point known to reveal maximal neurotoxic effects. The brain was embedded in a gelatin matrix, frozen, and serially sectioned at a thickness of 40 microm. The cupric silver method of de Olmos was used to stain frozen sections at 320-microm intervals. Using a color charged-couple device (CCD) camera and a macro lens, a series of 2-dimensional images, which encompassed the entire rostral to caudal extent of the brain, was captured. A computer program was written to define internal and external boundaries in these 2-dimensional images. Then, 3-dimensional reconstructions were generated on a Silicon Graphics workstation using IRIS "Explorer." The quality of the 3-dimensional reconstructions allowed for easy identification of various neural substructures while clearly revealing the exact location and extent of the resulting necrotic neurons that were positively identified by the cupric silver stain. This 3-dimensional lesion reconstruction method provides a powerful tool for conveying spatial information about the nature of neurotoxic lesions in the brain. In addition, it may be used to investigate further dose-response relationships and the effects of other neurotoxicants.

Region-specific astrogliosis in brains of mice heterozygous for mutations in the neurofibromatosis type 1 (Nf1) tumor suppressor.

Brains from human neurofibromatosis type 1 (NF1) patients show increased expression of glial fibrillary acidic protein (GFAP), consistent with activation of astrocytes (M.L. Nordlund, T.A. Rizvi, C.I. Brannan, N. Ratner, Neurofibromin expression and astrogliosis in neurofibromatosis (type 1) brains, J. Neuropathol. Exp. Neurology 54 (1995) 588-600). We analyzed brains from transgenic mice in which the Nf1 gene was targeted by homologous recombination. We show here that, in all heterozygous mice analyzed, there are increased numbers of astrocytes expressing high levels of GFAP in medial regions of the periaqueductal gray and in the nucleus accumbens. More subtle, but significant, changes in the number of GFAP positive astrocytes were observed in the hippocampus in 60% of mutant mice analyzed. Astrocytes with elevated GFAP were present at 1 month, 2 months, 6 months and 12 months after birth. Most brain regions, including the cerebellum, basal ganglia, cerebral cortex, hypothalamus, thalamus, cortical amygdaloid area, and white matter tracts did not show any gliotic changes. No evidence of degenerating neurons was found using de Olmos' cupric silver stain. We conclude that Nf1/nf1 mice provide a model to study astrogliosis associated with neurofibromatosis type 1.

Neuronal degeneration in canine narcolepsy.

Narcolepsy is a lifelong illness characterized by persistent sleepiness, hypnagogic hallucinations, and episodes of motor paralysis called cataplexy. We have tested the hypothesis that a transient neurodegenerative process is linked to symptom onset. Using the amino-cupric silver stain on brain sections from canine narcoleptics, we found elevated levels of axonal degeneration in the amygdala, basal forebrain (including the nucleus of the diagonal band, substantia innominata, and preoptic region), entopeduncular nucleus, and medial septal region. Reactive neuronal somata, an indicator of neuronal pathology, were found in the ventral amygdala. Axonal degeneration was maximal at 2-4 months of age. The number of reactive cells was maximal at 1 month of age. These degenerative changes precede or coincide with symptom onset. The forebrain degeneration that we have observed can explain the major symptoms of narcolepsy.

Imaging of boron in tissue at the cellular level for boron neutron capture therapy.

Glioblastoma multiforme, and other tumors involving the brain, are undergoing experimental treatment with a promising new technique: boron neutron capture therapy (BNCT). BNCT relies on the capture of thermal neutrons by boron deposited biochemically in the tumor and the subsequent fission of the boron into energetic lithium ions and alpha particles. An important requirement for improved BNCT is the development of more selective boron delivery mechanisms. The ability to image the boron concentration in tissue sections and even inside individual cells would be an important aid in the development of these delivery mechanisms. We have compared both sputter-initiated resonance ionization microprobe (SIRIMP), which combines resonance ionization with a high-energy pulsed focused sputter ion beam and mass spectrometric detection of ions, with laser atomization resonance ionization microprobe (LARIMP), which uses a laser pulse instead of an ion pulse for the atomization process, to determine their characteristics in locating and quantifying boron concentrations as a function of position in tissues obtained from a rat which had been infused with a BNCT drug. The data show that the SIRIMP/LARIMP techniques are well suited for quantitative and ultrasensitive imaging of boron trace element concentrations in biological tissue sections. The LARIMP mode could be used to quickly determine the spatial boron concentration with intercellular resolution over large areas down to the low nanograms-per-gram level, while the SIRIMP mode could be used to determine the spatial boron concentration and its variability in intracellular areas.

Distribution of bismuth in the brain after intraperitoneal dosing of bismuth subnitrate in mice: implications for routes of entry of xenobiotic metals into the brain.

Bismuth (Bi) can produce neurotoxic effects in both humans and animals under certain dosing conditions, but little else is known about the effects of Bi in the brain. In the present study we determined the distribution of Bi in the brains of adult female Swiss-Webster mice 4, 7, 14, 21 and 28 days after a single 2500 mg/kg i.p. injection of Bi subnitrate (BSN), which establishes a depot of absorbable Bi and produces morphological signs of neurotoxicity. Sections of brains were processed by autometallographic (AMG) procedures that produced silver grains at the site of Bi localization (AMGBi). Ventricular dilation was observed in all BSN-dosed mice. Among treated mice there were marked interanimal differences in the absolute amount of AMGBi, but consistent regional and cellular patterns of AMGBi were observed. AMGBi was observed in many cell types in brain regions adjacent to fenestrated blood vessels of the circumventricular organs (CVOs) and olfactory epithelium. Prominent intrasomal AMGBi was observed in nuclei containing large cell bodies, including cranial motor neurons innervating somatic muscle, lateral vestibular and red nucleus and pontine/medullary reticular nuclei. In the hypothalamus, the supraoptic and paraventricular nuclei demonstrated the densest AMGBi. In the cerebellum, Purkinje and granule cell layers with the densest AMGBi were in folia adjacent to the fourth ventricle. In the hippocampus, AMGBi was densest in the fasciola cinerum, polymorph cells of the dentate gyrus, and pyramidal cell layer of the CA3 regions. Neuropil of subcortical auditory nuclei (cochlear nucleus, trapezoid body, lateral lemniscus and nucleus of lateral lemniscus, medial geniculate nucleus and inferior colliculus) had a high density of AMGBi. Among nonneuronal cells, ependyma and meninges lining the ventricular and subarachnoid spaces were labeled extensively. Glial labeling was prominent adjacent to CVOs, in subependymal regions, and in fiber tracts. Presumptive perivascular cells lining large blood vessels had extremely dense AMGBi as early as 4 days after dosing. Smaller blood vessels had moderate AMGBi. However, in regions (e.g. cerebral cortex, striatum) known to have low brain Bi levels after i.p. dosing, vascular deposits accounted for most of the AMGBi. Several animals had foci of AMGBi which suggested that vascular or perivascular aberrations may have contributed to the unusually dense accumulations. The results of the present studies indicate that Bi accumulates predictably in certain regions and cell types. The pattern of regions and cells with the highest AMGBi accumulations is very similar to pattern reported for other xenobiotic metals (i.e. mercury, silver, gold), and supports the hypothesis that these metals may share some mechanisms for entry, distribution and storage in the brain.

Integrated evaluation of central nervous system lesions: stains for neurons, astrocytes, and microglia reveal the spatial and temporal features of MK-801-induced neuronal necrosis in the rat cerebral cortex.

Routinely processed, hematoxylin and eosin (H&E)-stained slides are typically used to assess the morphologic integrity of the central nervous system in neurotoxicity safety studies. However, the value of special stains for improving neuropathologic evaluations during the assessment of neurotoxicity has been emphasized in the neuroscience literature and by regulatory agencies. The primary objective of the present study was to characterize the spatial and temporal changes in neurons, astrocytes, and microglia after dizocilpine maleate (MK-801)-induced focal neuronal necrosis in the posterior cingulate/retrosplenial (PC/RS) cortex of the rat. A secondary objective was to evaluate the application of special stains and a novel sectioning procedure for detecting neurotoxicity. Sixty adult male Sprague-Dawley rats were treated with sterile water vehicle or 10 mg/kg MK-801 and perfused through the left ventricle (pumped at 65 mm Hg pressure) with 10% neutral buffered formalin or 4% paraformaldehyde at 4 hr and on days 1, 3, 7, 14, and 28 after treatment. For light microscopic evaluation, brain sections were stained with H&E, a special cupric-silver (CS) stain that selectively impregnates degenerating neurons and makes them readily evident, glial fibrillary acidic protein (GFAP) immunohistochemistry for astrocytes, and Griffonia simplicifolia isolectin B4(GSA) histochemistry for microglia. Brains perfusion-fixed with 4% paraformaldehyde were prepared for CS staining with a novel frozen-sectioning procedure for multiple embedding in a composite gelatin block. In H&E sections from treated rats, necrotic nerve cell bodies were observed in PC/RS cortical layers 3 and 4 on days 1, 3, 7, and 14, but not on day 28. These necrotic neurons required high magnification for detection (x20 objective, x10 ocular). In contrast, degenerating neurons selectively stained with CS were observed in the same location as necrotic neurons seen with H&E but at low magnification (x2 objective, x10 ocular). Cupric-silver staining showed details not seen with H&E, including dendritic and axonal degeneration with progressive fragmentation. Beginning on day 3, GFAP immunohistochemistry revealed hypertrophic astrocytes in a diffuse pattern throughout the region of cell body necrosis, a change that persisted throughout the study. However, GSA lectin histochemistry identified a few reactive microglia on day 1 in a multifocal pattern throughout the region of cell body necrosis. Reactive microglia were observed on days 3, 7, and 14, but not on day 28. Glial changes observed with H&E staining were limited to an increase in the cellularity of glial cell nuclei in the area of neuronal necrosis. This study provides a comprehensive and integrated view of the temporal changes occurring in neurons, astrocytes, and microglia during acute neurotoxic injury. Moreover, advantages for using new staining and sectioning methodologies to enhance the toxicologic evaluation of the central nervous system are demonstrated.

Activation of astrocytes during epileptogenesis in the absence of neuronal degeneration.

The issue of whether neuronal degeneration is a primary factor in activation of astrocytes during epileptogenesis was addressed using the kindling model of epilepsy. No degenerative changes specific to the kindling process were observed in brain sections from kindled animals, sampled from the olfactory bulbs through to cerebellum and processed with the degeneration-sensitive cupric silver stain. Also, examination of lectin-stained sections did not reveal any reactive microglia. At the same time, reactive astrocytes, as judged by an increase in glial fibrillary acidic protein immunoreactivity and a de novo vimentin immunoreactivity, were prominent in amygdala, piriform cortex, entorhinal cortex and hippocampus. These results suggest that loss of neurones is not a prerequisite for establishment of epilepsy-prone state, that seizures of short duration do not necessarily result in neuronal death, and that in kindling, astrocytes are activated by factors that are not related to neuronal degeneration, but which are likely associated with abnormal neuronal activity.

Neuroexcitatory and neurotoxic actions of the amnesic shellfish poison, domoic acid.

We have investigated the action of domoic acid in the mouse brain following systemic exposure. Domoic acid increased c-fos mRNA within 15 min and its translational product (c-Fos) within 1 h. c-Fos immunoreactivity was most prominent in the hippocampal formation, lateral septal nucleus, olfactory bulb, area postrema and the nucleus of the solitary tract. We next examined irreversible toxic effects of domoic acid. Domoic acid caused extensive degeneration in CA1-2 of the hippocampus, lateral septal nucleus and olfactory bulb. No degeneration was evident in the dentate gyrus or brain stem. These studies demonstrate that domoic acid has only neuroexcitatory effects on brain stem regions associated with visceral function whereas it has permanent neurotoxic effects on brain regions associated with memory formation.

Phylogeny through brain traits: more characters for the analysis of mammalian evolution.

We have assembled data on nine brain traits, in addition to the fifteen we have previously described, which provide new evidence for assessing mammalian relationships. States of these characters are tabulated as they occur in each of 152 mammalian species, providing data in numerically ordered form, useful for multiple analyses of phylogenetic relationships in programs which take into account variations in several different characters simultaneously. Derived states of each of the nine traits are characteristic of certain restricted groups of mammals; (1) mirroring of the complete SI body representation in isocortex (anthropoid primates); (2) loss of the accessory olfactory bulbs (sirenians, cetaceans, most bats, catarrhine primates); (3) Rindenkerne, clumps of cell bodies in layer 6 of cerebral cortex (sirenians); (4) posteriorly-pointing digits in the SI body representation (bats, both mega- and micro-); (5) equivalent tectopetal connections to the anterior colliculus of one side from both retinas, rather than predominantly from the contralateral retina (primates and megabats); (6) loss of lamination in dorsal cochlear nuclei (anthropoid primates, bats, seals, sirenians, cetaceans); (7) separation of claustrum from cerebral cortex (diprotodont marsupials, carnivores, artiodactyls, perissodactyls, hyracoids, cetaceans and primates), (8) presence of a complete secondary (SII) somatic sensory region of cerebral cortex (therians-all extant mammals other than monotremes), and (9) presence of a distinct external cuneate nucleus among the nuclei of the dorsal columns (all mammalian groups except monotremes and sirenians). Two examples of phylogenetic trees derived from these data are presented. These sample trees maintain the segregation of the monotremes and the marsupials, and the basic dichotomy of placentals seen in our earlier trees based entirely on brain data. They also show: an orderly sequence of bifurcations (rather than the commonly seen multifurcation near the base of the radiation) in the reconstruction of placental relationships; extremes of derivation for the Cetacea, the Chiroptera, and the Sirenia (in concordance with trees based on other data); a ferungulate association of Carnivora, Perissodactyla, Artiodactyla, Hyracoidea and Sirenia; and an assemblage of related Dermoptera, Primates, Scandentia, and Chiroptera which in this model also includes Insectivora and Macroscelidea. Analyses based on brain characters can reinforce conclusions based on other data, while at the same time introducing new ideas about relationships. Neural traits provide a source of data independent of those commonly used in phylogenetic analysis, and are extremely valuable for testing old hypotheses and for introducing new ones.(ABSTRACT TRUNCATED AT 400 WORDS)

Dissimilar patterns of degeneration in brain following four different addictive stimulants.

Patterns of neural degeneration were compared following continuous administration of four drugs of addiction, each of which induces model psychoses in chronic addicts. D-amphetamine (D-Amph), cocaine (Coc), or phencyclidine (PCP) were administered continuously over a 5-day period. Both D-Amph and Coc induced pronounced degeneration in fasciculus retroflexus, but only D-Amph further induced substantial degeneration in striatum. Continuous PCP produced entirely different degeneration largely confined to the posterior entorhinal cortex, ventral dentate gyrus, and cingulate cortex. Methamphetamine (Meth) administered in the very high dose but less prolonged drug regimen often employed in studies of dopamine toxicity induced pronounced degeneration in striatum, but widespread degeneration in many other regions as well. These results indicate that drugs of abuse with psychotomimetic properties induce distinctively different patterns of neural degeneration, a finding with implications for theories of addiction and psychosis. They predict two different anatomical loci for alterations in psychosis: fasciculus retroflexus and ventral parahippocampus and hippocampus.

Strategies for assessing neurotoxicity.

The ultimate end point among the various measures of neurotoxicity is the death of neurons, since they do not regenerate. Experiments that accurately assess this degree of neurotoxicity must utilize a sensitive means of detection and control for the factors that are variables in the expression of neurotoxicity. Neurotoxic substances have the characteristic trait that each selectively destroys specific populations or subpopulations of neurons. The mechanisms underlying this selectivity are generally unknown. Without a priori knowledge of where in the brain to look, it is prudent to employ a histological procedure that imparts an easily detected marker to degenerating neuronal components. For example, the so-called "reduced silver" stains specifically impregnate degenerating neural elements. From studies of known neurotoxins, several factors have been shown to influence neurotoxicity: 1) species and strains; 2) age; 3) sex; 4) dose rate (acute vs. chronic); 5) survival time; and 6) sampling interval in the brain. Failure to include these factors in a screening paradigm could yield false-negative results and, thereby, invalidate extrapolations made to man.