Acetylcholinesterase enzyme localization in the amygdala: a comparative histochemical and ultrastructural study

The distribution of acetylcholinesterase enzyme was studied in the amygdala of some rodents, subprimates and several primates. The cytoarchitecture of the amygdala has presented various problems to anatomists, including the question as to how many nuclear groups and subgroups should be identified. Among the mammals examined, the arrangement of the amygdaloid nuclei is remarkably uniform and no clear phylogenetic trend can be recognised. Although there are minor differences, there seems to be a general similarity between most mammals examined in so far as the distribution of cholinesterase is concerned. The staining is less intense in the brains of the monkeys examined. The sole exception to the rule, that cholinesterase distribution is slightly different from nucleus to nucleus in different animals, is the magnocellular part of the basal nucleus. This amygdaloid nucleus stains quite strongly in all animals examined. From these findings, and those of others studying the distribution of choline acetyltransferase, it was concluded that the basal amygdaloid nucleus is cholinergic and possible cholinoceptive. The ultrastructural investigations appear to confirm this point. This is particularly applicable to the magnocellular part of the basal amygdaloid nucleus.     

Expression of the carbohydrate epitope 3-fucosyl-N-acetyl-lactosamine (CD15) in the vertebrate cerebellar cortex

Summary The distribution of the carbohydrate epitope CD15 was investigated on paraffin sections of the brains of man and mammals (monkey, dog, rabbit, rat, mouse, dolphin), reptile, bird and fish by means of immunohistochemistry. This paper demonstrates a differential expression of the CD15 epitope in the cerebella of these various vertebrates. CD15 positivity was found on glial cells and neuronal structures. In adult brains two major distribution patterns were distinguished: one with very intense labelling of the molecular layer, for which the rat is representative, the other with very low immunoreactivity in this layer (mouse). Amongst the rodents (mouse, rat and rabbit), as well as the monkey and human, the positivity in the molecular layer could be attributed to Bergmann fibres of the Golgi epithelial cells. A typical parasagittal band pattern, present in the mouse molecular layer for CD15, which is absent in rat and rabbit molecular layer, is present during human cerebellar development. CD15 positivity on neuronal structures is found on parallel fibres in the developing human, on the lower stellate cells in the dog, and in climbing fibres of the dolphin and, presumably, the catfish too. Moreover, within the parrot cerebellum, large CD15-positive mossy fibre-like endings are found just at the infraplexiform layer.     

Quantitative alpha-ketoglutarate dehydrogenase activity staining in brain sections and in cultured cells

The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, as well as in thiamine-deficient (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cellular or regional changes in enzyme activity within the brain. To overcome this limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in cultured cells. The assay was linear with time and was highly specific for KGDHC. The dark-blue reaction product (formazan) formed a pattern that was consistent with mitochondrial localization. Treatment of the cultured cells with both reversible and irreversible inhibitors decreased formazan production, whereas conventional enzyme assays on cell lysates only revealed loss of KGDHC activity with irreversible inhibitors. The activity staining was also linear with time and highly specific for KGDHC activity in mouse brain sections. Staining occurred throughout the brain, and discrete neuronal populations exhibited particularly intense staining. The pattern of staining differed markedly from the distribution of KGDHC protein by immunocytochemistry. Generalized decreases in the intensity of activity staining that occurred in the TD brains compared to controls were comparable with the loss of KGDHC activity by conventional enzyme assay. Thus, the present study introduces a new histochemical method to measure KGDHC activity at the cellular and regional level, which will be useful to determine changes of in situ enzyme activity.     

Immuno-electron microscopical demonstration of vasopressin and oxytocin synapses in the limbic system of the rat

Summary The extensive distribution of exohypothalamic vasopressin or oxytocin containing nerve fibres is thought to be the anatomical basis for the involvement of these neuropeptides in central processes. Following light microscopic observations suggesting that these fibres terminate on other neurons, the present study was undertaken to demonstrate the existence of such endings in the limbic system, which is one of the main target areas for these peptides. For immunoelectron microscopy glutaraldehyde-paraformaldehyde perfused brains of male Wistar rats and Brattleboro rats, homozygous for diabetes insipidus, with and without postfixation in OsO₄, were used. Post-embedding staining revealed false positive reaction product on all dense core vesicles, e.g., in the lateral septum. With pre-embedding staining, however, intense and specific reactions were observed for both vasopressin and oxytocin at their sites of production, as well as the neurohypophysis and in the extrahypothalamic limbic brain regions.In the lateral septum and habenular nucleus only vasopressin-containing synapses could be demonstrated, while in the medial nucleus of the amygdala synapses containing either vasopressin or oxytocin were observed. These peptide containing synapses do not seem to differ in any fundamental way from the classical transmitter-containing synapses in the brain.Supported by the Foundation for Medical Research FUNGOThe authors wish to thank Prof. Dr. A.H.M. Lohman for having made the vibratome available, and Miss C. de Raay for her expert technical assistance     

Peripheral and central olfactory tuning in a moth

Animals can be innately attracted to certain odorants. Because these attractants are particularly salient, they might be expected to induce relatively strong responses throughout the olfactory pathway, helping animals detect the most relevant odors but limiting flexibility to respond to other odors. Alternatively, specific neural wiring might link innately preferred odors to appropriate behaviors without a need for intensity biases. How nonpheromonal attractants are processed by the general olfactory system remains largely unknown. In the moth Manduca sexta, we studied this with a set of innately preferred host plant odors and other, neutral odors. Electroantennogram recordings showed that, as a population, olfactory receptor neurons (ORNs) did not respond with greater intensity to host plant odors, and further local field potential recordings showed that no specific amplification of signals induced by host plant odors occurred between the first olfactory center and the second. Moreover, when odorants were mutually diluted to elicit equally intense output from the ORNs, moths were able to learn to associate all tested odorants equally well with food reward. Together, these results suggest that, although nonpheromonal host plant odors activate broadly distributed responses, they may be linked to attractive behaviors mainly through specific wiring in the brain.     

Differential distribution of protein kinase C isozymes in the various regions of brain

We have previously identified three types of protein kinase C (a Ca2+-activated phospholipid-dependent kinase) isozymes, designated types I, II, and III, from rat brain (Huang, K.-P., Nakabayashi, H., and Huang, F. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8535-8539). These enzymes are different in their elution profile from hydroxylapatite column, sites of autophosphorylation, and immunoreactivity toward two types of monoclonal antibodies. Now we describe the purification of similar protein kinase C isozymes from monkey brain and their regional distribution in the brain. These primate enzymes all have the same molecular weight of 82,000, and each type of isozyme cross-reacts with the purified monospecific antibodies against its corresponding rat brain counterpart isozyme. These purified antibodies were used to quantify the relative contents of three types of protein kinase C isozymes in various regions of rat and monkey brains. In rat brain, cerebellum contained a high level of the type I isozyme; cerebral cortex, thalamus, and corpus callosum were high in the type II enzyme; and olfactory bulb was highest in the type III enzyme. In monkey brain, the type I isozyme was found to be enriched in cerebellum, hippocampus, and amygdala; the type II enzyme was at very high level in caudate, frontal and motor cerebral cortices, substantia nigra, and thalamus; and the type III enzyme was at the highest level in olfactory bulb. These results indicate that protein kinase C isozymes are differentially distributed in various regions of rat and monkey brains and suggest a unique role for each isozyme in controlling the different neuronal functions in the brain.     

Distribution of axon diameters in cortical white matter: an electron-microscopic study on three human brains and a macaque

The aim of this study was to obtain information on the axonal diameters of cortico-cortical fibres in the human brain, connecting distant regions of the same hemisphere via the white matter. Samples for electron microscopy were taken from the region of the superior longitudinal fascicle and from the transitional white matter between temporal and frontal lobe where the uncinate and inferior occipitofrontal fascicle merge. We measured the inner diameter of cross sections of myelinated axons. For comparison with data from the literature on the human corpus callosum, we also took samples from that region. For comparison with well-fixed material, we also included samples from corresponding regions of a monkey brain (Macaca mulatta). Fibre diameters in human brains ranged from 0.16 to 9  \(\upmu \hbox {m}\) . Distributions of diameters were similar in the three systems of cortico-cortical fibres investigated, both in humans and the monkey, with most of the average values below 1  \(\upmu \) m diameter and a small population of much thicker fibres. Within individual human brains, the averages were larger in the superior longitudinal fascicle than in the transitional zone between temporal and frontal lobe. An asymmetry between left and right could be found in one of the human brains, as well as in the monkey brain. A correlation was also found between the thickness of the myelin sheath and the inner axon diameter for axons whose calibre was greater than about 0.6  \(\upmu \hbox {m}\) . The results are compared to white matter data in other mammals and are discussed with respect to conduction velocity, brain size, cognition, as well as diffusion weighted imaging studies.     

猕猴早期胚胎脑的发育与NF-κB的表达

目的检测NF-kB转录因子在早期猕猴脑的表达,探讨非人类灵长类猕猴脑发育与NF-kB的关系。方法应用单克隆抗体链菌素亲生物蛋白过氧化酶（SP）免疫组织化学方法及电子计算机图像分析技术,观察NF-kB的表达。结果NF-kB在早期猕猴脑各部均有表达。在40d胎脑NF-kB呈弱表达;在55d胎脑NF-kB表达较前者明显增强,特别是在下丘脑、尾状核、豆状核和丘脑近第3脑室旁的神经细胞内NF-kB呈强阳性表达。结论NF-kB参与早期猕猴脑发育的调控。     

Radioimmunoassay for brain natriuretic peptide (BNP) detection of BNP in canine brain

We established a highly sensitive and specific radioimmunoassay (RIA) for BNP. Our RIA detected BNP-like immunoreactivity (-LI) in the porcine and canine brains but did not detect BNP-LI in the human, monkey or rat brain. The widespread distribution of BNP-LI was demonstrated both in the porcine and canine brains, with the highest concentration in the medulla oblongata. In contrast, the highest concentration of ANP-LI determined simultaneously was in the midbrain and the olfactory bulb. High performance-gel permeation chromatography coupled with RIA revealed that the major component of BNP-LI was eluted at the position of synthetic BNP with a small molecular weight (3K). These results indicate that the RIA for BNP serves as a useful tool to investigate physiological roles of BNP.     

Immunohistochemical localisation of natriuretic peptides in the brains and hearts of the spiny dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa

Summary The avidin-biotin peroxidase technique was used to determine the distribution of natriuretic peptides in the hearts and brains of the dogfishSqualus acanthias and the Atlantic hagfishMyxine glutinosa. Three antisera were used: one raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide, but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); and the third raised against rat atrial natriuretic peptide (termed rat atrial natriuretic peptide-like immunoreactivity). Only natriuretic peptide-like immunoreactivity was observed in the heart ofS. acanthias which was most likely due to the antiserum cross-reacting with C-type natriuretic peptide. No immunoreactivity was found in theM. glutinosa heart. In the brain ofS. acanthias, natriuretic peptide-like immunoreactive fibres were located in many areas of the telencephalon, diencephalon, mesencephalon, rhombencephalon, and spinal cord. Extensive immunoreactivity was observed in the hypothalamo-hypophyseal tract and the neurointermediate lobe of the hypophysis. Natriuretic peptide-like immunoreactive perikarya were found in ventromedial regions of the telencephalon and in the nucleus preopticus. Most perikarya had short, thick processes which extended toward the ventricle. Another group of perikarya was observed in the rhombencephalon. Porcine brain natriuretic peptide-like immunoreactive fibres were observed in the telencephalon, diencephalon, mesencephalon, and rhombencephalon, but perikarya were only present in the preoptic area. In theM. glutinosa brain, natriuretic peptide-like immunoreactive fibres were present in all regions. Immunoreactive perikarya were observed in the pallium, primordium hippocampi, pars ventralis thalami, pars dorsalis thalami, nucleus diffusus hypothalami, nucleus profundus, nucleus tuberculi posterioris, and nucleus ventralis tegmenti. Procine brain natriuretic peptide-like immunoreactive perikarya and fibres had a similar, but less abundant distribution than natriuretic peptide-like immunoreactive structures. Although the chemical structures of natriuretic peptides in the brains of dogfish and hagfish are unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic peptide or porcine C-type natriuretic peptide. The presence of natriuretic peptides in the brain suggest they could be important neuromodulators and/or neurotransmitters. Furthermore, there appears to be divergence in the structural forms of natriuretic peptides in the hearts and brains of dogfish and hagfish.     

Immunocytochemistry and heterogeneity of rat brain vimentin

Summary In unfixed cryostat sections of the brains of early postnatal and adult rats, we screened for cells containing vimentin-positive intermediate filaments (VI⁺-IFs) by applying a panel of four monoclonal antibodies (Mabs VI-01, VI-02, VI-05 and VI-5B3) using indirect immunofluorescence. All of the Mabs stained VI⁺-IFs in the stromal part of the choroid plexus, in endothelial cells of blood vessels and in meninges in both adult and immature brains, although with varying strength (VI-5B3 and VI-01 stained more strongly than VI-05 and VI-02). In the brain parenchyma of adults, intense staining was mainly localized in ventricular ependymal cells (VI-5B3/VI-01>VI-02/VI-05) and fibrous astrocyte-like cells (FAs). In the immature brain, the ependymall cells were activated in appearance, with evidence of cell enlargement, greater spreading of VI⁺-IFs within the cytoplasma and more pronounced VI⁺ cytoplasmic protrusions into the brain parenchyma.VI⁺-FAs were found near the ependymal and meningead borders as well as in the white matter tracts of adult brain (VI-5B3/VI-01>VI-05>VI-02). In immature animals, VI⁺-FAs were less frequently encountered in the forebrain regions, except in and near the subependymal layer (in the adjacent parenchyma) as well as in submeningeal layers. Weaker staining was usually clicited by Mabs VI-02 and VI-05. In the cerebellum, Bergmann cell fibres were stained in both age groups. In adults, the most intense fluorescence usually occurred in segments close to the pia (VI-5B3/VI-01>VI-05>VI-02). In immature animals, the Bergmann cell fibres were less straight, less smooth and thicker, and were stained along their whole length by all Mabs except VI-02. In adults, VI⁺-FAs were observed in the internal granular layer (VI-5B3 and VI-01) and, relatively more often, in the white matter (VI-05). In immature animals, a quasi-continuous mesh-work of VI⁺ cells was detected at some sites of the cerebellum, especially when VI-01 and VI-5B3 were used. With maturation, reduced staining was produced by all VI Mabs in the choroid plexus. We have thus demonstrated that VI is a common molecular denominator of cerebrospinal-fluid and/or blood-washed cells as well as of glial fibers contacting these cells. The differences in the staining of VI⁺ cells by various Mabs probably reflect an immunological heterogeneity of VI⁺-IFs based on the varying accessibility of the individual VI epitopes. This might be due to alterations in the tertiary structure of VI caused, for instance, by phosphorylation or other posttranslational processes. The actual structural state of VI may explain the variations in the immunostainability of astrocytes and also in the staining obtained using classical impregnation methods within the brain and/or its regions.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Species-specific homogeneity of the primate Alu family of repeated DNA sequences

We have determined the base sequence of several cloned Alu family members from the DNAs of a new world monkey (owl monkey) and a prosimian (galago). The three owl monkey Alu family members reported here belong to a single 300 base pair consensus sequence which closely resembles the human Alu family consensus. The galago Alu family members can best be represented as belonging to either of two related but distinct consensus sequences. One of the two galago Alu family subgroups (Type I) more accurately resembles the human consensus sequence than does the other subgroup (Type II). In this work we compare base sequences of human and galago Type I Alu family members. There are several examples of species-specific differences between the human and Type I galago sequences indicating that the Alu family members are effectively homogenized within a species.     

Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains.

Immunohistochemical distribution and cellular localization of neurotrophins was investigated in adult monkey brains using antisera against nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Western blot analysis showed that each antibody specifically recognized appropriate bands of approximately 14.7 kDa, 14.2 kDa, 13.6 kDa, and 14.5 kDa, for NGF, BDNF, NT-3, and NT-4, respectively. These positions coincided with the molecular masses of the neurotrophins studied. Furthermore, sections exposed to primary antiserum preadsorbed with full-length NGF, BDNF, NT-3, and NT-4 exhibited no detectable immunoreactivity, demonstrating specificities of the antibodies against the tissues prepared from rhesus monkeys. The study provided a systematic report on the distribution of NGF, BDNF, NT-3, and NT-4 in the monkey brain. Varying intensity of immunostaining was observed in the somata and processes of a wide variety of neurons and glial cells in the cerebrum, cerebellum, hippocampus, and other regions of the brain. Neurons in some regions such as the cerebral cortex and the hippocampus, which stained for neurotrophins, also expressed neurotrophic factor mRNA. In some other brain regions, there was discrepancy of protein distribution and mRNA expression reported previously, indicating a retrograde or anterograde action mode of neurotrophins. Results of this study provide a morphological basis for the elucidation of the roles of NGF, BDNF, NT-3, and NT-4 in adult primate brains.     

Gregarious desert locusts have substantially larger brains with altered proportions compared with the solitarious phase

The behavioural demands of group living and foraging have been implicated in both evolutionary and plastic changes in brain size. Desert locusts show extreme phenotypic plasticity, allowing brain morphology to be related to very different lifestyles in one species. At low population densities, locusts occur in a solitarious phase that avoids other locusts and is cryptic in appearance and behaviour. Crowding triggers the transformation into the highly active gregarious phase, which aggregates into dense migratory swarms. We found that the brains of gregarious locusts have very different proportions and are also 30 per cent larger overall than in solitarious locusts. To address whether brain proportions change with size through nonlinear scaling (allometry), we conducted the first comprehensive major axis regression analysis of scaling relations in an insect brain. This revealed that phase differences in brain proportions arise from a combination of allometric effects and deviations from the allometric expectation (grade shifts). In consequence, gregarious locusts had a larger midbrain∶optic lobe ratio, a larger central complex and a 50 per cent larger ratio of the olfactory primary calyx to the first olfactory neuropile. Solitarious locusts invest more in low-level sensory processing, having disproportionally larger primary visual and olfactory neuropiles, possibly to gain sensitivity. The larger brains of gregarious locusts prioritize higher integration, which may support the behavioural demands of generalist foraging and living in dense and highly mobile swarms dominated by intense intraspecific competition.     

Quantitive studies of the reactions to horizontal angular accelerations in axolotls. II. head-turning reflexes in animals with a supernumerary pair of labyrinths.

In axolotls (Ambystoma mexicanun) the labyrinths and the associated parts of the medulla were doubled artificially. In these so-called tandem-heads the vestibular afferent fibres from both labyrinths on one side united within the medulla to form common bundles. The head-turning reflexes following impulse acceleration and during long-lasting acceleration were measured quantitatively and compared with those for normal animals. The form and the time-course of the reactions were almost identical in both groups. Tandem-heads showed a linear relationship between stimulus intensity and reaction strength, parallel to that in normal animals but with a greater reaction for a given stimulus. Consequent to this shift in the relationship, there was a significant decrease in the reaction threshold. The removal of one horizontal semicircular canal in tandem-heads proved that both pairs of labyrinths were functionally connected with the brain. It was suggested that during ontogenesis there exists a kind of specificity in the connexion of vestibular fibres. From the parallel shift of the intensity functions it was concluded that the input from both pairs of labyrinths in tandem-heads is not simply accumulate but compared with a reference parameter, which is also double in tandem-heads.     

Über Gefässpaare im Gehirn der Katze

Summary Paired vessels, consisting of one artery and one vein, have so far only been described in the brains of some lower vertebrates and in the brains of marsupials (opossum). However, as shown in this paper, similar vessels also occur in the medulla oblongata, midbrain, hypothalamus, septum pellucidum, hippocampus and in the olfactory lobe of the cat's brain. In the neocortex and in the cerebellum no such vessels could be traced.—Whereas some paired vessels are first surrounded by connective tissue (Virchow-Robin's space) and then by a layer of glial fibres, most of the vessels are only ensheathed by a coat of glial fibres. These and other histological details are described and some functional implications are discussed under the assumption that the paired vessels allow diffusion of blood gases between venous and arterial blood and vice versa.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

Immunohistochemical Distribution of Cytochrome P4501A in Larvae and Fingerlings of the Siberian Sturgeon, Acipenser baeri

In this paper we investigate by means of immunohistochemistry, the tissue distribution of constitutive cytochrome P4501A (CYP1A), from hatching until 30 days posthatching in developing Siberian sturgeon, Acipenser baeri. For this purpose, a polyclonal (BN-1) antiserum developed against a conservative sequence of piscine CYP1A and a monoclonal (C10-7) antiserum directed against cod CYP1A were used on paraffin-embedded samples. From hatching onwards, distinct CYP1A immunoreactivity was distinctly observed in the following tissues and cells: envelope of oil droplets, matrix and syncytium of the yolk-sac, sinusoids, biliary epithelial cells and hepatocytes. In the digestive tract, buccopharyngeal, oesophageal, gastric and intestinal epithelia, as well as the cytoplasm and brush border of enterocytes were CYP1A-positive. Interestingly, gastric glands and melanin-plug present within lumen of the digestive system were strongly immunoreactive. Kidney (epithelia of renal tubules), gills (pillar and endothelial cells), skin (epithelial cells), muscle fibres of heart and eye (retina) were positive. In brain, we observed a strong CYP1A staining in the developing telencephalon and especially in olfactory system, as well as in those nerve fibres running ventrally toward the posterior brain. A strong CYP1A staining was observed in vascular endothelia of all organs/tissues, especially in the liver. In general, the intensity of CYP1A immunostaining increased during larval development, suggesting besides its known metabolic function (endogenous and/or exogenous), a possible participation of this heme-protein in control of cell division, regulation of growth and differentiation.     

Silver Staining of Insect Central Nervous Systems by the Bodian Protargol Method

Improved fixation of ganglia of the central nervous system of Periplaneta americana and Schistocerca gregaria for silver staining by Power's (1943) modification of the Bodian protargol method is given by alcoholic Bouin aged for at least 40 days at 60° C. During impregnation of sections, increased copper and decreased pH give paler staining, more selective for nerve fibres. Prolonging impregnation from 24 to 48 hours weakens the stain and decreases selectivity. The intensity of the stain depends chiefly upon the amount of unreduced (developable) silver combined with the tissues; selectivity is determined mainly by the number and distribution of the reduced silver particles (‘nuclei’). In development, increased sodium sulphite gives more differentiation, increased hydroquinone gives less. Optimum developer composition depends upon impregnation, and thick sections need more differentiation than thinner ones. Within limits, change in one of the factors that control staining can be balanced by changes in others, but by suitable adjustment of the conditions the result can be varied from almost total staining of nerve fibres, for general neuroanatomy, to highly selective staining for tracing individual fibres.     

Histochemical distribution of non-haem iron in the human brain.

The detailed anatomical distribution of iron in the post-mortem human brain has been studied using Perl's and Turnbull's methods with the diaminobenzidine intensification procedure for the demonstration of non-haem Fe3+ and Fe2+, respectively. Attention to methodological procedures has revealed that even brief immersion of tissue in routinely used fixatives causes a reduction of staining intensity in areas of high iron content and, often, loss of staining in areas of low iron content. Optimal staining is obtained using frozen section briefly fixed for 5 min in 4% formalin and Perl's stain (Fe3+) with diaminobenzidine intensification. Highest levels of stainable iron were found in the extrapyramidal system with the globus pallidus, substantia nigra zona reticulata, red nucleus and myelinated fibres of the putamen showing highest staining reactivity. Moderate staining intensity with Perl's technique was found in the majority of forebrain, midbrain and cerebellar structures with the striatum, thalamus, cortex and deep white matter, substantia nigra zona compacta, and cerebellar cortex showing consistent staining patterns with intensification of Perl's stain. The brain-stem and spinal cord generally only showed staining with the intensification procedure and even this was of low intensity. Microscopically the non-heam iron appears to be found predominantly in glial cells as fine cytoplasmic granules which in heavily stained areas coalesce to fill the entire cell. Iron-positive granules appear to be free in the neuropil and also around blood vessels in the globus pallidus, striatum and substantia nigra. The neuropil shows a fibrous impregnation when stained for iron which is, in part, derived from glial processes, myelinated fibres and fibre bundles. Neurones, in general, show only very low reactivity for iron, and this is difficult to discern due, often, to the higher reactivity of the surrounding neuropil. In the globus pallidus and substantia nigra zona reticulata, neurones with highly stainable iron content are found with granular cytoplasmic iron reactivity similar to that seen in the local glial cells. Our results are comparable with those of early workers, but with the use of intensification extend the distribution of non-haem iron to areas previously reported as negative. No apparent correlation of iron staining with known neurotransmitter systems is seen and the predilection for the extrapyramidal system is not easily explained, though the non-haem iron in the brain appears to be as a storage form in the iron storage protein ferritin. The localization of iron in the brain provides a foundation for the study of iron in certain neurodegenerative diseases such as Parkinson's disease, where iron has been implicated in the pathogenesis.