Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation.

VIDEO ABSTRACT: The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry.

Cytoarchitectural characteristics of hippocampal CA1 pyramidal neurons of rats, four months after global cerebral ischemia and progesterone treatment.

PURPOSE: To analyze the cytoarchitectural characteristics of the remaining pyramidal neurons in the hippocampal CA1 subfield of rats, four months after global cerebral ischemia (GCI) and progesterone treatment. METHODS: Dendritic arborization, and density and shape of the dendritic spines of CA1 pyramidal neurons in brains of intact rats, or rats submitted 120 days earlier to GCI and treatment with progesterone (8 mg/kg) or its vehicle, at 15 min, and 2, 6, 24, 48, and 72 h after the onset of reperfusion, were analyzed in samples processed by a modified Golgi method. RESULTS: Few impregnated CA1 pyramidal neurons were identified in the ischemic vehicle-treated rats, with a short apical dendrite devoid of bifurcations and dendritic spines. In contrast, the remaining CA1 pyramidal neurons sampled from ischemic progesterone-treated rats showed sinuously branched dendrites with similar number of bifurcations and whole density of spines, and higher proportional density of mushroom spines than those in the intact group. CONCLUSIONS: These cytoarchitectural characteristics may underlie the long-term preservation of place learning and memory functions seen after ischemia and progesterone neuroprotective treatment, possibly compensating for the severe reduction in neuronal population.

Nerve cells immunoreactive for p62 in select hypothalamic and brainstem nuclei of controls and Parkinson's disease cases.

The protein p62 plays an important role in the proteasomal and/or autophagic clearance of misfolded and aggregation-prone proteins. Immunoreactivity for p62, however, not only characterizes pathological proteinaceous inclusions but also occurs in the form of homogeneous nerve cell labeling in brains of both healthy and diseased individuals, e.g., in the vagal dorsal motor nucleus and other subcortical nuclei. In sporadic Parkinson's disease (PD), the pathological process initially involves preganglionic neurons of the parasympathetic and sympathetic system and probably advances caudo-rostrally from there along the neuroaxis. Since all subsequently affected nuclei (lower raphe nuclei, magnocellular reticular formation, locus coeruleus, and central subnucleus of the amygdala) generate descending projections that terminate in the vagal dorsal motor nucleus and intermediolateral column, it has been conjectured that retrograde axonal transport and transsynaptic transmission of a pathogen contribute to the pathogenesis of PD. The hypothalamic paraventricular nucleus also sends projections to the preganglionic nuclei under consideration and, thus, should belong to the nuclei endangered by the pathological process. However, it remains uninvolved for the duration of the disorder. For this reason, we performed a retrospective study of the relevant nuclei in a cohort of 36 individuals, including 17 with clinically documented PD, one case with incidental Lewy body disease (ILBD), and 18 controls using p62-immunocytochemistry. Remarkably, the neurosecretory cells of the paraventricular nucleus were among the sites showing homogeneous p62-immunolabeling with the greatest consistency. Its p62-immunoreactive profile may indicate that the hypothalamic paraventricular nucleus is somehow capable of effectively metabolizing misfolded proteins and/or preventing their aggregation.

Profiles of serotonin receptors in the developing human thalamus.

The critical importance of the thalamus and its serotonergic innervation with respect to neuropsychiatric syndromes is increasingly recognized. This study investigates the localization of serotonin (5-hydroxytryptamine; 5-HT) receptors by immunohistochemistry in the thalamic nuclei of human fetuses aged 21 to 32 weeks of gestation. Results indicate that, already at 21 weeks of gestation, two 5-HT receptors are present in the dorsomedial nucleus of the developing thalamus: 5-HT2A receptors are localized in neurons and 5-HT2C receptors in fibers. By 31 and 32 weeks of gestation, 5-HT1A and 5-HT4 receptors are also detected in neuronal fibers of the same nucleus. At this later developmental stage, the percentage of 5-HT2A labeled neurons has significantly increased in the dorsomedial nucleus, and 5-HT2C positive neurons are observed in the centromedian and lateroventral thalamic nuclei as well. In contrast, neither neuronal cells nor fibers display any immunoreactivity for 5-HT3 or 5-HT6 receptors at any of the ages examined. Our observation that 5-HT1A, 5-HT2A, 5-HT2C and 5-HT4 receptors are present in the human thalamus prenatally indicates that 5-HT may play a role during fetal development. Disrupted development of the thalamic serotonergic system during this gestational period may contribute to the pathophysiology of neuropsychiatric disorders.

APLP1, Alzheimer's-like pathology and neurodegeneration in the frontal cortex of manganese-exposed non-human primates.

Chronic manganese (Mn) exposure produces a neurological syndrome with psychiatric, cognitive and parkinsonian features. Gene expression studies in the frontal cortex of Cynomolgus macaques exposed to different doses of Mn showed gene expression changes associated with cell cycle regulation, DNA repair, apoptosis, ubiquitin-proteasome system, protein folding, cholesterol homeostasis, axonal/vesicular transport and inflammation. Amyloid-beta (A-beta) precursor-like protein 1 (APLP1), a member of the amyloid precursor family, was the most highly up-regulated gene. Immunohistochemistry confirmed increased APLP1 expression and revealed the presence of A-beta diffuse plaques. Cortical neurons and white matter fibers from Mn-exposed animals exhibited accumulation of silver grains indicative of on-going degeneration. Cortical neurons also expressed nuclear hypertrophy, intracytoplasmic vacuoles, and apoptotis stigmata. The levels of p53 were increased in neurons and glial cells in Mn-exposed tissue. Analysis of another amyloidogenic protein, alpha-synuclein, also exhibited aggregation in the gray and white matter from Mn-exposed animals. In summary, chronic Mn exposure in non-human primates produces a cellular stress response leading to neurodegenerative changes, diffuse A-beta plaques and alpha-synuclein aggregation in the frontal cortex. These changes may help explain the cognitive and working memory deficits expressed by these animals.

Spontaneous recurrent seizures after status epilepticus induced by soman in Sprague-Dawley rats.

PURPOSE: Exposure to toxic levels of organophosphorus (OP) nerve agents can lead to seizures, respiratory failure, and, if untreated, death. The cholinesterase inhibitor soman belongs to the class of OP nerve agents and can cause status epilepticus (SE) and brain damage due to neuroexcitotoxicity. In the present study, electroencephalographic seizures are characterized through telemetry implants in rats exposed to soman, followed by treatment with therapeutics similar to those administered after nerve agent exposure. METHODS: Cortical electroencephalography (EEG), motor activity and body temperature were recorded continuously for 2 days preexposure and 15 days postexposure to verify the occurrence of spontaneous recurrent seizures (SRS) after soman exposure. RESULTS: Behavioral seizures were monitored and the latency to SE was 7.8 ± 4.0 min after exposure. Among the rats that showed SE, approximately 90% had prolonged seizures within the initial 3 days after soman exposure. Five percent of the rats developed stage 1 seizures, 16% stage 2, 23% stage 3, 18% stage 4, and 38% stage 5. Seventy-nine percent of the rats presented SE and epileptiform-like discharges several days after SE, and 28.9% of those with SE experienced electrographic SRS. The latency to the appearance of SRS ranged from 5-10 days. Fiber degeneration evaluated through silver staining revealed damage in cortical and subcortical areas directly correlated with SE. DISCUSSION: The presence of SRS after seizures induced by soman highlights the importance of quantifying SRS in studies where the objective is to find new therapeutics against soman-induced seizures.

Withania somnifera prevents morphine withdrawal-induced decrease in spine density in nucleus accumbens shell of rats: a confocal laser scanning microscopy study.

Opiate withdrawal is associated with morphological changes of dopamine neurons in the ventral tegmental area and with reduction of spine density of second-order dendrites of medium size spiny neurons in the nucleus accumbens shell but not core. Withania somnifera has long been used in the Middle East, Africa, and India as a remedy for different conditions and diseases and a growing body of evidence points to its beneficial effects on a number of experimental models of neurological disorders. Recently, many studies focused on the potential neuritic regeneration and synaptic reconstruction properties of its methanolic extract and its constituents (withanolides). This study investigates whether morphine withdrawal-induced spine reduction in the nucleus accumbens is affected by the administration of a Withania somnifera extract. To this end, rats were chronically treated with Withania somnifera extract along with morphine or saline and, upon spontaneous (1 and 3 days) or pharmacologically precipitated withdrawal, their brains were fixed in Golgi-Cox stain for confocal microscopic examination. In a separate group of animals, Withania somnifera extract was administered during three days of spontaneous withdrawal. Withania somnifera extract treatment reduced the severity of the withdrawal syndrome when given during chronic morphine but not during withdrawal. In addition, treatment with Withania somnifera extract during chronic morphine, but not during withdrawal, fully prevented the reduction of spine density in the nucleus accumbens shell in spontaneous and pharmacologically precipitated morphine withdrawal. These results indicate that pretreatment with Withania somnifera extract protects from the structural changes induced by morphine withdrawal potentially providing beneficial effects on the consequences related to this condition.

Neonatal fluoxetine exposure affects the neuronal structure in the somatosensory cortex and somatosensory-related behaviors in adolescent rats.

Selective serotonin reuptake inhibitor (SSRI)-type antidepressants are often prescribed to depressive pregnant women for their less adverse side effects. However, growing evidences have shown increased congenital malformations and poor neonatal adaptation in the perinatal SSRI-exposed human infants as well as animal pups. In this study, we examined the effects of early exposure of fluoxetine, the most popular SSRI-type antidepressant, on the developing somatosensory system. Physiological saline or fluoxetine (10 mg/kg) was subcutaneously injected into neonatal rats from P0 to P6. Somatosensory-related behaviors were examined in adolescence (P30-P35). Morphological features of the primary somatosensory cortex were checked at P7 and P35. The tactile and thermal perceptions as well as locomotor activity were affected by neonatal fluoxetine treatment. At the morphological level, the number of branch tips of thalamocortical afferents to the somatosensory cortex was reduced in the fluoxetine-treated rats. Furthermore, the spiny stellate neurons in the layer IV somatosensory cortex had reduced dendritic span and complexity with fewer branches, shorter dendritic length, and smaller dendritic field. The spine density of spiny stellate neurons was significantly reduced whereas the spine length of mushroom- and branched-type was increased. Taken together, these results indicate that neonatal fluoxetine administration has long-lasting effects on the function and structure in the somatosensory system. Sensory information processing may be disturbed in the neonatal fluoxetine-treated animals due to the structural deformation in the thalamocortical afferents and dendritic structures of the spiny stellate neurons in the layer IV somatosensory cortex.

Estradiol induces hypothalamic dendritic spines by enhancing glutamate release: a mechanism for organizational sex differences.

The naturally occurring sex difference in dendritic spine number on hypothalamic neurons offers a unique opportunity to investigate mechanisms establishing synaptic patterning during perinatal sensitive periods. A major advantage of the rat as a model of sexual differentiation is that treatment of neonatal females with estradiol will permanently induce the male phenotype. During the development of other systems, exuberant innervation is followed by activity-dependent pruning necessary for elimination of spurious synapses. In contrast, we demonstrate that estradiol-induced organization in the hypothalamus involves the induction of new synapses on dendritic spines. Activation of estrogen receptors by estradiol triggers a nongenomic activation of PI3 kinase that results in enhanced glutamate release from presynaptic neurons. Subsequent activation of ionotropic glutamate receptors activates MAP kinases, thereby inducing dendritic spine formation. These results reveal a transneuronal mechanism by which estradiol acts during a sensitive period to establish a profound and lasting sex difference in hypothalamic synaptic patterning.

Glutamate AMPA/kainate receptors, not GABA(A) receptors, mediate estradiol-induced sex differences in the hypothalamus.

Sex differences in brain morphology underlie physiological and behavioral differences between males and females. During the critical perinatal period for sexual differentiation in the rat, gonadal steroids act in a regionally specific manner to alter neuronal morphology. Using Golgi-Cox impregnation, we examined several parameters of neuronal morphology in postnatal day 2 (PN2) rats. We found that in the ventromedial nucleus of the hypothalamus (VMN) and in areas just dorsal and just lateral to the VMN that there was a sex difference in total dendritic spine number (males greater) that was abolished by treating female neonates with exogenous testosterone. Dendritic branching was similarly sexually differentiated and hormonally modulated in the VMN and dorsal to the VMN. We then used spinophilin, a protein that positively correlates with the amount of dendritic spines, to investigate the mechanisms underlying these sex differences. Estradiol, which mediates most aspects of masculinization and is the aromatized product of testosterone, increased spinophilin levels in female PN2 rats to that of males. Muscimol, an agonist at GABA(A) receptors, did not affect spinophilin protein levels in either male or female neonates. Kainic acid, an agonist at glutamatergic AMPA/kainate receptors, mimicked the effect of estradiol in females. Antagonizing AMPA/kainate receptors with NBQX prevented the estradiol-induced increase in spinophilin in females but did not affect spinophilin level in males.

Effect of N-methyl-d-aspartate receptor blockade on plasticity of frontal cortex after cholinergic deafferentation in rat.

Cholinergic projections from the nucleus basalis play a critical role in cortical plasticity. For instance, cholinergic deafferentation increases dendritic spine density and expression of the GluR1 subunit of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor in frontal cortex. Acetylcholine modulates glutamatergic activity in cortex, and the N-methyl-d-aspartate subtype of glutamate receptor plays a role in many forms of synaptic plasticity. To assess whether N-methyl-d-aspartate receptors mediate the increase in GluR1 and spine density resulting from cholinergic deafferentation, we examined the effect of N-methyl-d-aspartate receptor blockade on nucleus basalis lesion-induced upregulation of GluR1 and dendritic spines. Rats received unilateral sham or 192 IgG saporin lesions of the nucleus basalis. Half of the rats in each group were treated with the N-methyl-d-aspartate antagonist MK-801 or phosphate-buffered saline. Two weeks later, brains were processed for either immunohistochemical staining of the GluR1 subunit or Golgi histology. In layer II-III of frontal cortex, neuronal GluR1 expression was assessed using an unbiased stereological technique, and spine density was assessed on basilar branches of pyramidal neurons. GluR1 expression was increased after nucleus basalis lesion, but this increase was prevented with MK-801. Similarly, nucleus basalis-lesioned animals had significantly higher spine densities, and this effect was also prevented by treatment with MK-801. Thus, N-methyl-d-aspartate receptor blockade prevented both GluR1 and spine density upregulation following cholinergic deafferentation, suggesting that these effects are N-methyl-d-aspartate receptor-mediated.

Protection with estradiol in developmental models of apoptotic neurodegeneration.

Medical measures that bear no known danger for the adult brain may trigger active neuronal death in the developing brain. Pharmacological blockade of N-methyl-D-aspartate or activation of GABA(A) receptors, blockade of voltage-dependent sodium channels, and oxygen induce widespread apoptotic neurodegeneration during the period of rapid brain growth in rodents. Because such measures are often necessary in critically ill infants and toddlers, search for adjunctive neuroprotective strategies is warranted. We report that 17beta-estradiol ameliorates neurotoxicity of drugs that block N-methyl-D-aspartate receptors, activate GABA(A) receptors, or block voltage-gated sodium channels and reduces neurotoxicity of oxygen in the infant rat brain. This neuroprotective effect is reversed by tamoxifen and cannot be reproduced by 17alpha-estradiol. 17Beta-estradiol did not affect GABA(A) or N-methyl-D-aspartate currents in hippocampal neuronal cultures, indicating that direct modulation of neurotransmitter receptor/channel properties by this compound cannot explain neuroprotective effect. 17beta-Estradiol did, however, increase levels of phosphorylated extracellular signal-regulated kinase 1/2 and AKT, suggesting that activation of these prosurvival proteins may represent one mechanism for its neuroprotective action. 17Beta-estradiol and related compounds may be neuroprotective agents suitable for use in critically ill infants and toddlers. Its supplementation may particularly help to improve neurocognitive outcome in preterm infants who are prematurely deprived of maternal estrogen.

The lateral ventromedial thalamic nucleus spreads nociceptive signals from the whole body surface to layer I of the frontal cortex.

Neurons within the lateral ventromedial thalamic nucleus (VMl) convey selectively nociceptive information from all parts of the body. The present experiments were performed in rats and were designed to determine the organization of cortical projections from VMl neurons. In a first series of experiments, these cells were characterized electrophysiologically and individually labelled in a Golgi-like manner following juxtacellular electrophoresis of biotin-dextran. In a second experimental series, topical applications of the tracers fluorogold and tetramethylrhodamine-labelled dextran were placed into both the rostral-most and caudal areas of layer I of the dorsolateral frontal cortex, respectively. All VMl nociceptive neurons were fusiform and their full dendritic arborizations were bipolar, extending in the lateromedial axis. VMl cells are thus particularly well located to receive widespread nociceptive inputs via a brainstem link, viz. the medullary subnucleus reticularis dorsalis. VMl neurons driven by 'whole body' nociceptive receptive fields project to the rostral part of the layer I of the dorsolateral frontal cortex. These projections are widespread because double-labelling data showed a great number of VMl neurons labelled from both rostral and caudal dorsolateral cortices. The VMl comprises a homogeneous, organized subset of thalamic neurons that allow any signals of pain to modify cortical activity in a widespread manner, by interacting with the entire layer I of the dorsolateral neocortex.

Long-distance transport of L-ascorbic acid in potato.

BACKGROUND: Following on from recent advances in plant AsA biosynthesis there is increasing interest in elucidating the factors contributing to the L-ascorbic acid (AsA) content of edible crops. One main objective is to establish whether in sink organs such as fruits and tubers, AsA is synthesised in situ from imported photoassimilates or synthesised in source tissues and translocated via the phloem. In the current work we test the hypothesis that long-distance transport is involved in AsA accumulation within the potato tuber, the most significant source of AsA in the European diet. RESULTS: Using the EDTA exudation technique we confirm the presence of AsA in the phloem of potato plants and demonstrate a correlation between changes in the AsA content of source leaves and that of phloem exudates. Comparison of carboxyflourescein and AgNO3 staining is suggestive of symplastic unloading of AsA in developing tubers. This hypothesis was further supported by the changes in AsA distribution during tuber development which closely resembled those of imported photoassimilates. Manipulation of leaf AsA content by supply of precursors to source leaves resulted in increased AsA content of developing tubers. CONCLUSION: Our data provide strong support to the hypothesis that long-distance transport of AsA occurs in potato. We also show that phloem AsA content and AsA accumulation in sink organs can be directly increased via manipulation of AsA content in the foliage. We are now attempting to establish the quantitative contribution of imported AsA to overall AsA accumulation in developing potato tubers via transgenic approaches.

Nutritional iron deprivation attenuates kainate-induced neurotoxicity in rats: implications for involvement of iron in neurodegeneration.

There is evidence suggesting that oxidative stress contributes to kainate neurotoxicity. Since iron promotes oxidative stress, the present study explores how change in nutritional iron content modulates kainate-induced neurotoxicity. Rats received an iron-deficient diet (ID) from 22 days of age for 4 weeks. One control group received the same diet supplemented with iron and another control group received standard rodent diet. Cellular damage after subcutaneous kainate (10 mg/kg) was assessed by silver impregnation and gliosis by staining microglia. ID reduced cellular damage in piriform and entorhinal cortex, in thalamus, and in hippocampal layers CA1-3. ID also attenuated gliosis, except in the hippocampal CA1 layer. Given involvement of zinc in hippocampal neurotransmission and in oxidative stress, we tested for a possible interaction of nutritional iron with nutritional zinc. Rats were made iron-deficient and then assigned to supplementation with iron, zinc, or iron + zinc. Controls were continued on ID diet. After 2 weeks, rats were treated with kainate. Iron supplementation abolished the protective effect of ID in piriform and entorhinal cortex. In hippocampal CA1 and dorsal thalamus, neither iron nor zinc supplementation alone abolished the protective effect of ID against cellular damage. Iron + zinc supplementation abolished ID protection in dorsal thalamus, but not in reuniens nucleus. Kainate-induced gliosis in CA1 remained unaffected by nutritional treatments. Thus, in piriform and entorhinal cortex, nutritional iron has a major impact on cellular damage and gliosis. In hippocampal CA1, gliosis may associate with synaptic plasticity not modulated by nutritional iron, while cellular damage is sensitive to nutritional iron and zinc.

Cytoarchitecture of the avian optic tectum: neuronal substrate for cellular computation.

To analyse cellular computation in the vertebrate brain, a thorough knowledge of the underlying anatomy, physiology and connectivity of the neuronal substrate is essential. This review compiles data on one of the best known structures of the vertebrate brain, the optic tectum of birds. The functions of this structure are multifold, but can be attributed largely to orientation and the basic analysis of sensory data in a spatial context. In the tectum, a wealth of data on physiology and anatomy has been gathered over more than a century and provides an excellent background for computational studies. The analysis of the optic tectum is facilitated by several principles of organisation, including the retinotopic input and the highly laminated layout with separated input and output layers. Moreover, the molecular mechanisms guiding the development and connectivity have been analysed in detail. As the avian tectum and the mammalian superior colliculus are partly homologous, the cellular mechanisms unraveled in the tectum can also be transferred to the colliculus and thus contribute to the understanding of the vertebrate visual system in general.

Early (E12) cortical progenitors can change their fate upon heterotopic transplantation.

To help understand how the cortical map is set up during the early stages of corticogenesis, we have examined the developmental fate of embryonic day (E) 12 cortical progenitors in the rat. We have analysed the pattern of thalamic connections and cytoarchitectonic organization developed by progenitor cells removed at E12 from the presumptive parietal or occipital cortex and grafted into the parietal cortex of newborn hosts. Occipital progenitors grafted into the parietal cortex differentiated into neurons that developed reciprocal connections with the ventrobasal complex of the host thalamus. They could also form barrel-like structures, within which axons of the ventrobasal complex were distributed in dense patches. Some of these barrel-like structures were arranged in rows. Moreover, these progenitors failed to develop characteristic traits of occipital cortex cells as they did not establish connections with the dorsal lateral geniculate nucleus. We propose that cortical progenitors are not committed at E12 and, upon heterotopic transplantation, have the capacity to respond to local cues and to subsequently differentiate and maintain major phenotypic characteristics of neurons in their new environment. Only early progenitors are multipotent. By E13/E14, indeed, most cortical cells become irreversibly committed and upon heterotopic transplantation differentiate neurons with phenotypic characteristics of their cortical site of origin (Pinaudeau et al., 2000, Eur. J. Neurosci., 12, 2486-2496).

Silver stain removal using H2O2 for enhanced peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Hydrogen peroxide solutions are reported for the removal of silver stain from proteins isolated in polyacrylamide gels. Removal of silver stain prior to in-gel digestion is shown to enhance sensitivity and sequence coverage of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) peptide mass maps. The rate of silver removal using H2O2 is influenced by H2O2 concentration and increases with increasing pH. The presence of complexation reagents such as ammonia from mass spectrometry compatible ammonium bicarbonate solutions enhances the efficiency and speed of H2O2-mediated silver removal. H2O2-mediated silver removal using the described procedure does not appear to have any detrimental effects on proteins but is observed to produce a slightly elevated level of methionine oxidization over that usually observed in in-gel tryptic digestion.

Examining neocortical circuits: some background and facts.

The first definitive studies of where afferents to cerebral cortex terminate were made possible by the finding that as they degenerate axon terminals become electron dense. Gold toning of Golgi impregnated neurons allowed the postsynaptic targets of these afferents to be identified by electron microscopy and also allowed the termination sites of axons from a variety of types of cortical neurons to be ascertained, while the development of antibodies to GAD and to GABA made it possible to determine which types of cortical neurons are inhibitory. Subsequently the use of gold toned, Golgi impregnated material to examine neuronal connectivity was made redundant by the development of techniques that allowed the physiological properties of cortical neurons to be evaluated in neurons filled intracellularly with markers. Intracellular filling showed the axonal trees of cortical neurons are much more widespread than had been revealed by Golgi impregnations. As a result of numerous studies of the axons of identified neurons, we know a great deal about where most of the different types of neurons in cerebral cortex form their synapses, but on the other side of the picture there is a dearth of information about the origins of the inputs that specific types of cortical neurons receive. However, it is evident that each cortical neuron is the focus of input from many other neurons, and on the basis of the available data it is estimated that a single pyramidal cell in cortex receives its input from as many as 1,000 other excitatory neurons and as many as 75 inhibitory neurons.

Bismuth autometallography: protocol, specificity, and differentiation.

We provide a detailed protocol of the autometallographic bismuth technique and evaluate the specificity of the technique. We show by the multi-element technique "proton-induced X-ray microanalysis" (PIXE) that the autometallographic grains contain silver, bismuth, and sulfur, proving that autometallography can be used for specific tracing of bismuth bound as bismuth sulfide clusters in tissue sections from Bi-exposed animals or humans. In sections from animals exposed concurrently to selenium and bismuth, the autometallographic grains also contain selenium. This demonstrates that, if present in excess in the organisms, selenium will bind to exogenous bismuth, creating bismuth selenide clusters. As a further possible control for specificity and as a tool for differentiating among autometallographically detectable metals in sections containing more than one, we describe how bismuth sulfide clusters can be removed from Epon-embedded tissue sections by potassium cyanide.