Immunohistochemical detection of O6-ethyldeoxyguanosine in the rat brain after in vivo applications of N-ethyl-N-nitrosourea.

An immunohistochemical procedure was developed which allows the localization of the DNA lesion O6-ethyldeoxyguanosine (O6-EtdGuo) within tissues and organs. The method permits the detection of 24,000 residues of O6-EtdGuo per diploid nucleus. We have used this procedure to localize N-ethyl-N-nitrosourea (ENU)-induced O6-EtdGuo in the rat brain. Shortly after a single injection of ENU, we observed O6-EtdGuo in most or all of the rat brain nuclei. After repeated injections of small doses of ENU, with intervals of 1 or 2 weeks between the injections and between the last injection and sacrifice, we could demonstrate O6-EtdGuo only in part of the rat brain nuclei. Oligodendrocytes, granular neurons and endothelial cells, and part of the pyramidal neurons and astrocytes had accumulated O6-EtdGuo, while in all other cells this lesion was not detectable after repeated injections of small doses of ENU. We found no obvious correlation between the putative sensitivity of rat brain cells to tumor induction and the accumulation of O6-EtdGuo in their DNA.

Lack of association between depression and loss of neurons in the locus coeruleus in Alzheimer disease.

BACKGROUND: Depression, one of the most frequent psychiatric disturbances in Alzheimer disease (AD), is proposed to have its neurobiological basis in neuron loss in the noradrenergic locus coeruleus, although this is not the case in idiopathic depression. METHODS: We performed image analyzer-assisted morphometry of the locus coeruleus in 6 depressed, 6 transiently depressed, and 6 nondepressed patients with AD and in 8 control subjects, emphasizing longitudinal psychiatric evaluations and matching for the clinical and neuropathological severity of dementia. RESULTS: The mean (+/-SD) number of pigmented neurons in the locus coeruleus in controls (11 607+/-946) was higher than in patients with AD, regardless of being depressed (5165+/-928; P=.001), transiently depressed (5647+/-1163; P=.003), or nondepressed (3717+/-661; P=.001). No significant difference was found in the number of pigmented neurons between patients with AD who were depressed, transiently depressed, and nondepressed. Patients who had depression at the onset of AD had a higher pigmented neuron number than other patients with AD. CONCLUSIONS: We confirmed the loss of pigmented neurons in the locus coeruleus of patients with AD; however, no supplementary loss of pigmented neurons in the locus coeruleus was found in patients with depression and AD. This finding resembles the situation in idiopathic depression, but is in contrast with earlier studies on depression in AD.

Myofibrillar differences among mammalian skeletal muscle fibres at the ultrastructural level. A comparison of immunocytochemical and morphometrical parameters.

In the light microscope two types (I, II) of skeletal muscle fibres can be distinguished with antibodies against myosin isozymes. At the ultrastructural level a difference in Z-line width has led to muscle fibre classification. In this study we distinguish at the ultrastructural level between type I and type II fibres of the M. soleus and M. plantaris of adult mice using ultracryosections with immuno-ferritin and antisera against myosin isozymes. Muscle fibres of the M. plantaris are identified as type II fibres and the fibres of the M. soleus are divided in type I and type II fibres. In the immunologically identified fibres the filament overlap in the Z-line was measured. The type II fibres of the M. plantaris have narrow Z-lines, whereas type I and type II fibres of the M. soleus have wide Z-lines. We conclude that a classification of fibres based on Z-line width differs from the type I/type II classification. The antimyosin antibodies react exclusively with the A-band. In serial sections the myosin isozymes can be identified unambiguously. This is a prerequisite for further studies of myosin isozyme distribution in "mixed" muscle fibres.

Male-to-female transsexuals have female neuron numbers in a limbic nucleus.

Transsexuals experience themselves as being of the opposite sex, despite having the biological characteristics of one sex. A crucial question resulting from a previous brain study in male-to-female transsexuals was whether the reported difference according to gender identity in the central part of the bed nucleus of the stria terminalis (BSTc) was based on a neuronal difference in the BSTc itself or just a reflection of a difference in vasoactive intestinal polypeptide innervation from the amygdala, which was used as a marker. Therefore, we determined in 42 subjects the number of somatostatin-expressing neurons in the BSTc in relation to sex, sexual orientation, gender identity, and past or present hormonal status. Regardless of sexual orientation, men had almost twice as many somatostatin neurons as women (P < 0.006). The number of neurons in the BSTc of male-to-female transsexuals was similar to that of the females (P = 0.83). In contrast, the neuron number of a female-to-male transsexual was found to be in the male range. Hormone treatment or sex hormone level variations in adulthood did not seem to have influenced BSTc neuron numbers. The present findings of somatostatin neuronal sex differences in the BSTc and its sex reversal in the transsexual brain clearly support the paradigm that in transsexuals sexual differentiation of the brain and genitals may go into opposite directions and point to a neurobiological basis of gender identity disorder.

Development of the dopaminergic innervation in the prefrontal cortex of the rat.

The pre- and postnatal development of the dopaminergic innervation in the prefrontal cortex (PFC) of the rat is described from embryonic day 14 through postnatal day 90. By embryonic day 15 the dopamine (DA)-containing fibers reach the anlage of the lateral neocortex; 2 days later the first fibers have reached the subplate of the future prefrontal cortex. The process of entering the cortical plate starts just before birth. Prenatally, some dopaminergic fibers can be observed in the marginal zone of both the lateral and the medial wall of the hemisphere. Within 48 hours after birth a large number of dopaminergic fibers can be observed in the marginal zone, i.e., the future layer I, in some subareas of the PFC. A transient appearance of DA-positive fibers is noticed in the late embryonic and early postnatal periods especially in the marginal zone and possibly in the superficial layers of the pregenual cingulate cortex. Changes in the morphology of DA fibers at P4 suggest that the actual DA innervation starts at this age. From postnatal day 6 the different subareas of the PFC can be recognized according to the characteristics of the topographical distribution of the dopaminergic fibers. Until postnatal day 60 the density of the dopaminergic fibers continues to increase. No difference in density and topography was observed between postnatal days 60 and 90.

Differences in colocalization between Fos and PHI, GRP, VIP and VP in neurons of the rat suprachiasmatic nucleus after a light stimulus during the phase delay versus the phase advance period of the night.

Two groups of four rats each received a 15-minute light stimulus during the first part of the night (ZT14) and the second part (ZT19), respectively. After 45-60 minutes, the animals were killed by perfusion fixation. Adjacent Vibratome sections through the suprachiasmatic nucleus (SCN) were double-immunostained for the presence of peptide histidine isoleucine (PHI), gastrin releasing peptide (GRP) or vasoactive intestinal peptide (VIP) with Fos by using fluorophore-conjugated secondary antibodies. A few sections were triple-immunostained for PHI, GRP or VIP with vasopressin (VP) and Fos. Sections were analyzed with a confocal laser scanning microscope. It turned out that the ZT19 light stimulus induced 4.2 times more nuclear profiles in the SCN immunoreactive for Fos than the light stimulus given at ZT14. The SCN of control animals did not show any Fos immunoreactivity. After the ZT14 light stimulus, approximately 33% of the Fos profiles showed colocalization with a perikaryal profile immunoreactive for PHI, GRP or VIP, whereas at ZT19, this percentage had doubled to approximately 65%. After the light stimulus at ZT14, the relatively low Fos induction was numerically and proportionally most prominent in the PHI-immunoreactive perikarya. As compared with ZT14, the increase of Fos after the ZT19 light stimulus was most pronounced in the GRP-immunoreactive perikarya (21x) followed by VIP (15x) and PHI (5x). This outcome suggests that at least three different cell groups characterized by, respectively, PHI alone, GRP, and VIP fully or partly colocalized with PHI, play a prominent role during light-induced phase shifts: the PHI neurons during light-induced phase delays, the GRP and VIP/(PHI) neurons during light-induced phase advances.

The distribution of vasotocin and isotocin in the brain of the rainbow trout.

The distribution of vasotocin and isotocin in the brain of the rainbow trout Salmo gairdneri was investigated by the unlabeled antibody enzyme method, by using purified antisera against arginine vasotocin and isotocin. In the preoptic nucleus no clear differences were observed in the distribution of vasotocin- and isotocin-containing cells. Vasotocin and isotocin innervation was found in most brain regions, though in general isotocin fibers were much more abundant. The area dorsalis pars medialis of the telencephalon, the saccus dorsalis, and the basal part of the nucleus recessus posterioris were found to be innervated by vasotocin and scarcely by isotocin fibers. In the nucleus habenularis, the nucleus recessus lobus lateralis, the nucleus preglomerulosus pars medialis, and the tectum mesencephali isotocin fibers prevailed. These findings in the trout brain are compared with the vasopressin and oxytocin innervation of the rat brain.

Detection of antigens and antibodies by an immuno-peroxidase method applied on thin longitudinal sections of SDS-polyacrylamide gels.

This paper describes a method for detection of antigens in thin sections of SDS-polyacrylamide gels. This method, called SGIP, involves longitudinal sectioning of SDS gels, fixation of the proteins in the gels, removal of the SDS, incubation of the sections with an antiserum and detection of antigens by the indirect immuno-peroxidase technique. The method is useful for assessing the affinity spectrum of a given antiserum against a heterogeneous mixture of proteins, and for the detection of proteins in tissue homogenates or other protein mixtures by means of well defined antisera. By applying the method to serial sections from a single SDS-polyacrylamide gel, a dilution dependent reactivity of antisera is demonstrated.

Press-blotting on gelatin-coated nitrocellulose membranes. A method for sensitive quantitative immunodetection of peptides after gel isoelectric focusing.

A method is presented for the fixation of peptides in nitrocellulose membranes after isoelectric focusing on thin polyacrylamide gels. Focusing gels are covered with gelatin-coated nitrocellulose membrane. Using glutaraldehyde, focused peptides are covalently fixed onto this membrane. Fixed peptides are stained using the peroxidase-anti-peroxidase method and the immunoreaction is quantified by rendering the membrane transparent and measuring the optical density of the precipitated chromogen in each band. The effect of pore size and gelatin content of the membrane, glutaraldehyde concentration and fixation time on fixation efficiency and immunostaining has been investigated. Gelatin coating considerably increases the efficiency of glutaraldehyde fixation of peptides and greatly enhances antibody-binding. Consequently, sensitive quantitative immunodetection is possible and, depending on the antiserum, peptides are readily detected in quantities down to 10 pg.

Enkephalin immunoreactivity in synaptoid elements on glial cells in the rat neural lobe.

Opioid peptides were localized in fibres of the rat neural lobe using various immunocytochemical methods at the light- and electron-microscopical level. Leu-enkephalin immunoreactivity was present in beaded fibres distributed throughout the neural lobe. These fibres surround the neurohypophyseal glial cells (pituicytes) and make synaptoid contacts upon their soma and processes. The reaction product was localized both in dense-core vesicles of about 100 nm in diameter and diffusely spread over the cytoplasm. No arguments in support of the co-existence of enkephalins and the neurohypophyseal hormones vasopressin and oxytocin in the same terminal were found. It is suggested that pituicytes might mediate the inhibitory effect of opiod peptides on vasopressin and oxytocin release from the neural lobe.

Decreased neuronal activity in the nucleus basalis of Meynert in Alzheimer's disease as suggested by the size of the Golgi apparatus.

In order to study changes in neuronal activity in the nucleus basalis of Meynert in aging and Alzheimer's disease, we applied a polyclonal antibody directed against the Golgi apparatus on formalin-fixed, paraffin-embedded material. Subsequently, an image analysis system was used to measure the size of the Golgi apparatus in (i) all nucleus basalis neurons and also separately in (ii) the remaining large cells (perikaryonal diameter > 30 microns). A significant reduction of 49% in the size of the Golgi apparatus was found in the entire population of nucleus basalis neurons in Alzheimer's disease. Furthermore, although there was no significant decrease in the size of the persisting large neurons in the nucleus basalis of Meynert, a significantly decreased size of the Golgi apparatus was found in these neurons in Alzheimer's disease. These results suggest that the overall activity of nucleus basalis neurons is severely decreased in Alzheimer's disease. Furthermore, these data support the idea that atrophy and decreased activity are the main phenomena in the nucleus basalis in Alzheimer's disease; they also indicate that the size of the Golgi apparatus is a sensitive parameter to follow this process.

Quantitative estimation of estrogen and androgen receptor-immunoreactive cells in the forebrain of neonatally estrogen-deprived male rats.

Using quantitative immunocytochemical procedures, the total number of estrogen and androgen receptors was estimated in a large number of hypothalamic and limbic nuclei of male rats, in which brain estrogen formation was inhibited neonatally by treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione. The highest densities of estrogen receptor immunoreactivity were observed in the periventricular preoptic area and the medial preoptic area. Neonatally estrogen-deprived males showed a higher estrogen receptor immunoreactivity than control males in the periventricular preoptic area and the ventrolateral portion of the ventromedial nucleus of the hypothalamus, i.e. those brain areas in which sex differences have been reported, with female rats showing a greater estrogen binding capacity than male rats. The highest densities of androgen receptor immunoreactivity were found in the septohypothalamic nucleus, the medial preoptic area, the posterior division of the bed nucleus of the stria terminalis and the posterodorsal division of the medial amygdaloid nucleus. No significant differences in distribution or total numbers of androgen receptors were found between neonatally estrogen-deprived males and control males. These findings suggest that neonatal estrogens, derived from the neural aromatization of testosterone, are involved in the sexual differentiation of the estrogen receptor system in the periventricular preoptic area and the ventromedial hypothalamus. The role of neonatal estrogens in the development of the forebrain androgen receptor system is less clear.

Kindling induces time-dependent and regional specific changes in the [3H]muscimol binding in the rat hippocampus: a quantitative autoradiographic study.

To investigate possible changes in the GABAA receptor agonist site in the CA1 area and fascia dentata of rats kindled by stimulation of Schaffer collaterals, a quantitative autoradiographic study of the [3H]muscimol binding was carried out. Two kindled groups were studied, at 24 h (fully kindled stage) and at 28 days (long-term stage) after the last class V seizure. Several concentrations of [3H]muscimol were tested in the range of the high/intermediate (5-40 nM) and low-affinity (60-100 nM) binding sites. In the fully kindled group, the binding over the complete range of tested [3H]muscimol concentrations was significantly increased by 30-50% in the fascia dentata, while the binding was significantly decreased by 10-25% in the CA1 area. The high/intermediate-affinity binding was still significantly increased by 20-30% in the fascia dentata 28 days after the last seizure. In this long-term group there was still a significant decrease of 10-18% of the low-affinity binding in the CA1 area. These results show that kindling epileptogenesis induces long-lasting changes in the GABAA receptor agonist binding sites that are region specific. We hypothesize that the changes encountered at the fully kindled stage, i.e. increased binding in the fascia dentata and decreased binding in the CA1 area, may underly the electrophysiologically observed increased paired-pulse depression of field potentials in the former and the decreased paired-pulse depression in the latter area [Kamphuis et al. (1992) Neurosci. Lett. 141, 101-105; Kamphuis et al. (1988) Brain Res. 440, 205-215; Zhao and Leung (1991) Brain Res. 564, 220-229; Zhao and Leung (1992) Brain Res. 582, 163-167]. We conclude that the observed changes may not only contribute to the induction of kindling epileptogenesis but may also play a role in the maintenance of the kindled state.

The alpha-glucan-uridine diphosphate glucosyltransferase reation for the identification of glycogen-depleted muscle fibers.

This paper decribes the use of the alpha-glucan uridine di-phosphate glucosyl transferase reaction for enhancing the contrast between glycogen depleted and non-depleted muscle fibers in the periodic acid schiff (PAS) reaction. Muscle fiber glycogen was depleted by prolonged repetitive stimulation of single motor units of the extensor digitorum longus muscle from the rat.

An improved immunocytochemical staining method for large semi-thin plastic epon sections: application to GABA in rat cerebral cortex.

We describe here a procedure that significantly enhances the intensity and method-specificity of immunocytochemical staining in large mounted semi-thin plastic Epon sections. The procedure was developed for the detection of the neurotransmitter gamma-aminobutyric acid (GABA) in rat brain tissue fixed with glutaraldehyde, but it may also be helpful in unmasking other antigens under different conditions. In addition to some practical suggestions for improving the reproducibility of the staining procedure, we demonstrate that the crucial step in the procedure is pre-treatment of the deplasticized sections with proteinase-K before exposure to the first antibody. This leads to a high morphological resolution and an excellent immunocytochemical signal.

Quantification of antiserum reactivity in immunocytochemistry. Two new methods for measuring peroxidase activity on antigen-coupled beads incubated according to an immunocytoperoxidase method.

Antigens covalently coupled to agarose beads provide a matrix for an economical, sensitive, and quantitative immunocytochemical detection of antiserum bindings potencies. Despite some very powerful features (e.g., the ability to control the outcome of a solid phase adsorption on the same matrix), the use of this technique is not very widespread when compared with the other enzyme-linked immunosorbent assay (ELISA) techniques. The main reason for this is the necessity for rather laborious measurements of the immunocytochemical tracer on individual beads. A description of two new methods for the batch measurement of the peroxidase activity on immunoperoxidase incubated antigen-coupled beads is presented. The first method involves the measurement of the diaminobenzidine (DAB) extinction from a large number of beads with a scanning microspectrophotometer. In the second method, during the peroxidase reaction, the beads are incubated with o-phenyldiamine (OPD), which is soluble both in the reduced and oxidized form, whereby absorbance measurements of the supernatant of the beads in a normal spectrophotometer are possible. The sensitivity and the quantitative relation between bound first antibody and absorbance are compared for both methods. From the two immunoperoxidase procedures used (the three step peroxidase-antiperoxidase and the two-step peroxidase conjugate procedure) only the latter met the conditions for a quantitative (first) antibody assay.

Double immunolabeling of neuropeptides in the human hypothalamus as analyzed by confocal laser scanning fluorescence microscopy.

The main goal of this study was to develop a better light microscopic procedure for quantitative study of the cellular co-localization of neuropeptides in adult human brain tissue. To reach this goal, we opted for a method (proved to be optimal on rat brain) in which sections were double immunolabeled with two different fluorophore-conjugated secondary antibodies and analyzed with a confocal laser scanning fluorescence microscope. One of our main problems faced was a strong autofluorescence of the sections, mainly caused by lipofuscin granules normally present in adult human brain tissue, which made any analysis of specific fluorescence impossible. This problem could be solved by staining the sections after immunolabeling with the dye Sudan Black B, which completely blocked this autofluorescence. The complete optimized procedure that we eventually developed can be summarized as follows. After a relatively short fixation time (6-14 days) in 4% freshly depolymerized paraformaldehyde, the resected brain tissue can best be stored in a 30% sucrose solution supplemented with 0.05% NaN3 at 4C. Stored under these conditions, cryosections from the tissue still reveal good histology and allow successful immunocytochemical staining after a period of 6 months. Double immunolabeling is done by incubating cryo- or paraffin sections in a mixture of two primary antibodies directed against the targeted antigens, followed by incubation with two different fluorophore-conjugated secondary antibodies. Amplification with a biotinylated secondary antibody followed by fluorophore-conjugated streptavidin is possible. Finally, the sections are stained with Sudan Black B, mounted in plain 80% Tris-buffered glycerol, and studied by confocal laser scanning fluorescence microscopy. Sections processed in this way are well suited for qualitative and quantitative analyses of co-localized neuropeptides in human brain tissue.

Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G.

Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.

Specificity of oxytocin and vasopressin immunofluorescence.

A total inhibition of immunofluorescence could not be obtained using the technique of preincubating either anti-vasopressin or anti-oxytocin plasmas with their homologous antigens. An alternative test of specificity was therefore developed. Lysine-vasopressin (LVP), arginine-vasopressin (AVP) or oxytocin were covalently bound to CNB-activated agarose beads. These hormone-coupled beads were then either placed immediately on glass slides and treated in the same way as tissue sections for immunofluorescence, or pre-incubated in test-tubes with the antibodies and then prepared for immunofluorescence. Fluorescence was measured quantitatively using a Leitz orthoplan microscope with epi-illumination and a photometer attachment. Without pre-incubation the anti-oxytocin plasmas produced intensive fluorescence on those beads containing their homologous antigen (oxytocin) but only slight fluorescence with the heterologous antigens (AVP or LVP). Anti-vasopressin plasmas produced intensive fluorescence of AVP-, LVP- and oxytocin-containing beads. Because the neurohypophysial hormones were bound to agarose beads, antibodies binding to these hormones could be removed by simple centrifugation. Anti-oxytocin and anti-vasopressin lost their fluorescence capacity after pre-incubation with oxytocin- or vasopressin-containing beads respectively, showing that all the antibodies important for fluorescence were bound to homologous antigens. Pre-incubation of anti-oxytocin or anti-vasopressin with beads coupled to their heterologous hormones completely removed the components binding to the heterologous hormone, leaving antibodies that showed fluorescence only with oxytocin or vasopressin respectively. This purification showed that in contrast to the findings with homologous antigens, not all of the antibody population bound to its heterologous antigen. Using non-purified anti-vasopressin, immunofluorescence was observed in neurohypophyses of homozygous Brattleboro rats. This was evidently due to those antibodies which bind to oxytocin, since the fluorescence was abolished after pre-incubating the antibody-containing plasmas with oxytocin-coupled beads. Immunofluorescence was still observed, however, in the suprachiasmatic nucleus of both Wistar and heterozygous Brattleboro rats, if purified anti-vasopressin was used. This was probably due to either vasopressin storage or production in these hypothalamic cells.

Immunocytochemical localization of vasopressin-binding sites in the rat kidney.

The rat kidney and brain are major target organs for vasopressin (VP). A procedure was developed for immunocytochemical staining of VP and its binding sites in the kidney. This procedure involved preincubation of kidney sections with the ligand, followed by immunocytochemical detection of VP. The staining in renal tubules from Wistar rats was enhanced by preincubation of tissue sections with increasing concentrations of VP (6-6000 nmol/l). Staining was present in the epithelium of distal convolutions and collecting ducts (medullary and cortical portions) and more pronounced in the apical zone of the tubular epithelium. With high concentrations of VP in the preincubation, staining was also obtained in the thick ascending limb of the loop of Henle. There was no staining under any circumstances in proximal tubules. In the kidney of the Brattleboro rat homozygous for hypothalamic diabetes insipidus (DI) which congenitally lacks VP but responds to the peptide, exactly the same staining pattern was observed after preincubation with VP, but the maximal staining was less intense. The VP binding to the DI rat kidney, after 2 weeks treatment with VP (using Accurel implants), reached levels seen in the Wistar kidney after in-vitro preincubation with high doses of VP.