Arja A. Sluiter

Localization of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the human hypothalamus; age-dependent colocalization with vasopressin.

Immunocytochemical staining, using a monoclonal antibody against corticotropin-releasing hormone, was performed on hypothalami of 13 human subjects between 23 and 91 years of age who had not suffered from a primary neurological or psychiatric disease. Corticotropin-releasing hormone (CRH) immunoreactivity was present in neurons of the paraventricular nucleus (PVN) and in their fibers running to the median eminence. The CRH-positive neurons were scattered throughout the PVN, but in the rostral part relatively few cells were present. There were large individual differences in the number and staining intensity of CRH neurons in the PVN and in the staining intensity of the median eminence. These differences seemed not to be attributed to age, sex, postmortem delay, fixation time or hour of death. In the rat, too, no relationship was found between a postmortem delay of up to 24 h and CRH staining intensity of the median eminence. Since the distribution of CRH-immunoreactive neurons in the human PVN strongly overlap with vasopressin, colocalization of these peptides was investigated in a double label study and indeed found in subjects ranging between 43 and 91 years of age. However, cells staining for only one of the peptides were also observed. The vasopressin cells had a mean cellular profile area which was 2.3 times as large as the CRH cells and 2.2 times as large as the CRH and vasopressin containing neurons. In younger subjects (23-37 years of age) no colocalization of the two peptides was seen. The age-dependent colocalization of CRH with vasopressin is interpreted as a sign of increased activation of the CRH neurons with age.

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.

Cells in human postmortem brain tissue slices remain alive for several weeks in culture

Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue slices obtained by autopsy within 8 h after death can be maintained in vitro for extended periods (up to 78 days) and can be manipulated experimentally. We report for the first time that 1) neurons and glia in such cultures could be induced to express the reporter gene LacZ after transduction with adeno‐associated viral vectors and 2) cytochrome oxidase activity could be enhanced by the addition of pyruvate to the medium. These slice cultures offer new opportunities to study the cellular and molecular mechanisms of neurological and psychiatric diseases and new therapeutic strategies.— Verwer, R. W. H., Hermens, W. T. J. M. C. Dijkhuizen, P. A., ter Brake, O., Baker, R. E., Salehi, A., Sluiter, A. A., Kok, M. J. M., Müller, L. J., Verhaagen, J., Swaab, D. F. Cells in human postmortem brain tissue slices remain alive for several weeks in culture. FASEB J. 16, 54–60 (2002)

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.

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.

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.

The monoclonal antibody Alz-50, used to reveal cytoskeletal changes in Alzheimer's disease, also reacts with a large subpopulation of somatostatin neurons in the normal human hypothalamus and adjoining areas

The monoclonal antibody Alz-50 is directed against Alzheimers disease-related modified tau proteins and reveals cytoskeletal changes, i.e. neurofibrillary tangles and dystrophic neurites. The present study shows that, in the hypothalamus of non-demented control subjects, this same antibody gives a distinctive staining pattern of a subpopulation of somatostatin neurons and beaded fibres. Furthermore, Alz-50 occasionally recognizes somatostatin-containing cell bodies and dystrophic neurite-like fibers in the (neuritic) senile plaques of AD patients. These observations have direct consequences for the interpretation of Alz-50 staining in diagnostic usage and for the assessment of Alzheimers disease-like changes induced by beta-amyloid in experimental animal brains. On dot spotting, Alz-50 was found to bind to a number of fragments from the somatostatin precursor, of which somatostatin 15-28 stained best. Preadsorption of Alz-50 by somatostatin 15-28, as well as other specificity tests, failed, however, to provide any clue to the nature of the unknown compound(s) stained in the control hypothalamus.

Decreased hippocampal metabolic activity in Alzheimer patients is not reflected in the immunoreactivity of cytochrome oxidase subunits.

In the present study we have compared histochemically determined cytochrome oxidase activity with the levels of immunocytochemically stained cytochrome oxidase subunits (CO II and CO IV) and ATP synthase in the human hippocampus in relation with Alzheimers disease. Cytochrome oxidase activity was significantly reduced in all hippocampal areas of Alzheimer patients. The protein levels of subunits II and IV were not different between control subjects and Alzheimer patients. Additionally, it was observed that the active cytochrome oxidase is evenly distributed over both cell bodies and neuropil, while a relatively large pool of inactive enzyme or precursors is limited to the neuronal somata. Further, in Alzheimer patients the CO IV immunoreactivity decreased with age, whereas in control subjects it increased with age. Our results suggest that the assembly of cytochrome oxidase or the processing of its subunits may be impaired.

Distribution of Lys-γ2-melanocyte-stimulating hormone- (Lys-γ2-MSH)-like immunoreactivity in neuronal elements in the brain and peripheral tissues of the rat

Abstract Using an antiserum raised against Lys-γ 2 -melanocyte-stimulating hormone (Lys-γ 2 -MSH), with a high specificity for this peptide and its des-Lys derivative, γ 2 -MSH, we found Lys-γ 2 -MSH-like immunoreactivity to have a widespread distribution in the rat brain. In colchicine-treated rats, groups of immunopositive cell bodies were found in the intermediate and anterior lobes of the pituitary gland, in the hypothalamic arcuate nucleus and in the commissural part of the nucleus of the solitary tract (NTS). Immunopositive fibers were found to originate from the latter two cell body regions. The distribution of these fibers was similar to that of the pro-opiomelanocortin-containing cell bodies and projections as it has been described previously. Immunopositive terminals were found in brain regions containing neurons which have been shown to express mRNA for melanocortin receptors, though the distribution of Lys-γ 2 -MSH-like immunoreactivity is considerably more widespread than that of mRNA for the ‘γ-MSH receptor’ (the melanocortin MC 3 receptor), which has been reported to be mainly expressed in the hypothalamus. In the periphery Lys-γ 2 -MSH immunoreactivity was localized in the adrenal medulla and in neuronal fibers and varicosities in the heart. The vascular system, the bronchi and kidney were immunonegative. The occurrence of Lys-γ 2 -MSH immunoreactivity in many of the brain regions which are involved in cardiovascular regulation offers leads for further studies on the putative role of γ-MSHs in cardiovascular control. The occurrence in the rat heart of Lys-γ 2 -MSH-containing fibers suggests a role of the γ-MSHs in cardiac function.

Post-mortem brain tissue cultures from elderly control subjects and patients with a neurodegenerative disease

Aging may be viewed as a progressive loss of normal biological function. Due to complex genetic and environmental interactions, the sequence of functional impairment shows a high degree of individual variability. In humans life style and health care have an additional influence on the aging process. To study aging and age-related disorders of the human nervous system, brain tissue that has undergone aging and pathological alterations can provide valuable study material. Recently, we have shown that adult human postmortem brain tissue can be cultured and experimentally manipulated. This approach permits the study of cellular aspects of human neuronal aging and neurodegenerative processes and complements those existing research methods such as in vivo imaging (MRI, PET, etc.) and fixed or frozen postmortem brain tissue examination.